B1 revision targeted decks

Question 1

Histology

Describe features of neural tissue

Answer 1

• Cellular Elements
• Neuron (Nerve Cell)
• Neuroglial Cells
  – central neurglia (astrocyte,
  oligodendrocyte, microglia and ependymal
  cell)
  – peripheral neuroglia (schwann cell in
  nerve and ganglion satellite (capsular) cell
  in ganglion)
• Intercellular Substance: extremely small amount

CNS:
* Neurons and their processes
* Glial cells
– Oligodendrocytes: small round dense homogenous nucleus, cytoplasmic processes wrap axons to form myelin
– Astrocytes:processes, pale chromatin, round to oval nuclei, physical and metabolic support to neurons
– Microglia: elongated irregularly shaped nucleus, clumped chromatin, mesodermal origin, same origin as monocytes, fixed macrophage
– Ependymal cells: line ventricles, ciliated, cuboidal or low columnar, lack of tight junctions
* Collagen only found around
blood vessels
* Meninges –surrounds brain: dura mater, dense fibroelastic tissue
* Choroid plexus: capillaries and choroid cells with microvilli, arises wall of ventricles, makes CSF

Question 2

Histology

Describe connective tissue types and components

Answer 2

Ordinary connective tissues (connective tissue proper)
Loose ordinary connective tissue: mesenchyme, areolar, mucoid, reticular, adipose white and brown
Dense ordinary connective tissue - irregular or regular

Specialised connective tissues
Adipose tissue
Blood and blood forming tissues
Cartilage
Bone
Elastic tissue

*Cells - indigenous
*Fixed - Fibroblasts, Reticulocytes,
Adipocytes
*Wandering - Macrophages, Mast cells,
Eosinophils, Lymphocytes, Plasma cells

• • migratory
    *Neutrophils, Eosinophils
    *Lymphocytes, Monocytes
    *Extracellular matrix
    *Fibres:
    *Collagen
    *Reticular fibres
    *Elastic fibres
    *Ground substance
    *Proteoglycans, water, salts and other
    low molecular substances

Question 3

List and describe the features of the three types of muscle

Answer 3

Skeletal muscle:
Both types found in all human muscles in different proportions
Type 1: Slow contraction -
Aerobic, using oxidative phosphorylation, many mitochondria.
Cells contain large amount of myoglobin (O 2 storage - red
colour), many capillaries
Type 2: Fast contraction -
Anaerobic, using glycolysis, therefore rich in glycogen,
white colour
—
 Muscle fibres contain myofibrils
 Myofibrils are elongated cylindrical
structures made up of contractile proteins
 Cross striations of striated muscle is due to
the ordered arrangement of these
contractile proteins
— The dark A band is made of thick myosin
filaments
 The light I band is made of thin actin filaments
 The Z bands mark the anchor points of the
actin filaments
 Triads (terminal cisternae and T tubules) at A-I
junctions
—
 Not striated
Small cells spindle shaped, single central oval nucelus
still have actin and myosin, No Z, but dense bodies and attachement junctions
 Involuntary
 Under autonomic and hormonal control
 Visceral structures
 Enables continual contractions of low force
Synaptic control: unitary/multiple; phasic and tonic
—
 Elongated cells
 Central nuclei
 Branching fibres with intercalated discs
 Contractile proteins arranged similarly to skeletal
muscle
 Cross striations
 Contractile units: sarcomeres made of myosin
and actin

 Sarcoplasmic reticulum
and T tubules similar to
skeletal muscle
 T tubules are larger
and located at the level
of the Z disc
 Contractile proteins
arranged similarly to
skeletal muscle

Intercalated Discs
 Made of 3 types of membrane
contacts
 Fascia adherens
 site of actin filament insertion
 Desmosomes
 anchorage for intermediate
filaments
 Gap junctions
 pores with low electrical resistance
enabling ion and molecule transfer
between the cells → coordinated
contraction

Question 4

List the gap junctions between epithelial cells and briefly describe them

Answer 4

Three are different types of connecting junctions, that bind the cells together.

occluding junctions (zonula occludens or tight junctions)
adhering junctions (zonula adherens).
desmosomes (macula adherens). There are also ‘hemidesmosomes’ that lie on the basal membrane, to help stick the cells to the underlying basal lamina.
Gap junctions. These are communicating junctions. (also known as nexus, septate junction)

Question 5

List types of cartilage and describe the process of cartilage formation

Answer 5

• hyaline cartilage: articular cartilage, glassy blue
• fibrocartilage: white, eg IV discs
• elastic cartilage: yellow, found in ears

• Begins fifth week of life
• Precursor cells become rounded and form densely packed cellular
  masses, centres of chondrification. The cartilage-forming cells,
  chondroblasts, begin to secrete the components of the extracellular
  matrix of cartilage
• As amount of matrix increases chondroblasts become separated from
  each other
• Chondroblasts become isolated in small cavities within the matrix
  lacunae
• Chondroblasts differentiate into mature cartilage cells
  chondrocytes

Cartilage growth occurs by two mechanisms:
* Interstitial growth
* Appositional growth

Interstitial growth
* Chondroblasts within
existing cartilage divide and
form small groups of cells,
which produce matrix to
become separated from
each other by a thin
partition of matrix
* Interstitial growth occurs
mainly in immature
cartilage

Appositional growth
Mesenchymal cells surrounding
cartilage in the deep part of
perichondrium (or the chondrogenic
layer) differentiate into
chondroblasts. Appositional growth
also occurs in immature and mature
cartilage

Question 6

Pathology

Describe the components of tymour survival, and steps of malignant transformation

Answer 6

Two key basic components of a neoplasm:
- proliferating neoplastic cells - these determine the behaviour and outcome of the neoplasm
- stromal component/desmoplastic stroma - which is responsible for the neoplasm’s growth and evolution

1. Malignant transformation of target cells
   - cell cycle inhibitors work on G1 cell cycle checkpoint. Key cyclin here is cyclin D/CDK4
   - p53 or “guardian of the genome”, is key molecule at G2 cell cycle checkpoint. Often perturbation of p53 is associated with cancer development
2. Proliferation and accumulation of transformed cells
   Proliferation determines the rate of neoplasm growth.
   Transformed cells accumulate due to two factors:
   - inhibition of apoptosis ^[[[Pathology Lecture 5]]]
   - telomerase activity, which confers limitless replicative potential
3. Local invasion
   Invasion is a biological hallmark of malignant tumours, as defined as tissue moving into sites where they should not be.
   Invasion is facilitated by the loss of adhesion molecules on neoplastic cells ^[aka non-functional or absent desmosomes].
4. Distant metastasis
   Distant metastasis is defined as the presence of neoplastic implants at a site away from the primary tumour.

It is a hallmark of malignant neoplasms (along with invasion).

Question 7

Pathology

• Define the acute phase response, outline the cells involved

Answer 7

The acute phase response (APR) is a prominent systemic reaction of the organism to local or systemic disturbances in its homeostasis caused by infection, tissue injury, trauma or surgery, neoplastic growth or immunological disorders.
The acute-phase reaction characteristically involves fever, acceleration of peripheral leukocytes, circulating neutrophils and their precursors.
n response to injury, local inflammatory cells (neutrophil granulocytes and macrophages) secrete a number of cytokines into the bloodstream, most notable of which are the interleukins IL1, and IL6, and TNF-α. The liver responds by producing many acute-phase reactants. Positive acute-phase proteins serve (as part of the innate immune system) different physiological functions within the immune system. Some act to destroy or inhibit growth of microbes, e.g., C-reactive protein, mannose-binding protein,[3] complement factors, ferritin, ceruloplasmin, serum amyloid A and haptoglobin. Note EST correlates with CRP and other acute phase proteins, but is not direct—depends on elevation of fibrinogen.

Question 8

Pathology

List the features of malignancy

Answer 8

Cytological features:
- variable cell size and shape (cellular pleomorphism) ^[pleomorphism = variation in appearance]
- variable nuclear size and shape (nuclear pleomorphism)
- increased nuclear- cytoplasmic ratio (normal 1:4, 1:6 – ratio in neoplastic cells may approach 1:1)
- hyperchromatic nuclei (increased DNA, on haematoxylin and eosin, looks more ‘blue’)
- increased mitosis/abnormal mitosis
- tumour (neoplastic) giant cells

Architectural features:
- refers to how the cells relate to each other, the arrangement of cells with respect to each other
- another way to think about it is organisation
- in general, benign neoplasms are orderly, and benign neoplasms tend to grow by expansion, and is often associated with capsule, while malignant neoplasms are disordered, and malignant neoplasms infiltrate and ‘penetrate neighbouring tissues with hostile intent’

Question 9

Pathology

• Distinguish between granuloma and granulation tissue

Answer 9

Granulomatous inflammation is a special type of chronic inflammation.
A granuloma is a collection of activated (epithelioid) macrophages, often surrounded by T lymphocytes, sometimes with central necrosis.

• Granulomata are small - usually microscopic
• Granulomata form in order to try to contain an offending agent that is difficult to eradicate e.g. foreign material (sutures), some infections
• activated macrophages develop abundant cytoplasm and begin to resemble epithelial cells i.e. epithelioid histiocytes
• macrophages may fuse to form multinucleated giant cells

Formation of granulation tissue: fibroblasts, loose connective tissue, new blood vessels and interspersed leukocytes. Normal part of scar formation, general feature of chronic inflammation.

Question 10

Pathology

Describe processes of wound healing and repair

Answer 10

There are two pathways to repair:
- regeneration
- scar formation
Regeneration can take place in tissues with labile and stable tissues when tissue damage isn’t extensive.
Regeneration can occur via two mechanisms:
- proliferation of mature cells
- proliferation and differentiation of tissue stem cells

The restoration of tissue architecture can only occur if the tissue is generally structurally intact.

Three broad steps:
1. Priming via cytokine signals
2. Growth factor phase: stimulate gne expression, cell cycle enry and replication
3. Termination phase- back to quiescent state

Scarring:
1. Inflammation – leukocyte invasion
2. Angiogenesis due to gfs
3. Granulation tissue formation
4. Connective etissue deposition by fibroblasts eg ECM
5. Contraction of scar by myofibroblasts
6. Remodelling – stable fibrous scar

Can heal by primary intention: when the injury only involved the epithelial layer, with only focal BM disruption and connective tissue/cell death or secondary: cell and tissue loss is extensive.
Primary steps:

1. In the immediate: a blood clot forms
2. Within 24 hours, inflammation occurs, infiltration of PMNs
3. Within 24 to 48 hours, epithelial cells migrate from the edges of the wound
4. On day 3, macrophages predominate, and granulation tissue begins to migrate
5. On day 5, VEGF= peak neovascularisation (of leaky vessels), ECM deposition
6. After 1 week: continued collagen and ECM deposition, and vascular regression (strength decreased to 10% of normal)
7. After 1 month, an established scar is formed, with minimal inflammation, epidermis repaired, adnexal structures , i.e. appendages, lost
8. Onwards, scar matures and increases in strength i.e. 70-80% of normal
   Note in healing by secondary intention, much more granulation tissue is formed.

Note that the timeline of healing by secondary intention is similar to the processes involved in primary intention. The major differences include:
- larger clot
- more necrosis
- more inflammation
- more granulation tissue formed
- wound contraction is important

Question 11

Clinical disciplines/pathology/haematology

List the types of anaemia and the changes you would expect

Answer 11

Question 12

Physiology

List the causes of shock and responses by the body

Answer 12

Physiological shock is a state of cardiovascular dysfunction resulting in generalised inadequacy of oxygen delivery or DO2, relative to the metabolic requirements.

In other words, an imbalance.

1. Hypovolaemic
   The primary problem is:
   - intravascular volume loss: bleeding/lose excess fluid from gut or kidneys
   - Inadequate stretch of muscle fibres

• inadequate volume into heart i.e. pre-load
• Low stroke volume
• Low cardiac output
• DO2 = (SV x HR) x ([Hb] x SaO2 x 1.39) + PaO2 x 0.003
  Response:
• Tachycardia (CO = ↓SV x ↑HR)
• Vasoconstriction (BP = ↓CO x ↑TPR) (cold peripherally and pale)
• Sympathetic outflow (sweating)

Treatment:
- replace whichever fluid has been lost
- correct underlying problem e.g. thirst, diabetic ketoacidosis, fluid replacement, and insulin treatment

2. Cardiogenic shock
   - Failing heart (myocardial infarct, cardiomyopathy)
   - Too much stretch of myocardial fibre
   - Low stroke volume
   - Low cardiac output

DO2 = (SV x HR) x ([Hb] x SaO2 x 1.39) + PaO2 x 0.003

Response:
- Tachycardia (CO = ↓SV x ↑HR)
- Vasoconstriction (BP = ↓CO x ↑TPR)
(cold peripherally and pale)
- Sympathetic outflow (sweating)

Treatment:
- inotropes
- fluid restriction
- diuretics
- correct underlying problem (ischaemia, valve)

~Similarities between hypovolaemic and cardiogenic shock~

Primary problem:
- Hypovolaemia: inadequate filling of heart, low stroke volume
- Cardiogenic shock: low stroke volume (due to failing heart: contractility and valvular dysfunction)

Response:
- sympathetic outflow (sweating)
- tachycardia
- vasoconstriction (cold peripherally, tachycardiac, sweaty)

~ Differences~

• Hypovolaemia:
  
  • postural hypotension
  • invisible JVP or low CVP
  • evidence of loss: blood, negative fluid balance, diarrhea, vomiting
• Cardiogenic:
  
  • signs of heart failure: raised JVP, oedema (pulmonary and peripheral), gallop rhythm
  • reasons for heart failure: arrhythmia, ischaemia/infarct, VSD, valve failure

3. Septic shock
   Primary problem:

Initially:
- excessive cytokine release (infection, inflammation)
- peripheral vasodilation (dec. BP = CO x dec. TPR)
- reduced venous return to heart
- reduced stroke volume
- reduced cardiac output

More complicated:
- treatment includes supplementing with fluids
- improves preload and hence stroke volume
- already tachycardiac (temperature)
- improves cardiac outpit, often massively, in low resistance system
- huge DO2, but:
- still vasodilated (warm) with adequate pre-load, BP remains low
- and cells cannot utilise oxygen

Two stages:
1. decreased preload to heart (low cardiac output)
2. with fluids, large cardiac output, but low BP - need pressure gradient for there to be flow

Treatment:
- correct underlying problem
- volume resuscitation
- vasoconstriction
- antibodies

4. Obstructive shock
   Primary problem:
   Inadequate volume into the (left or right) heart

• Good systemic venous return
• Inadequate filling of the heart eg cardiac tamponade ^[cardiac tamponade** a dangerous situation in which there is a build-up of fluid around the heart within the pericardial sac. This causes compression of the heart, which is therefore unable to fill with blood adequately in order to pump effectively], pulmonary embolus
• Reduced Stroke Volume
• Reduced Cardiac Output

DO2 = (SV x HR) x ([Hb] x SaO2 x 1.39) + PaO2 x 0.003

Response:
- Tachycardia (CO = ↓SV x ↑HR)
- Vasoconstriction (BP = ↓CO x ↑TPR) (cold peripherally and pale)
- Sympathetic outflow (sweating)
- raised JVP
- pulsus pardoxus

Treatment:
Depends on aetiology
- cardiac tamponade: fill the right heart as much as possible, drain the fluid as quickly as possible
- pulmonary embolus: thrombolysis, ongoing anticoagulation

Question 13

Physiology

Describe the oxygen delivery equation, why oxygen is important, and list and describe the determinants of oxygen delivery

Answer 13

Oxygen is important in physiology because it is the terminal electron acceptor at the end of the electron transport chain in the mitochondria ([[Biochemistry Lecture 8]]). It is thus integral to aerobic respiration and the generation of (a lot of ) ATP.
In other words, oxygen is necessary for energy production.
Energy is generated by catabolising macromolecules i.e. sugars, fats and amino acids.
Catabolism of all three macromolecules consumes oxygen and produces or releases carbon dioxide. However, the ratio of oxygen consumed to carbon dioxide produced is different. This ratio can be expressed as the respiratory quotient (RQ).

DO = C0 Xaoc
= svhr * (Hb conc sao21.39 + paO20.003)

Question 14

Biochemistry

List and describe the steps of cholesterol synthesis

Answer 14

There are five main steps to cholesterol synthesis:
1. Mevalonate synthesis in the cytosol: 3 moleciles molecules of acetyl CoA used
- Reaction sequence identical to KB synthesis
- But reactions are cytosolic and HMG- CRA reductase catalyses the committed step
- Enzyme attached to ER membrane
- Irreversible reaction
- Regulated step of pathway
note: this is essentially the reverse of ketone body synthesis (swapping out lyase for reductase)
2. Isoprene formation or activation. 2 isoprenoids are formed, and this has an energy cost
3. activated isoprenes are used in the condensation to squalene (6 used).
4. Squalene undergoes ring closure, this occurs in the ER
5. Cholesterol is formed

Question 15

Describe the link between gluconeogenesis and ketogenesis

Answer 15

The carbon skeletons of amino acids may proceed to form either glucose or ketone bodies (a fuel source that brain cells can use in starvation). In other words the C skeleton can be metabolised for energy release.

Carbon skeletons that lead to glucose are said to be glucogenic e.g. alanine. Carbon skeletons that lead to ketone bodies are said to be ketognenic.

note that there are also ‘mixed’ amino acids i.e. Phe, Trp and Tyr.

Question 16

Describe glucose storage

Answer 16

Storage of carbohydrates for energy provision

The ‘normal’ blood glucose of a healthy adult is between 3.6 to 5.8 mM.
Post meal levels may rise to 7.8 mM in non-diabetics. The recommended post-meal level for healthy adults is less than 10 mM. ^[the calculation for these values in terms of amount: 75 kg male, blood volume of 5L, 5.5 mM corresponds to 5 g of glucose]

In order to maintain this range of glucose concentrations, there must be a mechanism for glucose uptake (storage) and mobilisation.

The liver is the organ primarily responsible for glucose uptake. If the concentration of glucose falls below 4 mM, there is low hepatic uptake. However, if the concentration increases higher than 7mM, there is increased hepatic uptake.

(note that between 4 and 7 mM, not much uptake?)

The properties of uptake are controlled by the characteristics of GLUT-2 transporter proteins. In other words it constitutes a key regulatory mechanism. [[Physiology Lecture 2]].

The key players of glucose storage
GLUT-2 works to uptake glucose into hepatocytes (ergo, GLUT-2 is found in the liver). GLUT-2 engages in facilitated (passive) transport. It is a ?low affinity, and high capacity process, and is insulin insensitive.
Notably, GLUT-2 is not strictly glucose specific, and can transport galactose and fructose.

Enzymes, glucokinase in the liver and hexokinase elsewhere (notably in the muscle), phosphorylate glucose to glucose-6-phosphate. Not that although glucokinase has higher affinity (high Km) for glucose compared to hexokinase, their main purpose is the same: to maintain the glucose gradient into the cell by ensuring glucose goes into the appropriate cell.

Question 17

List and describe the bypass reactions of gluconeogenesis

Answer 17

REACTION 1:
* pyruvate carboxylase enzyme
* ATP dependent reaction
* carboxylation of pyruvate
* mitochondrial reaction

REACTION 2:
* PEP carboxy-kinase enzyme
* ATP dependent reaction
* cytosolic reaction

PFK1
* Phosphofructokinase-1
* pace-setting of glycolysis
* reaction driven to completion
* reverse reaction unfavourable
F1,6bPase
* Fructose-1,6-bis-phosphatase
* de-phosphorylation

Not a simple reversible reaction
Step requires distinct enzyme to glycolysis
Reciprocal regulation of enzyme activity

The final release

Energy precludes the reverse of the hexokinase/glucokinase reaction
(ATP dependent)
Energy considerations again!
The enzyme is glucose-6-phosphatase
G6-Pase is found only in liver/kidney
These tissues participate in gluconeogenesis and provide
glucose for peripheral tissues
Glucose (but not glucose-6-phosphate) can leave cells via GLUT

Question 18

List some fates of cholesterol

Answer 18

There are many utilizations of cholesterol and its derivatives in the body.

Esterification
Synthesised cholesterol is exported to peripheral tissues, the transport of synthesized cholesterol occurs from the liver via lipoproteins. The primary storage particles for cholesterol are LDLs. Cholesterol is converted to an ester form, which increases its hydrophobicity. ACAT is an enzyme found in the liver, which adds a fatty acid to cholesterol to form cholesterol esters. L-CAT transfers an acyl group from PC which are found in HDL particles.

Bile components i.e., using fat to digest fat
Cholic acid or bile acids are polar versions of cholesterol. They are synthesized in the liver and are stored in the gallbladder and then released to bile. Colonic acid or bile acids have detergents like properties which worked to break down or emulsify fatty acids. Thus, they have a role in solubilising dietary lipids.

Glycolic acid
Bile salts are conjugated bile acids. They are hydrophilic and are found in the small intestine. Glycine and taurine are frequent conjugates. Bile salts also have detergent like properties and work to solubilise dietary lipids.

Hormones
Progesterone is a steroid precursor. It prepares the uterus for implantation and prevents ovulation. Cortisol is another hormone derived from cholesterol. It promotes gluconeogenesis and suppresses inflammation. Testosterone is another cholesterol derived hormone which promotes male sex development and maintains characteristics. Estradiol is a another cholesterol derived hormone which promotes female sex development and maintains characteristics.

Question 19

Describe the process of glycolysis and maintenance

Answer 19

The glycolytic pathway is the primary reaction involved in the catabolism (oxidation) of glucose.
In summary:
![[Pasted image 20230315133112.png]]
Glycolysis occurs in the cytoplasm of the cells.
It is the partial oxidation of glucose, yielding two 3C molecules known as pyruvate. It also generates 2 ATP (net) per glucose, and also generates the reduced co-factor NADH (2 per molecule of glucose).

Glycolysis links to the TCA cycle for complete glucose oxidation. It also links to the anabolic pentose phosphate pathway.

The glycolytic pathway is responsive to the cellular state and to hormones.
The pathway occurs under both aerobic and anaerobic conditions. It is a vital pathway for erythrocytes(how does it do this?) as well as the brain.
The complete pathway is shown below
![[Pasted image 20230315133506.png]]
Some important takeaways from this:
- The first step, catalysed by hexokinase, is an energy dependent step i.e. requires ATP (recall also that the enzyme in the liver is glucokinase)
- The third step, catalysed by PFK-1, is a energy dependent step. It is also the first committed step of glycolysis, and is thus highly regulated
- At step 4, 3 carbon compounds are produced from fructose-1,6-bisphosphate (‘snapped in half’) which are inter-convertible
- At step 5, the generation of 1,3 bisphosphoglycerate, catalysed by GA3DPH, generates a reduced cofactor NADH, which links to TCA cycle
(note that low NAD+ concentration limits glycolysis, i.e. energy has been produced?)
- The production of 3-phosphoglycerate generates ATP (2 per molecule of glucose, 1 per 3C compound). This balances the earlier loss of ATP
- The final step which converts PEP to pyruvate also generates ATP(2 per molecule of glucose, 1 per 3C compound). It is catalysed by PK, and this step is also highly regulated.

In order for glycolysis to continue, we need a continual supply of NAD. Thus NADH generated in glycolysis keeps it going through the TCA cycle, regenerating NAD which returns to glycolysis
n.b. this process requires the presence of oxygen

Under aerobic conditions, NAD+ generated via the TCA cycle is returned to cytoplasm via shuttle system from mitochondria. NADH is generated from the conversion of GA3P to 1,3bPG and enters the TCA cycle, and the cycle repeats.

However, under anaerobic conditions, NAD+ must be generated via a different mechanism. Instead of entering the TCA cycle via the acetyl CoA ?link reaction, pyruvate is shunted* to lactate by LDH, this regenerates NAD+ which returns to the GA3P->1,3bPG reaction, and then lactate is recycled by ?liver.
![[Pasted image 20230315134738.png]]

Note: glycolysis is a linear metabolic pathway, with three metabolic pools. These pools represent inter-convertible intermediates. The reactions linking these pools e.g. F6P to F16bP, 1,3bPG to 3PG, and PEP to pyruvate, are key steps and are controlled.

Question 20

Describe fatty acid translocation and catabolism

Answer 20

Three main steps are involved:
1. Acyl CoA conversion i.e. activated fatty acid converted to acyl carnitine by action of CAT1. This conversion or modification is required so that it can be translocated across the membrane
2. Acyl carnitine transported into the mitochondrial matrix by an antiporter (acyl carnitine in, carnitine out)
3. Acyl CoA liberation i.e. the regeneration of acyl CoA with CoASH, by action of CAT2. The acyl CoA can then go on to b-oxidation. This also liberates carnitine which can be transported back
—
There are four main steps that comprise fatty acid catabolism:
1. Oxidation (which reduces FAD to FADH2)
2. Hydration (addition with H20)
3. Oxidation (which reduces NAD+ to NADH)
4. Thiolation (via action of CoASH)
Can cycle through this process multiple times in order to get more NADH and FADH2

A focus on β-oxidation
This process occurs entirely within the mitochondrial matrix. It is termed β-oxidation simply because the β-carbon is the one that gets oxidised.
The entire reaction effectively strips 2 carbons per cycle, and these are liberated as acetyl-CoA ^[this can go on to enter the TCA cycle, generating energy via downstream oxidative phosphorylation). A new (n-2) acyl-CoA rejoins the process, and continues until a single 2C acetyl CoA molecule is produced, which can enter the TCA cycle.
The two reduced cofactors FADH2 and NADH can also go on to enter the TCA cycle to undergo oxidation (and also to generate more energy for the cell)

note that from a single fatty acid molecule: n/2 acetyl CoA molecules, (n/2 -2) FADH2 and NADH are generated, all of which can go on to the TCA cycle and generate energy. This makes fatty acids a very energy dense molecule, when compared with carbohydrates and proteins i.e. it is a more energy efficient process (higher yield) ^[glucose yields about 36 ATP, a 16C fatty acid will generate 129 ATP]

Question 21

Biochemistry

Describe the steps of ketone body synthesis and the utilisation of KBs

HI

Answer 21

Mitochondrial enzymes of the hepatocytes engage in KB synthesis. Synthesis is constant.

• Two Acetyl- CoA molecules condense to form acetoacetyl CoA by the action of thiolase, which removes a CoASH.
• HMG-CoA synthase then catalyses the second step, converting acetoacetylCoA to HMG CoA, using the input of an acetyl-CoA and the removal of a CoASH group
  HMG-CoA lyase removes an aetyl-CoA group in order to synthesise aceto-acetate
  aceto-acetate can then be converted to acetone (by removal of Co2, non-enzymatically) ^[see also: https://www.ncbi.nlm.nih.gov/books/NBK493179/] or b-hydroxy-butyrate (using an NADH, and the action of b-hydroxy-butyrate dehydrogenase)

The process of ketone bodies as a fuel is a truncated i.e. 3step process, which also occurs in the mitochondria.
1. Once KBs reach extra-hepatic tissues, b-hydroxy-butyrate is converted back to acetoacetate by b-hydroxy-butyrate dehydrogenase (This generates an NADH)
2. Aceto-acetate is then converted back to aceto-acetyl CoA by keto-acyl-CoA transferase (this also converts succinyl-CoA to succinate) ^[[Biochemistry Lecture 7]]
3. Aceto-acetate CoA is then broken down into two acetyl-CoA molecules by thiolase
4. These acetyl-CoA molecules can then enter the TCA cycle and oxidative phosphorylation for energy i.e. ATP generation

note: virtually all enzymes involved in KB utilisation are the same as in synthesis, ‘moving backwards’ from KBs to acetyl-CoA. Only key difference is keto acyl-CoA transferase; this is an enzyme not found in the liver as liver is an exporter not a user. ^[note a potential exam question]

Question 22

Describe glucose mobilisation

Answer 22

There are three main processes of the storage and mobilisation of carbohydrates, they include:
- glycogenesis (GG): this occurs following meals. Glucose is stored as glycogen.
- glycogenolysis (GGL): the rapid mobilisation of glucose from storage
- gluconeogenesis (GNG): the synthesis of glucose during prolonged fasting (starvation)
These processes ensure that there is sufficient glucose supply to the brain, while also utilising and scavenging glucose precursors.

~ A closer look at glycogen~
Glycogen, as previously described, is the glucose storage form in (animal) cells. It is a polymer of glucose linked together by a -1,4- glycosidic bonds. At 8 to 10 residue spacing along the chain, the glycogen molecule has branch points, which are connected to the main chain via a -1,6- glycosidic bonds.
The initial residues are joined on the glycogenin primer (which is an enzyme).

There are two key enzymes in the build-up and breakdown of glycogen.
Glycogen synthase catalyses of glucose addition i.e. glycogen synthesis. Addition occurs via UDP-glucose. Note that glycogen synthase cannot generate the a -1,6 glycosidic bond, as so a branching enzymes adds a segment of a polysaccharide (consisting of 6-7 glucose molecules).

Glycogen phosphorylase catalyses glycogen degradation. The reaction involves phosphorolytic cleavage. Similar to glycogen synthase, a separate enzyme, a debranching enzymes is needed to tackle the a-1,6 glycosidic bond.

Question 23

Describe the entry of carbohydrates, amino acids and FAs into TCA

Answer 23

The entry of carbohydrates into the TCA cycle is influenced by hormonal signals i.e. adrenaline and glucagon.
Entry is also controlled by cellular redox status ^[potential exam question]: a decrease in the ATP/ADP ratio and NADH/NAD ratio ^[a high ATP/NADH signals that there is no need for GL and carbohydrate catabolism, there is enough].
The targets for control include:
- glycogen phosphorylase
- glycogen synthase
- hexokinase
- PFK-1 (catalyses the rate-limiting step in glycolysis and change concentration of ATP)
- pyruvate kinase

Mitochondrial entry: glycolytic products
Glycolysis ends at the formation of pyruvate ^[assuming that glycolysis proceeded under anaerobic conditions], and pyruvate enters the mitochondria via a carrier (a hydroxyl antiporter). Lactate may also be shunted back to pyruvate.

![[Pasted image 20230325222800.png]]
note: aCoA comes from glycolysis and b-oxidation; reaction is a favourable reaction.

PDH catalyses the reaction, and is not actullay a single enzyme but rather an enzyme complex (5 in one). The reaction releases energy i.e. is exergonic and therefore is essentially irreversible.
PDH requires TPP, lipoic acid and FAD as cofactors, and is inhibited by ATP, acetyl-CoA and NADH.

The carbohydrate link reaction
This occurs after glycolysis, links pyruvate to acetyl-CoA.
![[Pasted image 20230325223125.png]]
The link reaction consists of four steps:
1. Decarboxylation involves assistance (a TPP prosthetic)
2. Oxidation–transfers electrons to lipoamide
3. Transfer reaction which utilises CoASH
4. Oxidation transfers electrons to FAD, and to NADH, and regenerate lipoamide ^[a small cycle in the linear path]
The reaction is highly regulated by both energy status and hormones e.g. high ATP, NADH, energy status (if high, no need for link reaction).

A focus on β-oxidation ^[note implications for altered metabolism in cancer, see x]
This process occurs entirely within the mitochondrial matrix. It is termed β-oxidation simply because the β-carbon is the one that gets oxidised.
The entire reaction effectively strips 2 carbons per cycle, and these are liberated as acetyl-CoA ^[this can go on to enter the TCA cycle, generating energy via downstream oxidative phosphorylation). A new (n-2) acyl-CoA rejoins the process, and continues until a single 2C acetyl CoA molecule is produced, which can enter the TCA cycle.
The two reduced cofactors FADH2 and NADH can also go on to enter the TCA cycle to undergo oxidation (and also to generate more energy for the cell)
—
recall the dynamic nature of amino acid ‘pool’.
Amino acids and TCA cycle intermediates are interchangeable.
Entry may be via many points; several enter via pyruvate

Question 24

Biochemistry

Describe the synthesis of fatty acids

Answer 24

Entry to fatty acid synthesis occurs after the commitment step. It involves:
- 1: transfer of malonyl-CoA with ACP
- 2: condensation: losing CO2 and ACP, and joining either acetyl-ACP (if it is the first molecule in the chain) or acyl-ACP if joining a growing chain.
- 3: Reduction: which involves the formation of an oxidised cofactor NADP*
- 4: Dehydration
- 5: Reduction: which again results in the formation of an oxidised cofactor NADP*
note that in steps 3-5, a double bond is lost. This is to ensure the production of a linear, stable, unbranched, saturated hydrocarbon chain
- 6: resetting i.e. repeating the cycle with a new malonyl-CoA

Question 25

Distinguish between GNG and GL

Answer 25

The three irreversible steps of GL are bypassed using enzymes unique to GNG:
- PEP to Pyr, instead of PK. Two steps. PC converts Pyr to OAA, OAA is converted to PEP by PEP-Carboxylase
- instead of PFK-1 converting F6P to F16P, F1,6-Pase
- instead of GK, G-6-Phosphatase (going G6P to Gluc)

the seven reversible reactions are shared between GNG and GL

Question 26

Describe lipid movement

Answer 26

All lipids have poor solubility in aqueous solutions. Nevertheless, lipids are essential for cellular function and structure, and are huge energy source (thus dietary absorption is vital).

The amphiphilicity of lipids is a crucial element of their solubility.

The structures that fatty acids and lipids form in an aqueous solution is determined by its physical and chemical properties.

The complexity of lipid transport
:
Depending on their source and end, the pathways used to take up as well as mobilise lipids will differ.
Dietary lipids (FAs, TAGs, DGs) are taken up via the exogenous pathway: absorbed via emulsification and uptake into lipid storage (either in adipose tissue or hepatic storage).
Synthesised lipids from the liver are taken up via the endogenous pathway into lipid storage.

Mobilisation is undertaken by lipases, under the influence of hormones.

Lipoproteins: lipid transport vehicles
Lipoproteins are “ball”-shaped structures, where the hydrophilic heads of phospholipids, arranged in a surface hemi-leaflet, stick out, facing the aqueous environment, while hydrophobic tails point inward, away from the aqueous environment.
The content of lipoproteins is comprised of central TAGs (and cholesterol esters) ^[termed a hydrophobic core], which make up 80% of the mass.
Surface proteins, called apolipoproteins (APLs) interact with (mitochondrial) receptors and activate lipases (i.e. favouring mobilisation). The cholesterol-esters found in the core of lipoproteins are responsible for ‘picking up’ cholesterol and returning it to the liver.

There are several forms of lipoproteins, which can be ranked in terms of size (descending order): chylomicrons, LDL (colloquially bad cholesterol), VLDL, and HDL .
note: plasma lipoprotein particles are key to solving the solubility issue of lipids

Key takeaways from this figure:

• signals in the GI tract causes TAGs and FAs to be broken down and transported
• apoproteins allows for binding and mobilising of FAs
• HDL is a link, a “hoover” that picks up cholesterol and returns it
• VLDL, IDL and LDL differ in terms of density (i.e. )
• peripheral tissue i.e. near muscles

Mobilisation of fat stores
There are several steps to the mobilisation of fat stores:
1. Adipose tissue stores fat (approximately
3. Met Stimulus leads to release of hormones - adrenaline and glucagon
4. Signal transduction occurs at the adipocytes
5. This then leads to activation of hormone sensitive lipase
6. Which then leads to hydrolysis of fatty acids from TAGs
7. FAs then enter the blood and bind to serum albumin
8. Delivery of FAs to peripheral tissues for catabolism

Question 27

Describe the urea cycle

Answer 27

Ammonia is highly toxic to humans. Ammonium ions also exhibit neurotoxicity.
As a consequence, both ammonium and ammonia must be excreted.

Ammonia, on the other hand, is converted to urea for excretion and accounts for the bulk of nitrogen excretion.

-Urea contains 2 nitrogens linked using CO2
The synthesis of urea requires a multi-step pathway. This is mediated by the urea cycle (or ornithine cycle).
This process occurs almost entirely in hepatocytes ( #liver), and is split between the cytosol and the mitochondrial matrix. Urea is then excreted from the body via the #kidney

Question 28

Biochemistry

Describe the processes of protein metabolism and amino acid synthesis

Answer 28

• Protein metabolism
  Polypeptides by pepsin, short peptide by trypsin in small intestine – di/tri by amino-peptidases, uptake by H-linked secondary transport into circulation, amino acids taken up by Na linked secondary transport
• Amino acid synthesis
• all amino acids undergo transamination
• this is a reaction that changes an amino acid to an α-keto acid
• The reaction is oxidative deamination i.e. removal of an ammonia group in the presence of oxygen, which is transferred to another α’keto acid to produce another amino acid
• The α-keto acid can be used for the production of energy, biosynthesis or recycling (see below, aKGs)

As previously described, protein degradation has a number of fates. Amino acids may either be recycled or transformed. The amine moiety of amino acids is usually excreted (i.e. nitrogen is excreted), while the carbon skeleton is used for biosynthesis and energy production.
Cellular funnels are amino acid transferases; these are enzymes that catalyse the transfer of amino group to an α-keto acid.
The typical “acceptor” α-keto acid is usually αKG which funnels amino acids into glutamate. other acceptors can include oxaloacetate] or pyruvate. These will generate Asp or Ala, respectively. This shunting is described as flux

Question 29

Biochemistry

Discuss the dual roles of HMG-CoA

Answer 29

Formation of cholesterol or KB synthesis/acetylCoA when diverted from TCA for GNG
- Two Acetyl- CoA molecules condense to form acetoacetyl CoA by the action of thiolase, which removes a CoASH.
- HMG-CoA synthase then catalyses the second step, converting acetoacetylCoA to HMG CoA, using the input of an acetyl-CoA and the removal of a CoASH group
- HMG-CoA lyase removes an aetyl-CoA group in order to synthesise aceto-acetate
- aceto-acetate can then be converted to acetone (by removal of Co2, non-enzymatically) ^[see also: https://www.ncbi.nlm.nih.gov/books/NBK493179/] or b-hydroxy-butyrate (using an NADH, and the action of b-hydroxy-butyrate dehydrogenase)
Or see above from choelsterol

Question 30

Define chromosomes and describe them

Answer 30

DNA is a long polymer (almost 2 m in length). In order to fit into the nucleus of cells it must be wound tightly. To achieve this, DNA is wrapped around proteins called histones. Eight histones and the DNA that is wrapped around them form a condensed region of DNA termed nucleosomes, and a chain of nucleosomes comprises a linear structure known as the chromosome. This process of winding and packaging brings the DNA polymer down from a molecule with a 2nm width to 1400 nm (densely packaged), and a fraction of its original length.

Each species has a stable number of chromosomes in the cells of its organisms. Humans have 46 chromosomes in their karyotype, i.e. the complete set of chromosomes. Chromosomes can be divided into sex chromosomes, or allosomes e.g. X and Y sex chromosomes, or autosomes (all other non-sex chromosomes).

Each chromosome is comprised of a pair of sister chromatids. The ends of chromosomes (or sister chromatids) are known as telomeres. The point at which both sister chromatids meet is known as the centromere. The regions of the sister chromatids above the centromere region are known as the short arms (p), the regions below are known as long arms (q).

Question 31

List the regulatory points of the cell cycle

Answer 31

The cell cycle refers to the phases of cell division.
It is a highly regulated process
Dysregulaton can lead to aberrant cell growth and can result in tumorigenesis.
One way in which dysregulation occurs is by the perturbation of cell cycle checkpoints.
The restriction or R point: a molecular complex that dictates G1 phase arrest or progression to S phase. It scans if conditiosn are favourable for DNA replication and cell division.

The internal and external environment is evaluated at R point.
Passing the R point involdes the actiovaton of cyclin p\proteins eg Cyclin D BY WAY of CDK4 or 6 binding.
If the R pointe is passed, then cells are generally committed to enter S phase. i.e. no going back

note: up to this point in G1, G1 and preparation for DNA synthesis is mitogen-independent, ie does not need biomeolecule promoting cell division eg growth factors

The G1/S checkpoint

iN OTHER WORDS it is a failsafe checkpoint, if issues are not detected earlier at restriction chekpoint.
Consists of molecular machinery e.g. CDK2 and cyclin E(overexpression, tumorigenesis, prognostic marker)
checksthe integrity of the genome.

This is important, as differences in the genome of teh resultant daughter cells would be an issue*

induces cell death i.e. apoptosis if DNA damage is detecte, prior to start of S phase
This stage of G1 is mitogen dependent

• G2/M checkpoint: associated with Cyclom A and CDK1. It ensures that chromosomes have correctly attached to the mitotic spindle.
• Molecular operators at the checkpoint can stall the kinetochore and arest cell division if aberrant chromosomal attachments are detected.

Question 32

Describe factors that result in deviations of Hardy Weinberg equilibrium

Answer 32

There are some assumptions inherent in the Hardy Weinberg equation
- these are often not complete met in reality
- thus the predicted frequencies do not always directly correspond to the real frequencies
Assumptions listed below:
1. Random mating i.e. there is no preference for certain genotypes to mate with each other
2. No mutation i.e. the allele frequencies in the population remain constant from one generation to the next because there is no new mutation or deletion of alleles
- i.e. it does not consider subsequent genetic variation
- the frequency of p and q will always add up to 1
3. no migration
- the population is closed and therefore there is no gene flow i.e. transfer of alleles between populations
4. random selection
- no selective advantage and disadvantage for a subset of individual from the population (i.e. sexual selection)
5. no genetic drift
- no founder effect i.e. a new population is established from a larger population
- no population bottlenecks i.e. rapid reduction in population size leading to a loss of genetic diversity

Question 33

Define the following terms: founder effect, bottlenecks, negative selection and selective advantage

Answer 33

Negative selection
- refers to a genotype diminishing over time if a genotype is lethal
- e.g. if qq is lethal, over time, q allele frequencies approaches zero, and becomes extinguished
An example of negative selection is DMD or Duchenne Muscular dystrophy:
- DMD is a recessive X-linked disease
- it affects 1/3000 males and is caused by a mutation in the dystrophin gene
- results include
- structural component of muscle cells
- mutations lead to muscle weakness and wasting
- unable to reproduce i.e. reduced reproductive fitness
- the DMD gene is the largest gene on the X chromosome and thus is vulnerable to mutations
- you would expect as males die, incidence of DMD would decrease with each subsequent generation i.e. negative selection
- but this does not occur, and incidence (and allele frequency remains constant)

The heterozygous genotype “Ss” confers a selective advantage:
- the Ss individual is resistant to malaria
- and has no anaemia i.e. no sickle cell disease
- note: HbS, and malaria incidence, co-occur ^[i.e. additional evidence for the selective advantage that the Ss genotype confers].

The founder effect
is a small group of individuals i.e. founders that establish a new population.
The genetic composition of the new population is altered, as compared to the original population. There will be over-representation of allele frequencies i.e. some alleles may become more common due to change events, and under-representation of other alleles.

Population bottlenecks
refer to a sharp reduction in the size of a population due to a random and catastrophic event.

• This is not only an example of founder effect, but also genetic drift ^[genetic drift]: the tendency for variations to occur in the genetic composition of small isolated inbreeding populations by chance. Such populations become genetically rather different from the original population from which they were derived. (Ox.)]

Question 34

Genetics

Describe chromosomal abnormalities and provide examples

Answer 34

Aneuploidy refers to a numerical chromosomal abnormality; in other words, extra or fewer copies of homologous chromosomes.

Examples include:
- monosomy i.e. when one of two homologous chromosomes is missing. An example of this is monosomy 21. This is an incredibly rare disease. incompatible with life. ^[fyinterest: https://doi.org/10.1016/j.gene.2012.08.041]
- Uniparental disomy i.e. when two pairs of a homologous chromosome ^[(or part of a chromosome)] are inherited from one parent. Most UPDs result in no abnormalities in phenotype; however, if it occurs in meiosis II, rare recessive disorders may manifest in the child ^[ UPD should be suspected especially in cases where only one parent is a carrier of a recessive disorder, another parent isn’t, and the disease manifests in the child]. UPD can have a role in the etiology of imprinting disorders ^[read more?, and include a brief bit of mechanism]
- trisomy where three copies of a homologous chromosome are present. The classic case of trisomy is trisomy 21, which is the main cause of Down syndrome
Euploidy refers to alterations of complete sets of chromosomes ^[i.e. A euploidy can be either a chromosome loss or a gain in the chromosome sets.].
Di/tri/tetraploidy refers to gametes containing 2n, 3n, and 4n chromosomes; otherwise known as polyploidy, while monoploidy refers to the presence of only half of the normal number of chromosomes.
aneuploidies of autosomes have been discussed. However, these can also occur in sex chromosomes. Two more common examples include:
- Turner syndrome and Klinefleter syndrome

There are many types of chromosomal rearrangements include single chromosome structural changes: deletions (loss), duplications (gain), and inversion, and two chromosome structural changes: insertion and translocation (note that these are copy number neutral changes, along with inversion).
Duplications and deletions are a result of misalignment of homologous chromosomes. The result after crossing over is two unequal chromosomes: one with a duplication and one with a deletion i.e. one loss one gain.

Inversions occur when a chromosome loops in on itself, gaps are created and thus rejoined, resulting in an inverted sequence.
Translocations can either be reciprocal i.e. between non-homologous or any two chromosomes; or can be Robertsonian i.e. the joining of two non-homologous chromosomes

Question 35

Genetics

Describe the modes of inheritance and provide examples

Answer 35

An inherited disease/disorder = presence of disease attributable to inheritance of a particular allele from a parent.
There are five basic modes of inheritance for single-gene diseases: autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, and mitochondrial.
Also codominance: expression of both, phenotype blend e.g. ABO
Mitochondrial: mother to offspring

AD and X-linked dominance shows “balance”
Recessive traits show skipping between generations.

Question 36

Describe RNA processing

Answer 36

RNA processing
Transcription is the process of producing a single stranded RNA molecule from the double-stranded DNA template. There are three main steps of transcription:
- initiation: the enzyme RNA polymerase binds to a DNA promoter region ^[appreciate: site to bind and start transcription encoded by RNA], encoded by TATAAT, which is recognised by the RNA polymerase protein complex, and RNA polymerase begins to unwind ^[does it do this itself? yes] the DNA double helix
- elongation (start from small and get to big RNA molecule): RNA polymerase moves in a 3’ to 5’ direction along the template strand of DNA and synthesises a pre-mRNA, catalysing its production in the 5’ to 3’ direction
- termination: RNA polymerases release the pre-mRNA at the transcription termination site ^[appreciate: site to end transcription encoded by RNA]
‘reads from 3 to 5, writes from 5 to 3’

NOTE: prior to translation, the pre-mRNA undergoes post-transcriptional RNA processing. This mainly involves splicing, which is the process of removal of introns (non-coding regions) from pre-mRNA by specialised enzymes, leaving behind only the coding regions or exons. Introns are usually degraded during RNA processing but can be retained and give rise to regulatory RNAs.

The overall purpose of splicing is to increase the functional diversity of RNAs. In this way multiple proteins and modifications of the same protein can be generated from the same gene, which can carry out various functions in different cell types ^[examples: Fas receptor isoforms, membrane bound, increased in skin cells exposed to sun, presumed protective role against cancer; development of complex organisms such as humans; IgM (first antibody in response to antigen), roles in autophagy [[Cell Biology Lecture 3]], apotosis, protein localisation, enzyme activities, lignad interactions, TF activity and mRNA abundance…see intro of https://doi.org/10.3389/fpls.2019.00708]

Question 37

Describe examples of oncogenes and how they contribute to cancer

Answer 37

Oncognes are gnees that have the potential to cause malignancy when mutated.
An example of an oncogene in Myc
Mutations in the myc gene result in gain of function transforming it from a proto-oncogne into an oncogene

Question 38

Describe patterns of mitochondrial DNA inheritance

Answer 38

Leber’s Hereditary Optic Neuropathy (LHON)
* Increase prevalence in maternal relatives
* Chain of transmission ends when a father
is affected
* Pattern of maternal inheritance

• Mitochondria
• 100000 mitochondria/egg
• 100 mitochondria/sperm
• Highly debated if any mtDNA is transferred to offspring from the father
• At best it is 1:1000 (possibly negligible)
• Incidences of paternal leakage of mtDNA (Schwatz and Vissing (2002) – muscle
  tissue).
• Therefore only maternal mitochondrial inheritance occurs

Mechanisms of paternal mtDNA degradation: active degradation
* Most of the mitochondria in the sperm are located in the tail
* After fertilisation the tail of the sperm is directed towards degradation
mediated by endocytic pathways
* Exponential cell division results in the dilution of the mitochondria
originating from the embryo

Question 39

Describe the process of mitochondrial genome replication

Answer 39

1. Twinkle (DNA helicase) binds to OH region of the mtDNA and unwinds the DNA duplex
2. DNA polymerase gamma (POLG) synthesizes the H strand continuously
3. As the H strand is copied, Mitochondrial single-stranded DNA-binding protein (mtSSB) binds to the
   parental H strand and prevents its degradation and binding to the L-strand to reform the DNA dumplex
4. When POLG arrives at the OL region, a stem loop is form and prevents the binding of mtSSB.
5. RNA primers synthesized by POLRMT bind to the stem loop thus allowing the initiation of transcription of
   the Light Chain by POLG
6. RNA primers are removed, and synthesis is completed at the primer binding regions.

Question 40

Describe importance of DNA replication, the components involved, and the steps of DNA replication

Answer 40

There is a continuous need for cell division as they continually grow old and die, or are otherwise lost, and must be replaced. DNA replication is the process of producing an exact copy of genetic material from a template. DNA replication must occur with every cell division in order to ensure that each subsequent cell contains the same amount of DNA.

NOTE: DNA polymerase can only add bases in the 5’ to 3’ direction.

The replication machinery consists of a few components. They are:
- helicases: which unwind the DNA
- primases: which synthesise short RNA primers that serve as starting points for DNA synthesis
- DNA polymerase: enzymes that move along the strand in a 5’ to 3’ direction and synthesise a new complementary strand base by base
- on the leading strand i.e. the 3’ to 5’ strand, synthesis is continuous
- on the lagging strand i.e. the 5’ to 3’ strand, synthesis is discontinuous and forms short Okazaki fragments
- the gaps between the Okazaki fragments are filled in by DNA polymerases and are then linked by ligase

Question 41

Describe post translational histone modifications

Answer 41

The aims of post-translational histone modifications are three, they aim to:
- alter the net charge of histones
- alter inter-nucleosomal interactions
- provide a platform for chromatin-binding proteins that alter chromatin compaction

Examples of post-translational modification:
- acetylation
- methylation
- phosphorylation
- sumoylation
- ubiquination

Question 42

Describe the components of the histone complex

Answer 42

Histones
Histones are highly expressed proteins found in eukaryotic cell nuclei, that package and order DNA.
Like all proteins, histones can have variants, altering their normal function.
The various types of histones include:
- H2A, H2B, H3 and H4 are core histones
- H1/H5 are linker histones

The histone complex that binds DNA contains:
- 2x H3-H4 dimers
- 2x H2A-H2B dimers
- 8 histones total i.e. an octamer

Variants will bind to different regions of the DNA and therefore alter gene expression.

Chromatin is the combination, or complex, of DNA and proteins, that together make up the contents of the nucleus of the cell.
- heterochromatin - dense (genes are silent)
- euchromatin - light (genes are active)

The accessibility of the genes (dependent on packaging of the chromatin) is directly related to the level of their expression/activity:
- if gene accessible i.e. genetic material is naked duplex DNA, not packaged, it is considered active, and it is highly expressed
- if gene is less accessible, it is still considered active, but expression is modulated
- if gene is inaccessible e.g. packaged tightly in mitotic chromosome, it is considered inactive, and there will be no expression of the gene

Side note on histone variants:
“Despite a conserved role for histones as general DNA packaging agents, it is now clear that another key function of these proteins is to confer variations in chromatin structure to ensure dynamic patterns of transcriptional regulation in eukaryotes.”

Question 43

Define and distinguish heterochromatin and euchromatin

Answer 43

Chromatin is the combination, or complex, of DNA and proteins, that together make up the contents of the nucleus of the cell.
- heterochromatin - dense (genes are silent)
- euchromatin - light (genes are active)

Question 44

Describe the mitochondrial genome

Answer 44

Genome is small
* 16.5kb
* Circular
* 37 genes – involved in
* 13 mRNAs
* 22 tRNAs
* 2 ribosomal RNAs
* Regulator regions (eg gene promoters)

• The 13 mRNAs encoded in the mt genome are transcribed and
  translated in the mitochondrial matrix
• All 13 proteins are part of the oxidative phosphorylation complexes
• ~1200 proteins are found in a
  mitochondria
• Most of them are encoded in the genome
  of the cell and are synthesised via
  transcription and translation
• Coordinated import of proteins translated
  in the cytoplasm (from nDNA) and
  protein synthesized by in the
  mitochondria (from mtDNA) is required
  to correctly assemble the electron
  transport chain.

Rest of necessary genes synthesised by nucleus of cell

Mitochondrial Genome
* 0.0005% of the genetic information
* Circular
* 2-10 genomes per mitochondria
(multiple mitochondria per cell; 100
– 10000 genomes per cell)
* Multiple copies of each gene
* 93% is coding region
* Low copying fidelity during
replication

Nuclear Genome
* 99.9995% of the genetic
information
* Linear
* 2 copies of each chromosomes
per cell
* 2 copies of each gene
* 3% coding region
* High copying fidelity during
replication

Question 45

Describe DNA methylation

Answer 45

DNA methylation involves the linking of methyl group to DNA via a covalent bond (a very strong bond).
DNA methylation is associated with gene silencing, and halts transcription.
DNA methylation is thought to play an important role in the silencing of tissue-specific genes, and has a role in determining cell fate.

Methylation of DNA occurs in regions called CpG islands.
CpG islands range from 1000 to 2000 bp in length, and contain multiple CpG sites.

Methyl groups are most commonly bound to cytosine, although they can also bind adenosine as well.

CpG islands
CpG islands are regions of DNA consisting of C-G repeat sequences, with phosphate groups.
They are also known as gene ‘hotspots’.
They are highly susceptible to methylation.

70-80% of CpG islands are methylated, in contrast to all cytosines, of which only 1% are methylated.

Note:
- acetylation is always associated with activation
- mono- methylation: activation
- di-methylation: mainly repression, also activation
- tri-methylation: a mix, depends on site of modification (i.e. histone protein variant and amino acid)

Question 46

Explain why mtDNA replication is prone to error

Answer 46

• Copying mtDNA is 100x less accurate than copying nDNA;
• POLG exhibits proofreading activity comparable to to DNA polymerases involved in
  nDNA replication;
• Mismatch repair activity in the mitochondrial is poorly understood
• Reasons for increased mt DNA replication errors:
• Gene mutations involving the mtDNA replication machinery
• Imbalanced pool of nucleotide pools available for DNA synthesis
• Increased susceptibility to oxidative damage
• Results form the formation of reactive oxygen species (ROS) during oxidative
  phosphorylation
• Electrons may escape the electron transport chain and react with O 2 in the mitochondrial
  matrix forming O 2- (superoxide)
• Superoxides damage both DNA (mtDNA and nDNA) and lipid membranes and other
  reactive oxygen species
• Positive feedback amplifies reactive oxygen species accumulation.
• Enzymes that mediate the conversion of superoxides to water
• Eg: Superoxide dismutase (MnSOD), Catalase, Gutathione peroxidase
• Non-enzymatic mechanism involve the presence of antioxidants – molecules capable of
  capturing ROS and rendering them inert

Question 47

Describe the Hardy-Weinberg equilibrium

Answer 47

Allele frequency
is a basic statistic.
It is the proportion of a particular allele “A” in a population, relative to all the other alleles at the same locus.

The frequency of A = number of allele A / total number of alleles at the same locus.

Applying the Hardy-Weinberg equation numerically

The Hardy Weinberg equilibrium is a mathematical model that describes a relationship between allele and genotype frequencies.

Allele frequencies are used in the Hardy Weinberg formula, to calculate the potential of the appearance of genotype frequencies.
Genotype frequencies drives phenotypes and disease probabilities.

Allele frequency:
p + q = 1
p = frequency of A
q = frequency of a

Genotype frequency:
p2 + 2pq +q2 =1
- where p2 is the homozygous dominant frequency - AA, or the probability of offspring inheriting A from one parent and A from another parent i.e. A x A = p x p = p2
- 2pq is the heterozygous frequency - Aa, i.e. the probability of inheriting one A from one parent, one a from another parent ^[note: 2 pq, because A can either be inherited from mother or father, and a can either be inherited from mother, or father]

An example:
A frequency= 0.6 i.e. (p=0.6)
a frequency = 0.4 (q=0.4)
AA - p2 = 0.6^2 = 0.36
Aa - 2pq = 2x0.6x0.4 =0.48
aa - q2 = 0.4^2 =0.16

The frequencies calculated with the Hardy-Weinberg equation are usually expressed as a decimal, but can also be expressed aas a percentage.

Question 48

Contrast meiosis and mitosis

Answer 48

Mitosis and meiosis
- Meiosis is a specialised type of cell division used by sex cell or gametes i.e. eggs and sperm
- gametes are the reproductive cells of an organism. Four are generated at the end of mitosis (contrast with two in mitosis), which are haploid (contain half the number of chromosomes contrast with diploid in mitosis)
- meiosis results in greater genetic diversity, resulting from crossover or homologous recombination (note: this occurs in prophase)

Question 49

Describe how measures to protect cells from cycling can actually generate cells suscpetible to malignant transformation

Answer 49

• senescence, or pausing of the cell cycle, usually occurs due to cellulat stress
• examples of such stress include, DNA damage and respose, oncogene activation, and telomera shortening and damgae
• stressors result in cell cycle arrest
• thus the cell acquires a senescent phenotype: increased ROS and oxidative damage, metabolic changes and morphological hanges, ad prolonged cell cycle arrest
• the propensity to become senescent and potentially cancerous increases with age, as expansion signals decline in favour of cell cycle arrest signals

balance between amount of damage and repair capabiliteis of the cell– just kill cell off…or senescence and potentially cancer down the line

Question 50

Describe mechanisms of DNA repair

Answer 50

Examples of correction mechanism include:
- exonucleolytic proofreading: DNA polymerase detects bond instability and excises mismatching nucleotides
- mismatch repair: single nucleotide corrections, made by dedicated enzymes
The proofreading capacity is essential to the functioning of cells. Deleting the proofreading capacity of DNA polymerase results in more cancers and early death (when studied in mice). Similar effects are also observed in humans and manifest as the following syndromes:
- Bloom Syndrome ^[rare disease, short stature, sun-sensitive rash, mild immune deficiency, marked susceptibility to cancer, BLM mutation, inherited in an autosomal recessive manner, marked increase prevalence in Ash Jewish population. Normal gene encodes for a helicase–when damaged it cannot detect errors. See also: https://rarediseases.org/rare-diseases/bloom-syndrome/]
- Werner Syndrome
- Rothmund-Thomson syndromes
- Lynch syndrome

By and large, loss of the proofreading capacity results in developmental abnormalities and increased cancer susceptibility. ^[see also : https://doi.org/10.1038/s41436-020-0828-z]

Question 51

Describe TSGs and provide an example

Answer 51

In normal cells P53 is kept at low levels and is short lived (half life 5-20 minutes).
In response to stress, such as DNA damage (UV, IR or chemical), oxidative stress,
osmotic shock and oncogene dysregulation P53 increases.
Causes:
DNA Repair
Arrests cell cycle at G1/S
Initiates Apoptosis
In cancer cells P53 becomes inactivated, deleted or non-functional.

most frequently mutated gene in cancer, over 50%, especially in affressive cancers eg TNBC

Question 52

Cell biology

Describe cell cycle and mitosis

Answer 52

Not all cells cycle or divide e.g. neurons.
All other cells are either mitotic (i.e. actively dividing) or are in G0 (resting phase: stopped or are not actively dividing; examples include neural cells, corneal epithelial cells i.e. loss with aging, and does not divide. can become a problem is density severely declined–in this case a transplant would be required) ^[note that there are two types: quiescent i.e. in a reversible state, and irreversible(senescent and differentiated)]
The cell cycle includes periods of dormancy, gaps, synthesis of DNA and division.

G (gap) 1: this phase starts at the end of telophase (of cell division), and is responsible for cell growth and normal metabolic functions
S phase: this is the synthesis phase, when DNA replication occurs
G (gap) 2: in this phase chromatin condenses and prepares for entry into prophase (beginning of cell division)
M (mitosis) phase: cell division

Mitosis
Cell division is facilitated by structural proteins called microtubules. Microtubules can be re-arranged by cells, and are used to move chromosomes during cell division.

Cell division or cytokinesis broadly consists of 5 steps:
- prophase: in which chromosomes condense, the centrosome forms and migrates laterally, and the mitotic spindle forms i.e. the re-arrangement of microtubules [^[this structure checks if chromosomes are ready and broadcasts this message to initiate cell division. Kinetochore functions include anchoring of chromosomes to MTs in the spindle, verification of anchoring, activation of the spindle checkpoint and participation in the generation of force to propel chromosome movement during cell division]]
- prometaphase: in which the nuclear envelope breaks down and chromosomes migrate towards the equator (the kinetochore describes the interaction of the centromere with microtubules)
- metaphase: in which chromosomes are aligned on the equator and the cell elongates
- anaphase: in which chromosomes divide into chromatids and move to opposite poles, and cell ingression at the equator forms a visible cleavage furrow
- telophase: in which the nuclear envelope reassembles, the cleavage furrow contracts further until cell scission occurs, forming two diploid (2n) cells i.e. same number of chromosomes as parental cells

Question 53

Cell bio

List the mechanisms of cell communication and signalling

Answer 53

There are several mechanisms of cell communication and cell signalling:
- autocrine: the cell expresses the receptor and secretes the ligand, i.e. ‘talking to yourself’
- juxtacrine: one cell has the ligand, kept on the surface, the other cell has the receptor, the two cells come into direct contact, ‘talking to your neighbour’
- paracrine: ligand is secreted by one cell but it does not enter circulation, and the targeted cell is reached by diffusion through the interstitial fluid, ‘talking to your group’
- endocrine: ligand is secreted by one cell and reaches the target cell via circulation, ‘broadcasting to the entire world’

Question 54

List the events underlying cell signalling by GPCRs

Answer 54

GPCRs are G-protein coupled receptors
They are transmembrane receptors
1. ligand bind to receptor e.g. adrenaline to an adrenergic receptor
2. G proteins which is bound to IC surface, releases one of its subunits with teh release of GDP and binding to GTP
3. subunits activates an enzyme e.g. AC, which inteurn activates a secondary messenger cAMP
4.cAMP activates other cellular enzymes, eg PKA - which generates effector molecules e.g. active transcription factors
5.this then initiates sthe cells reposne to inital stimulus e.g. translocation of transcription factoors to nucleus, initiating transcription

Question 55

Describe mechanisms of endocytosis

Answer 55

What sort of material is trafficked? Damaged organelles and unwanted cytosolic material. This is known as autophagy.

Additionally, material that is internalised from the extracellular space is sent to lysosomes, in a process known as endocytosis. An example of this is engulfed bacteria.

The key principles include:
- cytosolic material destined for degradation does not simply go to the lysosome
- it is first captured in a lipid vesicle, known as an autophagosome
- The autophagosome fuses with the lysosome when degradation is required
- Once fused, enzymes can work to break down contents
- This is an elegant system, as we do not want spill of acid hydrolytic enzymes

As for endocytosed material, it remains trapped in a lipid vesicle derived from the membrane. The vesicle undergoes a maturation process pre-degradation, becoming an ‘endosome’ with ligation* of ligands across vesicle in order to dictate where it goes ^[isn’t it going to the lysosome?notnecessarily…]

There are several modes of endocytosis, including:

1. Phagocytosis (mostly by inflammatory cells^[Immunology]). The purpose of phagocytosis is to destroy pathogens, and avoid causing a biological response. The mature vesicle, known as a phagosome, fuses with lysosomes in order to be disposed. This process can also be used to detect and destroy cancer cells *
2. Pinocytosis (‘cell drinking’) brings mostly absorbed fluids, and small hydrophilic molecules ^[are any drugs trafficked this way?] into the cell. This is because there is either no receptor available to mediate transport, or no favourable gradient. Pinocytosis is thus more efficient than waiting for fluids to diffuse across the membrane. A small amount of energy is produced in this process*. The mature vesicle (a pinosome?) binds to lysosomes for breakdown and release of components
3. Receptor-mediated endocytosis. This is most utilised method to internalise material. This process requires the binding of a specific ligand to a specific receptor. Examples of ligands that bind receptors include hormones, serum proteins, growth factors, ligands (and neurotransmitters?)
   
   • several steps are involved in receptor-mediated endocytosis
   • Receptor ligand binding occurs
   • The receptor-ligand complex diffuses laterally until it encounters clathrin coated pits
   • The receptor-ligand complexes accumulate at the pits
   • Special proteins e.g. clathrin or other adaptor proteins like dynamin curve the membrane, causing it to invaginate, and pinch off
   • The mature vesicle fuses with lysosomes to begin breakdown

Question 56

Describe microfilaments

Answer 56

Microfilaments are primary actors in cell division ^[[Cell Biology Lecture 2]]
Microfilaments are involved in gross movement of the cell, exocytosis, endocytosis ^[by increasing curvature it impacts cell structure], and cell motility. They mainly work by altering cell membrane shape*
The formation of microfilaments, like microtubules, is dynamic; it consists of polymerisation and depolymerisation.
The famous group of microfilaments are the actin family of functional proteins.
In mitosis, they work to form a contractile around cells in order to constrict. The dynamics of this process are controlled by cross-talk with microtubules
- This cross-talk is initiated by signalling which occurs along microtubules. It is an interconnected system, involves kinetochores* to change actin structure and form the contractile ring
- This process enables precise control of cell function e.g. endocytosis, exocytosis, cell division

Question 57

Describe intermediate filaments

Answer 57

Intermediate filaments provide structural support and resist stress in cells. Intermediate filaments can be thought of a spring mechanism, and are often found in epithelia^[[Histology Lecture 2]] . There are 70 ^[can be classified into 6 types; other examples not included in the lecture include neurofilaments found along vertebrate axons, and desmins which as found in sarcomeres and connect different cell organelles together in order to regulate sarcomere architecture] types of intermediate filaments including keratin ^[which is found in cells such as the glia [[Histology Lecture 5]] and in retinal cells, and increased when injury is incurred in these tissues]. Other examples of intermediate filaments include GFAP in glia and astrocytes, and lamins found in the cell nucleus ^[[Cell Biology Lecture 1]].

^[side note: intermediate filaments so called as they have diameter (10nm) between actin microfilaments (7nm), and microtubules(25nm), have coiled-coil structure as building blocks. They are also dynamic, like microfilaments and microtubules, but do not go ‘treadmilling’, do not have polarity, and do not have a nucleoside triphosphate binding site]

Question 58

Describe microtubules

Answer 58

Microtubules are the largest of the filaments. They are straw-shaped, and are dynamic i.e. they elongate and shorten. The microtubules have two ends (+) and (-), the (+) end elongates faster ^[and the minus end elongates slower]. The microtubules grow and shrink by addition and subtraction of dimer units comprised of α and β tubulin.

The primary function of microtubules is to assist in cell shape, motility, movement of chromosomes and organelles, and cytosolic cargo movement.

The organisation of microtubules is orchestrated by MTOC (there is usually one per cell, however in cell division ^[[Cell Biology Lecture 1]?] there may be two or more). Centrosomes organise microtubules i.e. regulate how long microtubules should be, when they should be engaged for and in which direction*
(note that centrosomes are comprised of two centrioles orientated at right angles to each other, each consisting of 9 microtubules in a cylindrical array)

The negative end of the microtubules is directed at centrosomes.

Question 59

Describe the role of gap junctions in cell communicaiton

Answer 59

Role of gap junctions in cell communication i.e. juxtacrine signalling

Intercellular channels are specialised and allow direct communication and exchange of small molecules (ions and metabolites) between adjacent cells.

The most common protein involved
are connexins and allow coordinated cellular responses, and synchronisation of cellular activities. This is important in embryological development (sending signals to instruct cell how to behave), muscle contraction and neurological response.

This mode of transport is very efficient for small molecules, especially for hydrophilic molecules. Examples include Ca2+, glutamate, secondary messengers and signalling molecules: ATP, IP3, cAMP, cGAMP, polyamines and glutathione.

Question 60

Pharmacology

Define the following terms: first pass metabolism, clearance, elimination half-life, volume of distribution, first and zero order reactions, bioavailability, drug metabolism, potency vs efficacy, agonists vs partial agonists, and reversible vs irreversible competitive antagonists

Answer 60

Clearance: Volume of blood cleared of drug per unit time
− What: Irreversible elimination of a drug, excretion of unchanged drug (e.g. in
urine), metabolism into different chemical
− Quantification - expressed as volume of blood cleared of drug per unit time (e.g.
L/h, mL/min)
Determines maintenance dose, and half life (with volume of distribution.

Maintenance dose (mg/h) = steady-state drug concentration (mg/L) x clearance (L/h)

Volume of distribution: A theoretical volume needed for distributing drug to produce the
drug’s measured plasma concentration.
− Quantification – Need to measure plasma drug concentration and know the dose
of drug administered. It is expressed as a volume (but it’s ‘fake’, just a relative #)
Volume of distribution (Vd) = Total of drug in body (mg)/[drug]plasma (mg/L)

Half-life (t1/2): Time taken for drug concentration in body to be reduced by half
− First-order kinetics: Rate of elimination is proportional to drug concentration
To check if drug follows first-order kinetics:
o half-life will not change by variations in dose for drugs - rate of
metabolism/excretion is changing depending on dose
o Going from 100 to 50% will have the same t1/2 as 50 to 25% and so on.
Ct (concentration at time t) = C0 (concentration at time 0) x e-kt
(k = Clearance/Vd)
Determining half-life: t1/2 = ln(2)/k

First pass effect: extraction and metabolism of orally ingested drugs before they
reach the systemic circulation
− Achieved by the liver (lesser extent by the gut wall)
− Significant variation between individuals with regard to the degree of the first pass
effect for drugs

Bioavailability: amount of drug reaching the systemic circulation (expressed as a
fraction of the total dose)
− Influenced by absorption
− Influenced by first pass clearance

Drug metabolism
− Objective - lipophilic to hydrophilic (water soluble so it can filter and be eliminated
in urine via kidneys)
− Phase I: Oxidation, hydrolysis, etc. producing hydroxyl, thiol, amino groups, etc.
(chop off a piece)
− Phase II: Conjugation of glucuronyl, sulphate, methyl (addition of different
chemical entities onto drug)
− Example
o Aspirin: chopped off and made to salicylic acid
o INH: phase II before phase I (CH3 group added before hydrolysis to add
OH group) - into isonicotinic acid

Zero order elimination
o Rate of metabolism stays the same irrespective of the dose

Potency (EC50): concentration needed to achieve half the maximum effect with the
drug.
Efficacy (Emax): how much effect with total drug

Partial agonists:
− On their own - cause a response that is smaller than that caused by a full agonist
− In the presence of a full agonist - reduce effect of the full agonist while
maintaining activity (to a lesser degree) of the receptor

Reversible competitive antagonism
− No effect on efficacy, but reduced apparent potency

Irreversible competitive antagonism
− Reduction in efficacy without a change in potency*
− Sometimes, potency and efficacy are reduced

Question 61

Microbiology

What diseases are caused by S. pyogenes?

Answer 61

Immune mediated disease i.e. disease that has deleterious effect on host
- rheumatic fever
- group A streptococcus
- results in inflammatory changes in heat
- M protein mimics the heart muscle, such that M protein antibodies cross react with tthe heart and damage the heart

• glomerulonephritis
  
  • immune complex deposition in glomeruli

**Localised disease **
● Pharyngitis
● Skin infections
- impetigo
- erysipelas
- cellulitis
- necrotising fasciitis

● Disseminated infections eg bacteraemia, post partum sepsis

Toxin mediated disease
- Scarlet fever
- (streptococcal toxin shock syndrome) toxic shock syndrome
- massive release of inflammatory cytokines from T cells leading to:
- hypotension/shock
- multi-organ failure
- disseminated intravascular coagulation
- also associated with Staphylcoccus aureus
- necrotising fasciitis

Question 62

Neurophysiology

Describe muscle reflexes

Answer 62

A spinal reflex is a relatively predictable, stereotyped and involuntary response to
a stimulus. A reflex arc is the basic circuit of a reflex, and is divided into:
− Afferent component (sensory receptors and axons carrying inputs to the CNS)
− Central component (synapses and (excitatory or inhibitory) interneurons to
process the inputs)
− Efferent component (motor unit carrying command signals to muscle fibres).

− Inputs to alpha motor neurons
o There are 3 main sources of input to the alpha motor neuron
i. Command
ii. Sensory (afferent) inputs
iii. Interneurons (excitatory and inhibitory) i

Afferent inputters include:
▪ Proprioceptors are receptors that provide information of location and position in space, and include:
* Muscle spindles (stretch receptors): muscle stretch/length
* Golgi tendon organs: joint position and load/forces in tendons
* Mechanoreceptors: angle, velocity and movement of joints

o Central processing coordinates sensory inputs and effector outputs with the use of spinal interneurons (both excitatory and inhibitory). These interneurons form networks that coordinate motor programs in response
to complex inputs
o Efferent outputs = output effectors (motor neurons) from ventral spinal
cord that respond to the sensory feedback or central commands
▪ Alpha motor neurons
▪ Gamma motor neurons - gives input to muscle spindles

Question 63

Neurophysiology

• List the steps of electrical conduction/AP

Answer 63

1. At resting membrane potential, voltage-gated Na+ channels and voltage-gated K+ channels. Thus their conductance is 0 i.e. GNa+= G K+ = 0
   The NA/K ATPase and K+ leak channels maintain the resting membrane potential (Vm).
2. If the membrane potential depolarises to -55 mV (i.e. a slight depolarisation from RMP= -60mV), voltage-gated Na+ channels open quickly. This causes an influx of Na+ which makes Vm more positive.
   This explosive increase in Na+ conductance or G Na+ rapidly moves the Vm towards the Nernst potential for Na+ or E Na+.
3. As the Vm approaches E Na+, the motive force on Na+ drops.
   This slows its influx.
   At the same time as the voltage-gated Na+ channels opened, the K+ channels also started to open, but much more slowly. ^[likely to conformational differences between the two channels i.e. a rapid rearrangement of protein domain required for pore activation, see https://doi.org/10.1016/j.neuron.2013.05.036]
   This increase in K+ conductance, G K+, causes K+ to flow out of the cell, slowing the upswing of the Vm.
4. Voltage-gated Na+ channels start to inactivate which completely stops the G Na+ and more voltage-gated K+ channels open.
   Together these two actions lead to the repolarisation of the membrane.
5. The slow kinetics of the closing of the voltage-gated K+ channels leads to hyper-polarisation of the membrane, and results in the relative refractory period:
   
   • the relative refractory period is a period in which a stronger than normal stimulus is required to cause an action potential
6. The eventual closing of voltage gated K+ channels returns the Vm to resting state i.e. RMP. Inactivation fates on Na+ channels re-open

• The absolute refractory period is the time when a second action potential cannot be initiated
• The relative refractory period is the period where a second action potential can occur, but requires a larger stimulus

Question 64

Anatomy

Describe the actions of the muscles of the eye

Answer 64

During near vision, it engages in accommodation.
- Muscles in the ciliary body attach to ‘scleral spur’
- When muscle contracts ciliary body is drawn anteriorly, closer to spur. This reduces tension in the zonular fibres, allowing the lens to bulge/relax.
- A more rounded lens bends light more effectively in order to focus on near objects.

The action of the orbicularis oculi muscle is to draw the eyelids together (using the palpebral part), and draw skin inwards (forming characteristic crow’s feet).
Note: orbicularis oculi also helps open eyes.

It also works to elevate the upper eyelid, in other words, to open the eye.
The superior tarsal muscle opens eyes even more, accentuates this action..

The action of the levator palpebrae superioris is to elevate the upper eyelid, in other words, to open the eye.

Levator palpebrae superioris - moves the eyelid
Extraocular muscles: 4 rectus (superior, inferior, lateral and medial), 2 oblique (superior and inferior) - moves the eye

In simple english*:
- superior oblique muscle moves eyeball down and in (medially and downward)
- inferior oblique muscle moves eyeball up and in (medially and upward)
- lateral rectus moves laterally
- medial rectus moves medially
- superior rectus looks up and out (lateral and upward)
- inferior rectus looks down and out? (lateral and downward)

Question 65

What are some examples of specific microscopic techniques?

Answer 65

• Histology/cytology:
  this is the cornerstone of anatomic pathology and the basis of morphologic assessment
• Special stains:
  they are used to visualise and distinguish between various substances and strictires via chemical reactions, causing visible colour changes e.g. Haematoxylin and eosin, (and?, and?)

1. note that these special stains are adjunct to H & E staining
2. reticulin stain (black reticular collagen fibres), fontana masson (stains melanin black)/masson trichome (col=blue), Giemsa (blue, NA stain), Periodic acid schiff, perl’s/prussian (blue iron), Congo red (id’ing amyloids, anionic dye)

• Immunohistochemistry (IHC):
  this is used to identify myriad proteins via affinity reactions with (specific) immunoglobulins

to ascertain whether the proteins are normal or abnormal.

This occurs via an antigen antibody affinity reaction, where the antigen is a protein on the surface of cells in the tissue, and antibody is an immunoglobulin:

antibodies with a strong affinity for the target protein will stick to the tissue
these antibodies can then be detected using a secondary antibody which is labelled with either a colour change molecule and fluorescent molecule – secondary antibodies have high affinity for the tails of other antibodies

• Electron microscopy:
  ultrastructural analysis, is important in diagnosis of certain diseases e.g. renal glomerular diseases, ciliopathies, deposit diseases
• In situ hybridisation (ISH):
  used to identify specific genetic sequences (e.g. viral sequences* *, human gene sequences)
• Immunofluorescence:
  used to diagnose diseases that involve deposits of specfic classes of immunoglobulin and complement proteins (e.g. fluorescent antibodies have affinity for immunoglobulins and complement proteins)

Question 66

What are the components of an acute inflammatory response?

Answer 66

1. Vessels and endothelial cells
2. Circulating blood cells (red and white)
3. Tissue cells e.g. connective tissue cells
4. Inflammatory mediators, released by a varitey of cell types but mainly leukocytes e.g. neutrophils and monocytes
   in order to control the inflmamtory response eiher by enhancement or inhibition

Question 67

define sepsis and severe sepsis

Answer 67

sepsis= systemic response to infection (SIRS and infection)
severe= sepsis with multiple organ dysfunction syndrome or MODS /hypo or hyperfusion

septic shock =
sepsis + persistent
hypotension/hypoperfusion

Question 68

Define and distinguish between ulcer and abscess

Answer 68

Ulcer: ocal
defect of surface
of an organ or
tissue due to
shedding of
surface inflamed
necrotic tissue –
loss of epithelium
and basement
membrane

Can be acute or chronic. Acute: loss penetrates mucosa and submucosa; chronic penetrates layers of mucosa, subcosa, muscalris, scarring may also be present

Abscess - Abscess

Abscess histology: central pus with
reactive zone of granulation tissue and
fibrosis
ABSCESS = localised collection of pus
within tissue or a space (cf- cellulitis –
diffuse)

Question 69

List the steps of acute inflammation

Answer 69

1) Recognition of offending agent–> redness and heat
Macrophages and other sentinel cells in tissues e.g. DCs and mast cells, recognisiton of necrotic tissue, microbes etc

Recognition and Binding of agents ➔ downstream effects that
initiate and amplify inflammatory response

Type of leukocytes attracted to an initial injurious stimulus varies
* Majority of acute inflammatory reactions attract NEUTROPHILS first,
followed by MONOCYTES/MACROPHAGES

2) Recruitment of leukocytes and inflammatory
mediators to site –> redness and swelling, due to dilatation of vessels, increased permeability and exudate, oedema/ LOF

3) Removal (Elimination) of offending agent => swelling, neutros and macros removal

1) Recognition and binding of culprit to
phagocytic receptors

2) Engulfment

3) Intracellular destruction/killing of
debris/microbes
- Reactive Oxygen Species

4) Regulation (Control and termination) of
inflammatory response

Complex interplay of factors determines cessation
of acute inflammatory response
– Offending agents are removed
– Mediators have short half lives, or are
degraded
– Neutrophils have short half lives
– Anti-inflammatory mediators are released
– Neural impulses

5) Repair of tissue damage

Question 70

List the cellular contributions to acute inflammation

Answer 70

• Polymorphs (PMN’s, neutrophils): first to arrive; engulf, kill (free
  radicals) and digest (enzymic) microbes
• Mast cells: when activated (Complement) secrete mediators
• Monocytes and macrophages: late arrivals, secrete cytokines
  and chemokines, engulf cell debris, microbes
• Platelets: prostaglandin/leukotriene synthesis, free radicals, pro-
  inflammatory
• Vascular endothelial cells: contraction/ relaxation (nitric oxide).
  Also important in repair (angiogenesis)
• Neurons: release compounds including Substance P (pain), kinins
• Epithelial cells, and connective tissue cells can secrete
  mediators of inflammatory respo

Question 71

Distinguish between acute and chronic inflammation

Answer 71

• the onset of acute inflammation is fast, whereas chronic inflammation is slow
• the cellular infiltrate in acute inflammation is mainly neutrophils, while the infiltrate in chronic inflammation is monocytes/macrophages and lymphocytes
• tissue injury and fibrosis is usually mild and self-limited in acute inflammation, but is often severe and progressive in chronic inflammation
• local and systemic signs are prominent in acute inflammation but less so in chronic inflammation

Question 72

Thromboembolic disease

Answer 72

• Intravascular thrombosis due to a breakdown in
  normal haemostatic mechanisms.
• May occur in arterial or venous systems.
• Arterial thromboses are largely predisposed to by atherosclerosis, plaque rupture and platelet
  activation under high shear stress.
• Venous thromboembolism is predisposed to by venous stasis and changes in coagulation constituents of blood

Question 73

Describe the abnormalities of Virchow’s Triad in regards to arterial thrombosis and venous thrombosis

Answer 73

Virchow’s triad has three components:
- blood flow
- blood factors or constituents
- vessel wall

In arterial thrombosis:
- vessel wall has atherosclerosis
- blood flow is turbulent
- blood factors: qualitative platelet abnormalities

In venous thrombosis:
- vessel wall has trauma
- blood flow: stasis
- blood factors: hypercoaguability - either due to deficiency of inhibitors, or increased clotting factors

Question 74

Define thrombosis

Answer 74

Thrombosis is the pathological sealing of blood vessels, in reposnse to endothelial damage, stasis and procoagulants. It occurs due to a breakdown in normal haemostatic mechanisms

Question 75

Describe the pathology of arterial thrombosis

Answer 75

• Usually occlusive at sites of atherosclerosis with plaque rupture
• Often in coronary, cerebral and femoral arteries
• Grey to white and friable
• Composed of platelets, fibrin, red cells
• In larger vessels they may have macroscopically visible
  lines (of Zahn). These are composed of platelet rich and
  red cell rich areas
• Arterial thrombi in heart chambers and aorta are termed mural thrombi (due to atrial fibrillation wall motion abnormalities or aneurysms)

Question 76

Describe the intrinsic and extrinsic pathways of clotting

Answer 76

The Extrinsic Pathway is the dominant method of generating a haemostatic plug in vivo.

1. Arteriolar vasoconstriction: mediated by reflex neurogenic mechanisms and augmented by the local secretion of factors such as Endothelin (the most potent vasoconstrictor known). The effect is transient, however, and bleeding would resume if not for activation of the platelet and coagulation systems.
2. Primary haemostasis: Exposure of subendothelial extracellular matrix (ECM) facilitates platelet adherence and activation. Von Willebrand factor is a large glycoprotein present in blood plasma, megakaryocytes, and subendothelial connective tissue. vWF is important in platelet adhesion to wound sites, works by binding other proteins.
3. Secondary haemostasis: Tissue factor (aka Factor III, Thromboplastin) is a membrane-bound procoagulant on subendothelial connective tissue. It binds to Factor VII, and this complex cleaves Factor X to Xa. Factor Xa also forms a complex, which converts Prothrobmin (Factor II) to Thrombin. Thrombin cleaves circulating Fibrinogen into insoluble Fibrin, creating a fibrin meshwork, and also induces additional platelet recruitment and activation. This consolidates the initial platelet plug.
4. Thrombis and Anti-thrombotic events: Polymerized fibrin and platelet aggregates form a solid, permanent plug to prevent any further haemorrhage. At this stage, counter-regulatory mechanisms are set into motion to limit the haemostatic plug to the site of injury.

The Intrinsic Pathway serves as a positive feedback loop to amplify the production of thrombin initiated by the extrinsic pathway:

1. Thrombin feedback: The extrinsic pathway produces very small amounts of thrombin, which activiates Factors V, VIII, and XI. This leads to the production of more thrombin, in a positive feedback loop.
2. Clot formation: Thrombin cleaves fibrinogen to fibrin. Fibrin monomers bind to each other and become permanently cross-linked with the aid of factor XIIIa (also activated by thrombin) to form a clot.

Question 77

Describe pathophysiology of atherosclerosis

Answer 77

Pathophysiology of artherosclerosis/atherosclerosis
* Formation of inflammatory intimal fibrous
plaques with a central lipid rich core
* Primarily affects elastic arteries and
medium to large muscular arteries
* Multifocal

Atherosclerosis development
* Local endothelial injury (possibly due to haemodynamic disturbances, exacerbated by hyperlipidaemia), leads to – expression of adhesion molecules (eg Vascular cell adhesion molecule 1 :VCAM-1)

– Extravasation of lipids
* Monocytes & T cells recruited (by for example Monocyte chemotactic protein 1 from endothelial, smooth muscle cells and monocytes)
* Chronic inflammation and accumulation of oxidised LDL
* Oxidised LDL is a potent chemo-attractant for monocytes
* Migration of monocytes into the vessel wall, which develop into foamy (lipid laden) macrophages, engulfing lipid via scavenger receptors (rather than LDL receptors) ~ fatty streak

• Lipid accumulation and intimal thickening
• Cytokines lead to migration of smooth muscle cells into the intima
• Proliferation of smooth muscle in the intima and
  accumulation of collagen and proteoglycans - fibrous plaque
• Calcification in connective tissue elements and
  weakening of vessel wall
• Connective tissue on the intimal surface forms a fibrous cap
• Advanced lesions develop neovascularisation with a central lipid rich ,often necrotic core. Haemorrhage from these weak vasa vasorum may occur into plaque
  ~
  Note: Atherosclerosis triggers arterial thrombosis

Question 78

Describe hypertrophy, hyperplasia, metaplasia with examples

Answer 78

Hypertrophy is defined as an increase in the size of cells due to increased intracellular components or proteins; it results in increased organ size or weight.

Hypertrophy can be physiologic or pathologic in nature.

Physiologic examples of hypertrophy include:
- skeletal muscle hypertrophy (as in body building or fitness training)
- smooth muscle hypertrophy of uterus (pregnancy)

Pathologic examples of hypertrophy include:
- cardiac hypertrophy (it is initially adaptive to respond to the cardiac muscle’s increased workload, but eventually becomes maladaptive, and can also be accompanied by cardiac failure, or sudden death ^[eventual inability to cope, leads to regressive changes and loss of contractile elements and cell death])

Hyperplasia is defined as an increase in the number of cells in an organ/tissue due to proliferation of mature cells and/or stem cells.
It regresses if the initial stimulus is removed.
It may result in increased organ size and weight.

Like hypertrophy, hyperplasia may be physiologic or pathologic in nature.

Examples of physiologic hyperplasia include:
- breast (during puberty and pregnancy)
- liver (following partial resection or hepatectomy) - see also [[Pathology Lecture 6]]
- bone marrow (i.e. following blood loss, haemolysis)

Pathologic examples of hyperplasia:
- endometrial hyperplasia, or the thickening of endometrium inappropriately due to unopposed oestrogenic stimuli –> a precursor to some endometrial carcinomas
- benign prostatic hyperplasia or a common inappropriate enlargement of prostate gland due to androgenic influences
- some viral infections e.g. warts (HPV)

Common stimuli between pathologic and physiologic hyperplasia include: hormones, GFs, post organ damage/resection

-

Metaplasia is defined as one differentiated cell type being replaced by another cell type. This occurs to better withstand an environment.
The mechanism of cell type replacement is due to reprogramming of tissue stem cell differentiation NOT replacement of one mature cell by another mature cell.

Like hypertrophy and hyperplasia, metaplasia can be physiologic or pathologic in nature.

Physiologic examples of metaplasia include:
- formation of the cervical transformation zone during puberty (from glandular columnar to squamous epithelial cells) - see [[Histology Lecture 2]]

Pathologic examples of metaplasia include:
- columnar to squamous epithelial cells:
- bronchi (e.g. in smokers, vitamin A deficiency)
![[Pasted image 20230527192450.png]]
- bladder (e.g. stones, Schistosomiasis)
- ducts of salivary glands/pancreas/biliary tract (e.g. stones) ^[recall from anatomy prac, structures of head and neck: you can have stones in salivary glands; probably due to salt content]
- squamous to columnar epithelial cells:
- Barrett’s oesophagus (e.g. GERD)

Note: with continued injury, pathologic metaplasia may be the setting in which dysplasia and malignant transformation occur e.g. smoker’s bronchus

Question 79

Define and describe necrosis and apoptosis

Answer 79

Necrosis is a form of lethal injury in which there is:
- significant membrane damage, with leakage of cell components
- denaturation of intracellular proteins
- enzymatic digestion of cell constituents

Necrotic tissue incites an inflammatory response with time.

NOTE: Necrosis is always pathologic

Apoptosis is a form of cell death.
It results from a tightly regulated cellular “suicide programme” i.e. programmed cell death.
Cellular enzymes known as caspases digest DNA and cytoplasmic proteins, whilst preserving membrane integrity.

Small packages of apoptotic bodies are rapidly cleared by phagocytes.

NOTE: Apoptosis does not incite a visible inflammatory response

Unlike necrosis, apoptosis may be physiologic or pathologic

–
The mitochondrial pathway is the major mechanism of apoptosis.
It is initiated when mitochondrial permeability increases, releasing pro-apoptotic molecules into the cytoplasm.
The BCL2 family of proteins is key to the mitochondrial pathway. It consists of:
- anti-apoptotic proteins (e.g. Bcl-2)
- pro-apoptotic proteins (e.g. Bax, Bak)
- sensor proteins - which sense cell damage (e.g. of the BH3-only proteins: BAD, BIM, BID, Puma, Noxa)

• anti-apoptotic proteins are promoted by survival signals e.g. growth factors
• in a viable cell, this prevents apoptosis form occurring
• when cell damage occurs (i.e. irradiation and DNA damage, loss of survival signal, protein misfolding/ER stress) sensor proteins (BH3-only proteins) detect this and perform two key actions:
  - antagonise anti-apoptotic proteins i.e. BCL2
  - activate pro-apoptotic proteins i.e. BAX and BAK
• once activated, pro-apoptotic proteins form channels in the mitochondria, leading to the release or leakage of molecules, such as cytochrome C
• cytochrome C and other proteins activate caspases (initiator –> executioner) that in turn activate endonucleases to fragment the cell’s nucleus, and the breakdown of the cytoskeleton
  Eventually, the apoptosing cell forms cytoplasmic blebs that bud off and become apoptotic bodies, containing the fragments of the nucleus and the other contents of the cytoplasm.
  —
  The death-ligand pathway is the less common apoptotic pathway.
  It is initiated by the binding of a ligand to a plasma membrane death receptor e.g. TNF family of receptors.

The binding activates the intracytoplasmic death domain, which activates caspases (initiator caspases, which in turn activate executioner caspases, activating endonucleases to break down the nucleus, and breakdown of cytoskeleton, cytoplasmic blebbing, forming apoptotic bodies).

Irreversible cell death results in cell death. The two forms of cell death are necrosis and apoptosis.

In necrosis:
- the cell becomes enlarged (swelling)
- nucleus undergoes a series of changes: from pyknosis (nucleus thickens into a dense mass) to karyorrhexis (nuclear membrane ruptures and nucleus begins fragmenting) to karyolysis (complete dissolution of cell nucleus due to enzymatic degradation)
- plasma membrane is disrupted
- cellular contents digested by enzymes, and may leak out of cell
- adjacent inflammation is frequent
- necrosis is invariably pathologic

In apoptosis:
- the cell size reduces (shrinkage)
- nucleus undergoes fragmentation into nucleosome-sized fragments
- plasma membrane is intact, but the structure is altered, especially the orientation of the lipids
- cellular contents maybe intact, but may be released in apoptotic bodies
- no adjacent inflammation associated with apoptosis
- often physiologic (as a means of eliminating unwanted cells), although it may be pathologic after some forms of cell injury, especially DNA damage

Question 80

Describe passage across blood capillaries, describe the effect of capillary type on drug passage

Answer 80

Capillaries in different organs display wide variation in drug permeability

Rate at which drugs leave the blood will depend on :
1. Size
2. Sol
3. Degree of protein binding

• continuous: Many capillaries are continuous capillaries
  Only allow diffusion of water and small solutes through intercellular clefts
  e.g. skeletal and smooth muscle, connective tissues, lungs
• fenestrated:
  More permeable than continuous capillaries
  Allow rapid exchange of fluid and solutes as large as small peptides
  e.g. kidneys, villi of small intestine, choroid plexus
• sinusoids:
  Wider and more winding than other capillaries
  Incomplete basement membrane, large fenestrations, very large clefts
  Allow large proteins to pass through
  e.g. liver, spleen

Question 81

Describe the four compartment model of drug distribution

Answer 81

Fur compartments: blood/vrg, muscle, fat, vrp is fourth

Order of drug distribution follows the degree
of perfusion of the different “groups”
1st
2nd
3rd

Capacity to accumulate:
drug follows reverse order i.e. most in fat, then muscle, then VRGs

Question 82

Distribution to foetus

Answer 82

Foetus can be considered as a special type of
compartment into which drugs can distribute
Drugs cross the placenta by:
* Simple diffusion
* Active transport
* Pinocytosis
* Filtration

Active transport can also prevent some drugs from crossing placenta

Passive diffusion of drugs across the placenta is determined by the drugs:
* molecular weight
* pKa
* lipid solubility
* protein binding
Drugs with molecular weight > 500 Da do not cross the placenta well

Question 83

List the four types of receptors targeted by drugs

Answer 83

1. Ligand-gated ion channels
2. GPCRs
3. Kinase linked and related receptors
4. Nuclear receptors

All are examples of transmembrane signalling mechanisms

Question 84

Describe transporters

Answer 84

Transporters are required to allow passage across cell membranes when passive diffusion is not possible

Transporters factilitate transport across cell membranes.

Passive transport (diffusion) can occur with carrier-mediated transporters.

But, the most common form of transport is active transporters

Hydrolysis of ATP (adenosine triphosphate) creates energy to actively pump a substance across a membrane against an electrochemical gradient

Secondary Active Transporters:

Transporter uses the energy created by transporting an ion down it’s electrochmical gradient to transport another molecule simultaneously against it’s electrochemical gradient.

Symporter:  A transporter that moves both molecules across the membrane in the same direction
Antiporter:  Molecules move across the membrane in opposite directions

Question 85

Describe ion channels

Answer 85

Proteins embedded in cell membrane that control the flow of ions into and out of the cell

Ions can only pass through an ion channel down their electrochemical gradient from a compartment of higher ion concentration to a compartment containing lower ion concentration

The rate of flow through the channel is very high

*Drugs target ion channels (eg)

Amiloride (weak diuretic - blocks sodium channels)
Verapamil (Calcium channel blocker)

Question 86

Describe ligand gated ion channels

Answer 86

Type 1 - Ligand gated ion channel (Membrane bound channels)

Activated when a ligand binds to a specific site on the protein. May be induced to open or close.

Rapid response (milliseconds)

Responsible for functions such as:

Central and peripheral synaptic transmission mediated by neurotransmitters

Example: Nicotinic (acetylcholine) receptors; GABA receptors

Question 87

Describe GPCRs

Answer 87

At least 800 different types

Mediate response of hormones and neurotransmitters

Stimulate GTP binding protein which modulate intracellular second messenger

Takes seconds to respond

Effector can be a channel or enzyme

Example: Muscarinic (acetylcholine); beta (adreno); opioid

Question 88

Describe kinase linked transmembrane recpeors

Answer 88

Response takes hours

Receptors are linked to processes that alter gene transcription and protein synthesis

Example:

Tyrosine kinase receptor
Insulin
Growth factors
Cytokine receptors

Question 89

Describe nuclear receptors

Answer 89

Regulate gene transcription

Require binding of various other molecules prior to translocation to the nucleus (interact with specific response elements on genes)

Response time is hours (new proteins are synthesised)

Example:

Steroid hormone receptors

Question 90

Describe the changes that receptors can undergo

Answer 90

Tachyphylaxis:

Diminished response of receptor after repeated exposure to the same concentration of drug

Desensitisation:

Decreased response of the receptor-second messenger system

Downregulation:

A decrease in the number of receptors
Can contribute to desensitisation and loss of response

Upregulation:

An increase in receptor number
Can cause receptor hypersensitivity
Often occurs after chronic use of drugs that block receptors à drug removed à patient experiences ↑ response to stimuli

Question 91

Compare and contrast the innate and adaptive immune systems

Answer 91

The innate immune system is evolutionarily older, and present in older organsisms.
It relies on receptors and molecules that recognise specific patterns on pathogens: this classifies the pathogenes as foreign,
Examples of receptors include the toll like receptors.
When receptors are present on the surface of he cells, they are the same wherever they are expressed and are the same between different people.

They induce the same response the first and 100th time they recognise their pathogen.
In other words, the innate immune system has no memory.
Many cells carry the same receptor i.e. it is non-specific.

The adaptive system on the other hand is only present in higher vertebrates.
It relies on unique receptors on lymphocytes i.e. BCRs and TCRs to identify soecific pathofens.
Everyone has lumphocytes with different receptors and they are unique to each person– i.e. specific.
The adaptive immune system has memory i.e. can remmeber past pathogens and gives a faster better stonger response with repeated challenges e.g. memory B cells.
The receotors are unique to single cells, therefore takes time to activate and proliferate. innate immune system is by contrast much quicker due to its non-specific nature

Similarities:
- are designed to identify foreign substances i.e. separate from self
- activation of receptoos leads to to activation of signalling pathways resulting in activation of otehr parts os the immune system

Differences
- antigen reeptors in innate immune system identical in all people vs in adaptove immune system: unique to each individual
- antigen receptors of adaptove immune stystem eg TCRs and BCRs can eb modofied durng the immune response
- there is memoty, that wer habe responded to a particular foregin substance inthe adaptive immune sysem

Question 92

What are the two types of patterns that activated pattern recognition receptors?

Answer 92

PAMPs or pathogen associated molecular patterns.
Examples include: LPS of gram negative bacterial cells (TLR4), LTA and peptidoglycan (generally gram positive)

And DAMPs or damage associated molecular patterns.
Examples include: cellular debris released from damaged cells, extracellular ATP, cytoplasmic or extracellular DNA — typically intracellular

Pathogen recognition receptors e.g. TLRs are found on epithelial and immune cells e.g. macrphages.
When PRRs detect somethign they recognise they rleae signals meaning danger e.g. cytokines and chmokines.

Question 93

Describe the process of antigen presentation of T cells

Answer 93

• to show antigen to T cells cells need to break down proteins to peptides
• if a skin cell is showing a peptide to a T cell it will put it on an MHC I molecuels
• if DC or other APC (B, macro, follicular T) is showing a bacterial or viral protein to a T cell it will put it on an MHC class II
• these protein sare onyly on professional antigen ppresenting cells (in turn, T cells help fight patho (1))
• TCR only recognises peptids and MHC toghether, not separately
• it is your MHC molecules that dermine compatible organ donor

Question 94

Describe the role of follicular T helper cells

Answer 94

T follicular helper cells (Tfh) are a specialized subset of CD4+ T cells.

hey play a critical role in protective immunity helping B cells produce antibody against foreign pathogens. Tfh are located in secondary lymphoid organs (SLOs), including the tonsil, spleen and lymph nodes. These organs contain numerous lymphocytes, separated into defined T and B cell zones. Uniquely, Tfh are found in the B cell zone and spend the majority of their time in close interactions with B cells. In addition to SLOs, Tfh can also be identified in the circulation.

Tfh play an essential role in the formation of germinal centres (GCs), which are distinct structures that form within the B cell zones of SLOs during an ongoing immune response. B cells within GCs are known as GC B cells and undergo rapid proliferation and antibody diversification, allowing the production of many types of antibody, with greater affinity for their targets. GCs are also the site where B cells can differentiate into antibody secreting plasma cells and memory B cells which allow long lasting antibody production. Tfh direct this process by directly providing co-stimulation to the B cells via the co-stimulatory molecule CD40 interacting with CD40-ligand (CD40-L) on the B cell and by producing the cytokine IL-21 which drives B cell proliferation. Additional cytokine production by Tfh is able to determine the type of antibody produced.

In the absence of Tfh, GCs do not form and antibody defects are observed under typical situations.

Tfh are defined by expression of the transcription factor, B cell lymphoma 6 (Bcl6) and a number of cell surface markers including CXCR5.

Tfh function has been shown to be dysregulated in a number of diseases of excessive or insufficient antibody production.

Question 95

Describe dendritic cells

Answer 95

- the links between innate and adpative immunity
- take uop antigens in peripheral sites, usually by endocytosis nad process tem c=to cmmuicate with the rest o fthe immun esystem il.e. to present to other immune cells in a process known as antigen presentation

**ROLE: ALERT REST OF BODY TOO LOCAL PROBLEM BY ALEERTING THE CLOSEST LYMPH NODE

Question 96

Compare and contrast B and TCRs; Compare and contrast MHC class I and class II molecules

Answer 96

Similarities:
constant and variable regions
variables made by VDJ recombination- alpha or light V(D)J; beta or heavy by VDJ

each cell can only have one unique receptor
making a functional receptor essential for development of cells

Differences:
- BCR can bidn a number of diffrent structures or types of antigens eg prots lipids polysacchs
- TCR can also bind MHC peptide complex
- BCR can be modificed after antigen response, TCR CANNOT

Similarities
- cell surface receptors
- encoded in genome
- lots of variation in these genes between people
- MHC needs to have a peptide in groove to be stable and expressed on cell surface

Differences:
- MHC class I is expressed on all nucleated cells
- MHC class II on antigen presenting cells (professional; DC, B, macrophages)
- MHC class I present antigens found inside cells i.e. endogenous pathway of anitgen processsing
- MHC class II prsent antigene the cells has taken up prom outside i.e. endocytosed. THis is called the exogenous pathway of anitfen presenting
- MHC class I and peptide interacts with TCR on CD8+ T cells, MHC class II, and peptide with TCR on CD4+ T cells

Question 97

Order these processes: somatic hypermutation, class switch recombination, affinity maturation, VDJ recombiantion

Answer 97

• VDJ recombination: purpose is making functional BCR or TCR

Each gene segment (V, D, and J) has an adjacent Recombination Signal Sequence (RSS)

at the 3′ end of each V segment
at both ends of each D segment
at the 3′ end of each J segment
	
	he configuration prouced by RSS acts as a target for Recombinases. These are recognized by two proteins encoded by two Recombination Activating Genes:

RAG-1 and
RAG-2

The RAG-1 and RAG-2 proteins cut through both strands of DNA at the RSS forming double-stranded breaks (DSBs).

The cut ends are stitched together (ligated) to form:

• a coding joint (D-J or V-DJ for heavy chains; V-J for light chains)
• a signal joint (usually a loop of DNA deleting all the intervening DNA initially present between the 2 gene segments chosen)
• occurs in late-pro-B cell (H) and pre-B cell (L). Switch form immature to mature occurs when B cells that do not bind self antigen express d chain and membrane IgD with their IgM about the time they leave the marrow and become mature naive (resting) B cells (preBCR binds ligands, posotive; negative if it rsulsts in cell dath, self antigen).

The players first choose one each of the possible D and J cards and keep them together (by deleting the DNA sequences in between them).

DJ rearrangement occurs first

Then one of the many V cards is chosen, and this “card” is kept together with the D and J cards previously matched (again by deleting the DNA in between).

VDJ rearrangement occurs second
VDJ-C rearrangement occurs at last

rascription of Immature B cell DNA to RNA followed by RNA splicing of introns occur. This brings the Combined V(D)J together with “C” segment that encodes for Constant region of heavy or light chains and forms mRNA.

This is followed by Translation to produce proteins – specific heavy chain and light chain.

Can produce functional or non functional receptor.
Try again wit other locys - i.e. k or lambda. Allelic exclusion states that only one allele is expressed ie. active00 so nly one lighth and heavy is expressed. Allelic exclusion: Once a functional product has been achieved by one of the rearrangements, the cell shuts off the rearrangement and expression of the other allele on the homologous chromosome.

Improve affinity while retaining specificity for antigen:
- class switch recombination or isotype switching: from M to A G or E. The constant portion of heavy chain undergoes this process. \
- occurs after activation of B cell
- Recall: IgM predominates, frirst to be produced. pentameric strictire in blppd (Surfcea aresa and sites for mining, good and trapping and neutratlising antigen, and activating complement). Ig G1-4, complement, opson, neut. Ig A dimers, antigen trapping and neutralisation. IfE, boudn to Fc on masts basos and eosinos; allergy and parasite infections.
-
- Depends on which heavy chain constant egion fne is expressed– M to others. Switch regions in introns upsream of isotype genes guide AID other enzymes– create nicks, excise m, and repair joins togehter. CD4 T cells skew the immune response by producing certain cytokines.
- note : unlike other processes here– it is a rearrangement.

• somatic hypermutation: how B cells change specificity for antigen, introducing point muatutions in BCR seqquence- in V region i.e. weher it will bind antigen. AID- mismatch reparir and base excissibe repair.High rate. Occurs in dark zone, as centroblasts. Stop become centrocytes with expression of cell surface markers, move to light
• affinity maturation: follows somatic hypermuatuon, slects cels with BCR that bind strongest to antigen. occurs in lgiht zone. Full of FDCs exprssing antigen. Test binding. Bind–> survival signal–? cycle through somatic hypermutation, or cyclic re-entry. Keep competing with other cells for antigen– best fit survives
• Also necessary: prolonged T follucular helper cell contact– icosl and icos, uprefulatees CD40L, binds CD40, promotes survival by inhibitoon of apoptossis*

Question 98

DESCRIBE antigen recogntiion by Bs and Ts

Answer 98

• In the immune system,
  activation of cells occurs after at
  least two different signals to
  make sure that an unnecessary
  immune response doesn’t occur
• In lymph nodes, the dendritic
  cell activates the T cell most
  specific for the antigen that
  activated the dendritic cell
• The antigen that was brought
  can also bind directly to the
  most specific B cell activating
  the B cell leading to antibody
  production, and B cell activation
  to make antibodies can also be
  assisted by T cells specific for the
  pathogen

Question 99

What are the layers of skin?

Answer 99

• epidermis
  
  • subcutaneous tissue or subcutis
  • dermis

Question 100

Describe the layers of the epidermis

Answer 100

There are five main layers of the epidermis and they are, from superficial to deep:
- stratum corneum:
- comprised of 15-30 layers
- most superficial layer of the epidermis
- is only layer exposed to the outside environment
- the increased keratinisation/cornification gives it its name
- dry, dead layer with multiple functions including:
- mechanical immune barrier: helps prevent the penetration of microbes
- water regulation: prevents dehydration of underlying tissues
- mechanical protection: prevents damage to more delicate underlying layers
- cells in this layer are shed periodically and replaced by cells pushed up from granulosum ^[exc. palms and soles, lucidum]
- the entire layer is replaced every four to five weeks
- if the differentiation pathway is normal, final step of desquamation is orderly: individual cells separate as corneodesmosomes degrae and intercellular lipids are worn or washed away
- NOTE: there will be visible scaling if process is impaired by inflammation or genetic influences. as the keratinocytes do not detach properly
- stratum lucidum:
- a smooth and seemingly translaucent layer
- ONLY in thick skin of palms, soles and digits
- the keratinocytes of the stratum lucidum are flatteneed, and densely packed with eleidin
- eleidin is a clear protein derived from keratiohyaline – giving the cells ther transparent appeartance
- it is the lowest level of the terminally differentiated skin
- the strata luceum and corneum are essential to the skin’s barrier function
- N.B. cells in this layer are functionally dead with limited and degrading internal metabolism
- the cells of the lower stratum corneum and stratum lucidum are connected by a combination of desmosomal structures and hydrophobic bonds from the lipids that are extruded from the cells from the contents of their lamellar granules
- n.b. at this level, desmosomes are similar in structure and are termed corneodesmosomes
- stratum granulosum
- granular appearance due to accumulated cytoplasmic granules in preparation for final stages of keratinocyte differentiation. There are two main types of granules:
- keratohyaline granules: contain a number of proteins; the predominant one is filaggrin
- lamellar granules: so named for their lamellar appearance; containing a mix of lipids and proteases
- as cells differentiate further, the keratohyalin granules are processed, releasing their contents, including filaggrin
- filaggrin binds the keratin cytoskeleton together and collapses it into a dense protein wafer– which is visible as the stratum lucidum
- stratum spinosum:
- composed of 8 to 10 layers of keratinocytes
- the layer formed as a result of cell division in s. basale
- it is spiny in appearance (Artifact of staiing process) due to the protruding cell processes that join the cells via desmosomes
- the desmosomes, in turn, are linked to the keratinocyte cytoskeleton (a filamentous protein structure)
- keratin tetramers are integral to the keratinocyte cytoskeleton
- keratins are the dominant intermediate filament type in keratinocytes and have a mechanical function
- 54 known functional genes for keratin
- mutations lead to different pathologies: lower level keratins affect epidermal structural integrity, upper level keratins affect both epidermal integrity and regulation of growth and terminal differentiation
- stratum basale:
- a single layer of cells
- primarily made of basal cell: cuboidal shaped stem cell that is a precursor of the keratinocytes of the epidermis
- all of the keratinocytes form from this single layer, which are constantly undergoing mitosis to produce new cells
- as new cells are formed, existing cells are pushed superficially i.e. away from the stratum basale
- Two other cells types are found dispersed among the basal cells:
- Merkel cells which fucntions as a receptor and is responsible for stimualting sensory nerves, that the brain perceives as touch. Merkel cells are abundant on the surface of the hands and feet
- Melanocytes which produces mealnin and give hair and skin its colour and also helps protect livign cells of epidermis from UV damage
- ![[Pasted image 20230707171041.png]]
- Melanin is a dark brown/black pigment synthesised by melanocytes in organelles called melanosomes
- These are transported inot the keratinocytes that each mealnocyte suppprts
- the rate of melaninpriducton is regulated by chemical signals from keratinocytes, influenced by sunlight expoosre and cellular injury i.e. a suntan is a distress response to cellular injury
- the function of melanin is to protect keratinocytes from UB damage– by physically blocking light and as a free-radical absorber
- n.b. exc. sun exposure causes cumulative DNA damage that may resut in skin cnaner development

To remember: Can lions growl so brutally? ^[chatGPT input]

Question 101

Describe Merkel cells and melanocytes

Answer 101

Two other cells types are found dispersed among the basal cells:
- Merkel cells which fucntions as a receptor and is responsible for stimualting sensory nerves, that the brain perceives as touch. Merkel cells are abundant on the surface of the hands and feet
- Melanocytes which produces mealnin and give hair and skin its colour and also helps protect livign cells of epidermis from UV damage

Question 102

Describe the role of melanin

Answer 102

• Melanin is a dark brown/black pigment synthesised by melanocytes in organelles called melanosomes
  - These are transported inot the keratinocytes that each mealnocyte suppprts
  - the rate of melaninpriducton is regulated by chemical signals from keratinocytes, influenced by sunlight expoosre and cellular injury i.e. a suntan is a distress response to cellular injury
  - the function of melanin is to protect keratinocytes from UV damage– by physically blocking light and as a free-radical absorber
  - n.b. exc. sun exposure causes cumulative DNA damage that may resut in skin cnaner development

Question 103

Describe the dermis

Answer 103

The dermis is a layer of connective tissue that is home to a number of structures including the hair follicles, eccrine glands, sebaceous glands, blood vessels and nerves.
The dermis is composed a ground substance made primarily from glycosaminoglycans with various types of collagen and elastin fibres dispersed through it.

Like the epidermis, the dermis is made of layers:
- papillary dermis:
- forms a tight interlock with the epidermis above it
- ![[Pasted image 20230709114122.png]]
- reticular dermis:
- thicker, lower level of the dermis
- houses blood vessels and nerve fibres, and ‘other things’

To remember: “PR”

Collagen is the major structural component of the dermis.
Of the 13 types of collagen, 9 are present in the skin.
Type 1 is the most predominant (80%).

Elastin fibres form 3% of the skin’s dry weight. They are seen in both papillary and reticular dermis, but arranged slightly differently in the layers.
Elastin is responsible for the skin’s ability to stretch and recoil.

Question 104

Describe adnexal skin structures

Answer 104

The dermis has a number of adnexa:
- arrector pili muscle: a bundle of smooth muscle that connects the hair follicle to the connective tissue of the dermis. When it contracts it causes the hair to stand erect ^[This is important for thermoregulation in lower order fur covered animals.] It may assist the shiver response in humans, but otherwise largely vestigial.
- hair follicle: which can be divided into two parts
- upper portion consists of the infundibulum (the area between the opening of the sebaceous gland and the surface of the skin) and the isthmus (area between attachment of the arrector pili muscle and opening of the sebaceous gland)
- lower portion consists of the bulb and suprabulbar region
- the hair shaft is derived from various keratins and arises from the hair bulb. Composed of three main layers: cuticle, cortex, medulla
- growth cycle: hair grows in cycles. The rate at which it grows varies depending on site, androgen influence and external/nutritional factors. It is broken down into three main stages: anagen or active growth, catagen or rest, telogen or shedding (ACT)

sebaceous (oil) gland:
- produce oil or sebum.
- Sebaceous glands are usually associated with a hair follicle. Together the unit is called the pilo-sebaceous gland.
- There is a high concentration of sebaceous glands on the scalp, face, shoulders and chest.
- The sebaceous gland consists of the bulbous-like sebaceous gland and the sebaceous duct which connects with the hair follicle at the level of the infundibulum. **The sebaceous glands secrete by holocrine secretion i.e. the entire cell undergoes complete degradation releasing its contents into the gland’s lumen. **
- Sebum is a waxy substance composes of free fatty acids, wax and sterol esters, triglycerides and squalene.
- Sebum production appears to be highly androgen sensitive, increasing at puberty, and reducing later in life
- sweat gland:
- eccrine glands:
- most abundant sweat glands and can be found on any surface of the human body
- higher density on palms and soles
- the eccrine secretory unit consists of a coiled secretory section that drains into a long thin duct, whose apex opens onto the surface of the skin
- it secretes a sterile, dilute electrolyte substance that contains NaCl, K and HCO3
- eccrine sweat glands are activated by emotional and thermal stimuli
- their primary role is to maintain thermoregulation
- apocrine glands:
- within the axillae, anogenital, periumbilicus, and areolae
- the apocrine secretory unit consists of a coiled secretory section that drains into a short thick duct whose apex opens into the upper portion of the hair follicle
- apocrine glands secrete by merocrine secretion i.e. they accummulate their secretory product in their apices and release them in lipid bound vesicles into the gland’s lumen
- apocrine sweat is an oily substance that contains cholesterol, triglycerides, fatty acids, androgens, carbohydrates, and ammonia
- resident bacteria on the skin can degrade these substances resulting in bromhidrosis (body odour)
- ![[Pasted image 20230709120622.png]]

Question 105

Describe nails

Answer 105

The nail is a hard plate composed of keratin-rich onychocytes that sit upon the distal phalanx. It acts to protect the finger or toe but also plays a role in sensation. The nail plate is derived from the nail matrix which sits underneath the eponychium and is closely related to the joint.

![[Pasted image 20230709120945.png]]
![[Pasted image 20230709121057.png]]
There are several components of the nail:
- free edge of the nail : ‘free part’ of nail bed that protrudes beyond the end of the finger
- hyponychium: skin underneath the free edge of the distal nail plate
- onychodermal band: the pink transverse band at the distal end of the nail that marks detachment of the nail plate from the nail bed
- nail plate: ‘hard nail’. It is held onto the nail bed by a series of the longitudinal ridges that run from the lunula to the onychodermal band
- lateral nail fold: a fold in the skin of the finger along the side wall of the nail plate which protects the nail plate, and guides nail growth
- lunula: half moon shaped white patch over the proximal nail that demarcates the junction of the nail
- eponychium (or cuticle): a thin strip of epidermis that adheres to the nail to prevent inflammation of the nail matrix
- proximal nail fold: a double layer of skin overlying the proximal nail to help guide and mold it as it emerges from the nail matrix. It also helps protect the nail matrix
- nail bed: the underlying support structure that the nail plate moves along as it grows. It has longitudinal grooves that interlock with the under-surface of the nail plate, guiding and retaining it.