Frailty Flashcards

1
Q

What is RT-PCR used for?

A

Used to detect RNA expression in a sample, for example viral RNA or mRNA (to detect gene expression)

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2
Q

What are the steps of RT-PCR?

A

Purify the RNA
First strand synthesis: Use RT to convert RNA into double stranded cDNA
cDNA amplified by PCR

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3
Q

What is one step PCR? Also give one pro and one con

A

One step combines the steps of first strand synthesis and PCR amplification in a single tube
Minimises chance of contamination
Analysis limited to a few genes per sample

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4
Q

What is two step PCR? Also give one pro and one con

A

Reactions for first strand synthesis and PCR amplification take place in separate tubes
Allows for optimisation of reaction conditions
Increased chance of contamination

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5
Q

What is senescence?

A

Deterioration with age

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6
Q

What is cellular senescence?

A

Cells lose the ability to divide with age, so growth becomes halted

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7
Q

What causes cellular senescence?

A

Ageing causes telomere shortening
DNA damage detected by p53
p53 halts cell cycle at G1/S
Cells with arrested cell cycle will eventually apoptose

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8
Q

What is immunosenescence?

A

Deterioration of the immune system brought on by ageing

Includes inflammaging

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9
Q

What is inflammaging?

A

Chronic, low grade inflammation

Chronically elevated levels of circulating proinflammatory cytokines

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10
Q

What causes inflammaging?

A

Ageing, cellular senescence and ROS trigger NFkB signalling

Products of NFkB signalling cause inflammation

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11
Q

How can inflammaging increase the risk of cancer?

A

Triggers genetic mutations

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12
Q

What is apoptosis?

A

Programmed cell death

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13
Q

What is ELISA and what is it used for?

A

Enzyme Linked Immunosorbent Assay
Used to detect levels of protein/antigen/antibodies/glycoprotein in a biological sample
Can be used for pregnancy tests and detecting HIV

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14
Q

What are the steps of ELISA?

A
  1. Coat ELISA plate with capture antibody (raised to antigen of interest)
  2. Add sample - antigens captured by antibody
  3. Add enzyme-labelled detection antibody - binds to antigens bound to plate
  4. Add substrate to initiate chromogenic reaction
  5. Coloured product measured using plate reader
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15
Q

How can ELISA be used to detect apoptosis?

A

Apoptosis results in release of nucleosomes

Use ELISA to detect cytoplasmic nucleosomes in a sample

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16
Q

How is apoptosis initiated?

A

Cytokines activation JAK/STAT receptors, leads to activation of caspases

Bad protein binds to and forms a complex with Apoptosis Inhibitory Protein

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17
Q

What are caspases?

A

Protease enzymes that initiate apoptosis

Cysteine residue carries out protease activity

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18
Q

Where to tyrosine kinases phosphorylate?

A

Serine and Threonine residues

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19
Q

Steps of apoptosis

A

Small blebs form
Nucleus breaks down, DNA breaks down into smaller fragments
Organelles contained in blebs, still functioning
Breakdown into several apoptotic bodies

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20
Q

What is necrosis?

A

Non-programmed, premature cell death

Caused by infection, toxins, trauma

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21
Q

Steps of necrosis

A

Small blebs form
Structure of nucleus changes
Blebs fuse and become larger
Loss of cell membrane integrity; membrane ruptures (autolysis) and releases cell contents

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22
Q

What is autophagy?

A

Ordered degradation and recycling of cellular components

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23
Q

What is autophagy used for?

A

Maintain cellular energy levels during starvation
Degrade damaged organelles
Promote cell death

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24
Q

What are the 4 types of autophagy?

A

Macroautophagy
Microautophagy
Chaperone-mediated autophagy
Mitophagy

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25
Q

What is macroautophagy?

A

Phagophore engulfs organelles that are damaged or unused
Forms double membraned autophagosome
Autophagosome fuses with lysosome

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26
Q

What is microautophagy?

A

Direct engulfment of material into the lysosome, via invagination of the lysosomal membrane

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27
Q

What is chaperone-mediated autophagy?

A

Proteins with consensus motif (penta-peptide motif) recognised by Hsc70 chaperone protein
Substrate/chaperone complex taken to lysosomal membrane
Protein unfolds and translocated across lysosomal membrane

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28
Q

What is Hsc70?

A

Heat-shock protein

Produced by cell in response to stressful conditions

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29
Q

What is mitophagy?

A

Degradation of defective mitochondria

30
Q

What are ULK proteins?

A

Unc-51 Like Kinase

ULK1 and ULK2 activate downstream components of autophagy

31
Q

How is autophagy inhibited?

A

mTOR and AMPK phosphorylate ULK1 and ULK2

Inhibits ULK1 and ULK2 from initiating autophagy

32
Q

What are the genes called that regulate autophagy?

A

Atg genes

33
Q

How is autophagy induced?

A

Dephosphorylation of ULK1 and ULK2

34
Q

What is ubiquitination?

A

Ubiquitin ligase adds a ubiquitin tail onto a protein

This marks them to be degraded by a proteasome, or may alter their cellular location or activity

35
Q

What is a proteasome?

A

Protein complex that degrades unused proteins via proteolysis
Cylinder-like structure made of alpha and beta subunits
Proteins degraded in the hollow core

36
Q

What are the steps of the ubiquitin/proteasome pathway?

A
  1. Activation - Using ATP, Enzyme E1 activates ubiquitin
  2. Conjugation - Enzyme E2 transfers ubiquitin from E1 to E2
  3. Ligation - Enzyme E3 transfers ubiquitin onto the substrate
  4. Substrate degraded by proteasome
37
Q

What is the ubiquitin/proteasome pathway used for?

A

Degrade regulators of the cell cycle, regulators of apoptosis, transcription factors

38
Q

What is IGF-1 and what does it do?

A

Insulin-like growth factor 1
Binds to tyrosine kinase receptors
Initiates intracellular signalling pathways such as MAPK and PI3K
Causes cell growth, inhibits apoptosis and autophagy

39
Q

How is IGF-1 produced?

A

Produced by many cell types including hepatocytes

In response to growth hormone (GH)

40
Q

What is the somatotrophic axis?

A

Nutrients/amino acids are sensed
Hypothalamus release GHRH (growth hormone releasing hormone)
Anterior pituitary releases GH
Liver produces IGF-1

41
Q

What is the PI3K pathway?

A
  1. IGF-1 binds tyrosine kinase receptor
  2. Trans-auto phosphorylation
  3. Attracts SH2 domain of PI3K
  4. PI3K phosphorylates PIP2 –> PIP3
  5. PIP3 attracts PDK
  6. PDK phosphorylates Akt, activating it
42
Q

What is PIP2?

A

A membrane phospholipid

43
Q

Why can PIP3 attract PDK?

A

PIP3 can recruit proteins with a PH homology domain

44
Q

What are the 3 effects of Akt, once activated by phosphorylation?

A
  1. Akt inhibits ‘Bad’ protein, preventing apoptosis
  2. Activates mTORC1, leading to growth
  3. Inhibits transcription factor FOXO
45
Q

What is mTOR?

A

Mechanistic Target Of Rapamycin
Kinase protein
Forms complexes mTORC1 and mTORC2
Involved in regulation of many cellular processes

46
Q

What is the function of mTORC1?

A

Upregulates protein synthesis, cell growth ribosome production

47
Q

What is the function of mTORC2?

A

Activates Akt

Which then indirectly activates mTORC1

48
Q

What 2 things activate mTORC1?

A

Akt
and
Presence of calories/lipids/nutrients

49
Q

What is FOXO?

A

Activates genes involved in autophagy/ubiquitin proteosomal degradation/lysis/muscle atrophy

50
Q

What is Rheb?

A

Protein involved in how Akt activates mTORC1

51
Q

What is sarcopenia?

A

Skeletal muscle atrophy associated with ageing

52
Q

What is IRS-1 and what does it do?

A

Insulin Receptor Substrate-1

Transmits signals from insulin receptor to the PI3K and MAPK pathways

53
Q

How does IRS-1 work in the PI3K pathway?

A
  1. IGF-1 binds receptor
  2. Trans-auto phosphorylation
  3. Receptors phosphorylate IRS-1
  4. IRS-1 recruits SH2 domain of PI3K
  5. PI3K phosphorylates PIP2 –> PIP3
54
Q

What is myostatin?

A

Type of myokine - which is a cytokine released by muscle cells
Regulates (inhibits) muscle growth

55
Q

What is myostatin also known as?

A

Growth differentiation factor 8

56
Q

What receptors does myostatin bind to? (and what type of receptors are these)

A

Autocrine Activin type I and type II receptors

which are Tyrosine kinase receptors

57
Q

What happens if you lack myostatin?

A

You have hypertrophy and more muscle mass than usual

58
Q

What 2 things does myostatin do?

A
  1. Inhibits Akt, therefore reducing mTORC1 activity and reducing protein synthesis
  2. Leads to activation of genes that inhibit myogenesis, by halting the cell cycle at G1/S phase
59
Q

How does myostatin inhibit cell cycle progression?

A
  1. Released by myocytes
  2. Binds to activin type I and II receptors
  3. Receptors dimerise and trans auto phosphorylate
  4. Phosphorylation/Activation of SMAD2/3 transcription factors
  5. SMAD2/3 form complex with SMAD4
  6. Promotes expression of genes involved in atrophy, autophagy, proteasomal degradation
60
Q

What family is myostatin a member of?

A

TGF beta (transforming growth factor-beta) superfamily

61
Q

What is anabolism and catabolism?

A
Anabolism = creating molecules
Catabolism = Breaking down molecules
62
Q

What is the damage/error theory of ageing?

A

Cumulative damage from environment
Greater rate of oxygen basal metabolism = shorter lifespan
Free radical exposure causes oxidation of macromolecules

63
Q

What is the programmed theory of ageing?

A

Ageing follows biological timetable
Depends on expression of genes for maintenance, repair, defence systems
IGF-1 has key role

64
Q

What is a quiescent cell?

A

Cells in ‘G0’ state

They are not participating in the cell cycle but can do in the presence of mitogens

65
Q

What is thought to be the cause of Progeria conditions?

A

Faulty telomerase enzyme
Telomerase allows replication of telomere ends
Without this, telomeres will shorten with every cell division

66
Q

In which cells is telomerase present?

A

Stem cells, cancer cells

Not normal somatic cells

67
Q

What is the primary structure of a protein?

A

Sequence of amino acids

68
Q

What is the secondary structure of a protein?

A

Hydrogen bonds form between amino acids

Can form alpha helix, beta pleated sheet, U turn

69
Q

What is the tertiary structure of a protein?

A

Further folding, caused by formation of bond types

  • ionic bonds
  • hydrogen
  • disulfide
  • hydrophobic interactions
70
Q

What type of proteins assist with protein folding?

A

Molecular chaperones

They are heat shock proteins, as stressful conditions can cause proteins to fold incorrectly

71
Q

What is the quaternary structure of a protein?

A

Arrangement of many peptides, forming a multi-subunit protein
Subunits held together by non-covalent bonds

72
Q

Where does protein folding occur?

A

Endoplasmic reticulum