Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

1PATH3305 Medical Genetics > Lecture 5: Epigenetics > Flashcards

Lecture 5: Epigenetics Flashcards

1
Q

What does Epi mean?

A

Epi : from Greek – “on” or “upon”

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is Epigenetics?

A

Study of REVERSIBLE ‘HERITABLE’ changes in gene function that occur WITHOUT A CHANGE in the sequence of DNA.

i.e. “upon” or “in addition to” the genetic code

Genetic code in each cell is the same – EPIGENETIC CODE IS TISSUE AND CELL SPECIFIC

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Epigenetic: definition by Adrian Bird…without heritability

A

Adrian Bird proposed a modern definition that
avoids requirement for heritability:

“the structural adaptation of chromosomal
regions so as to register, signal or
perpetuate altered activity states”

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

define epigenetic - according to lecture

A

Epigenetics = study of the factors that cause STABLE &
HERITABLE, yet REVERSIBLE, changes in the way genes are expressed without changing their original DNA
sequence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Epigenetic changes are by…
+EXAMPLES

A

Epigenetic changes are by ADDING or SUBTRACTING various
CHEMICAL TAGS on DNA nucleotides/histones

EXAMPLES
* DNA methylation, histone acetylation/methylation +
OTHERS

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Defining Epigenetics:

Persistence of epigenetic marks.

A
  1. Alterations that last less than one cell cycle (green asterisk, a) do not
    qualify as epigenetic under the definition that strictly requires heritability.
  2. Whereas non-mutational changes that are transmitted from one cell to
    its daughters (red asterisk, b)
  3. or
    between generations of an organism
    (blue asterisk, c) do qualify.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Epigenetic processes: 3

A
  • Histone modification & chromatin remodeling
  • DNA methylation
  • Non-coding RNA-mediated regulation
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Epigenetic processes: examples = 5

A

1 * Cooling tulip bulbs before planting

2 * Maternal nurture

3 * Calico cats

4 * Honey bees – workers vs queens

5 * Host immunity e.g.
against retrovirus

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

what is chromosomes?

sizes of DNA TO CHROMOSOME…

A
  • DNA + protein (chromatin) = chromosomes
  • DNA DOUBLE HELIX = 2nm
  • NUCLEOSME CORE OF ‘8 HISTONE MOLECULES’
  • CHROMATOSOME
  • Histone H1 = 11nm
  • 30nm
  • 300nm
  • 250nm wide fibre
  • 700nm
  • chromosome = 1400nm

look at slide 9

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

DNA sequences are commonly compared to … epigenetics can be viewed similarly to..

A

DNA sequences are commonly compared to a text of written letters.

Epigenetics can be viewed
similarly to punctuation that can modify the meaning of text.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Cut And Paste.. LOOK at slide 12

A
  1. DNA methylation
  2. Histone Modification
  3. Small non-coding RNAs
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

A typical eukaryotic promoter
* Gene transcription: vs Histone proteins

Look at slide 13 image

A

‘Gene transcription’ in eukaryotes occurs in the context of DNA packaged
into chromatin

‘Histone proteins’ are important components of the “Epigenetic machinery”
that package genomic DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

The nucleotides of DNA?

VERSIONS OF CYTOSINE?

A

PURINES = Adenine + Guanosine

Pyrimidines = thymine, cytosine

– Cytosine (C)
— 5-methylcytosine (mC)
—- 5-Hydroxymethylcytosine (hmC)

LOOK AT CHEMICAL IMAGE ON SLIDE 14

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What are Histones? What is Histone H1? 4

A

1 * Nucleosomes arranged as an octamer of histone
proteins with protruding N-terminal ends.

2 – 147 bp of coiled DNA wrapped around the histones.

3 – Two each of the four core histones H2A, H2B, H3 and H4.

4 * Histone H1, the linker protein, is bound to DNA
between nucleosomes.

LOOK AT HISTONE IMAGE SLIDE 5

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Histone modifications and the histone code hypothesis: 4

A
  1. Modifications of the HISTONE TAILS act as EPIGENETIC MARKERS that control the expression
    or replication of chromosomal regions.
  2. The epigenetic marks in the histones are HERITABLE.
  3. The pattern of histone modifications (the histone code) can determine how
    histones behave.
  4. Modifications of the histone tails act as epigenetic marks that control the expression of chromosomal regions (controlled by transcription factors).
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Many histone tags work together to control histones. These include: 4

A
  1. Acetyl
  2. Phosphate
    3.Methyl
  3. Ubiquitin
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Active & repressive marks

A
  1. Different amino acids
    constituting HISTONE TAILS are represented along with the DIFFERENT COVALENT MODIFICATION SPECIFIC OF EACH RESIDUE.

2.ACTIVE MARKS are represented in
the SUPPER SECTION, and
REPRESSIVE MARKS in the LOWER SECTION

LOOK AT SLIDE 17 IMAGE

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Understanding Histone acetylation..8

A
  1. DNA is NEGATIVELY CHARGED , whilst HISTONES ARE POSITIVELY CHARGED
  2. Acetylation of histones OCCUR IN LYSINE RESIDUES OF HISTONE TAILS
  3. Acetylation NEUTRALISES THE POSITIVE CHARGE AND DECREASES THEIR AFFINITY FOR DNA.
  4. DNA IS LESS TIGHTLY WOUND AND PERMITS TRANSCRIPTION.
  5. “Acetylated lysine residues” = transcriptional ACTIVATION (gene
    expression)
  6. “Deacetylated lysine residues” = transcription REPRESSION (gene
    silencing)
  7. Histone acetylase (HAT) & histone deacetylase (HDAC) ENZYMES ADD/REMOVE ACETYL GROUPS
  8. HISTONES NEAR ACTIVE GENE ARE HYPERACETYLATED.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Histone de/acetylation

hdac vs hat

A

HDAC = for Deacetylation

HAT = ACETYLATION

LOOK AT IMAGE ON SLIDE 19

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Understanding DNA Methylation…

A

1 * A well studied example of an EPIGENETIC MECHANISM involved
in GENE REGULATION (AND OTHER PROCESSES)

2 *OCCURS IN BACTERIA (RESTRICTION ENZYME SITES), …NONE IN YEAST OR WORMS

3 * FOR VERTEBRATES=
- 5-methyl C residues within the CpG dinucleotide common (70-80% methylated CpG in mammals)

4 * CpG, CpNpG and CpHpH (H=A, T or C) methylation of C
COMM IN PLANTS (RARE IN ANIMALS)

    • 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine appear as intermediates of active DNA-demethylation
      during embryogenesis
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What appear as intermediates of active DNA-demethylation
during embryogenesis? 3

A

1….5-hydroxymethylcytosine,

2….. 5-formylcytosine,

3…. 5-carboxylcytosine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Understanding CpG DNA methylation… 4

A
  1. Forward equation:
    Cytosine — DNA methyltransferase (DNMT) —> 5 - Methylcytosine
    (AdoMet —–> AdoHcy)
  2. Reverse Equation: 5-methylcytosine —– DNA demethylase —–> Cytosine
  3. CpG RARE in most Vertebrate genomes —- convert to TpG
  4. CpG: p refers to the phosphate between bases
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Look at CpG DNA Methylation Equation …

A

slide 21

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Methylation - Normal Processes

CAN BE FOUND IN = 4

A

1 * embryonic development

2 * X chromosome inactivation

3 * Imprinting

4 * Gene silencing

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Conrad Waddington’s Epigenetic Landscape (1946)…

WHAT IS WADDINGTON LANDSCAPE?

A

Waddington landscape: A metaphor of development,
in which valleys and ridges illustrate the epigenetic
landscape that guides a PLURIPOTENT CELL to a WELL-DEFINED, DIFFERENTIATED STATE…., represented by a ball rolling down the landscape.

…Ball rolls down to spectator, corresponds to the developmental history of a particular part of the egg.
… first alternative = towards left or right.
… second alternative : along the former path..along the path to the left, the main channel continues leftwards but there is an alternative path which, however can only be reached over a threshold.

‘the complex system of interactions underlying the epigenetic landscape…”
- the pegs in the ground represent genes
- strings leading from them the chemical tendencies which the genes produce
- the modelling of epigenetic landscape, which slopes down from above one’s head towards the distance, is controlled by the power of numerous guy-ropes which are ultimately anchored the genes.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Stages of Conrad Waddington’s Epigenetic Landscape (1946)

A
  1. STEM CELLS
  2. NORMAL DEVELOPMENT OR AT NEOPLASIA
  3. NORMAL DEVLEOPMENT = DIFFERENTIATED STATES
  4. EPIGENETIC MODULATORS ..(HILLS AND VALLEYS)..
  5. EPIGENETIC MODIFIERS
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

Conrad Waddington’s
Epigenetic Landscape (1946)…LOOK AT DIAGRAM

A

SLIDE 24

28
Q

Epigenome remodeling
during embryogenesis…9

NEED TO LOOK AT SLIDE 25 AND UNDERSTAND DIAGRAM + IMAGES

A
  1. Oocyte + sperm
  2. Zygote
  3. Morula
  4. Blastocyte (inner cell mass –> ES)
  5. Embryo — PGCs —> Embryonic germ cells
  6. adult (male or female)
  7. germ cells

8… IN GERM CELLS = DNA methylation
… H327 METHYLATION
… Pluripotency -associated genes + developmental genes

  1. EMBRYO AND SOMATIC CELLS =
    …DNA methylation
    … H3K27 methylation
    … H3K4 methylation
    ….Pluripotency -associated genes + developmental genes
29
Q

Examples of epigenome remodeling during differentiation IN…4

A
  1. LYMPHOID/MYELOID DEVELOPMENT
  2. T-HELPER DIFFERENTIATION
  3. MUSCLE STEM CELL DIFFERENTIATION
  4. NEURAL STEM DIFFERENTIATION
30
Q

Look at how Epigenome remodeling
during embryogenesis…LYMPHOID/MYELOID DEVELOPMENT

A

DRAW, LABEL AND UNDERSTAND SLIDE 26

31
Q

Epigenome remodeling
during embryogenesis…IN T-HELPER DIFFERENTIATION

A

DRAW, LABEL AND UNDERSTAND SLIDE 26

32
Q

Epigenome remodeling
during embryogenesis… IN MUSLCE STEM CELL DIFFERENTIATION

A

DRAW, LABEL AND UNDERSTAND SLIDE 27

33
Q

Epigenome remodeling
during embryogenesis… IN NEURAL STEM CELL DIFFERENTIATION

A

DRAW, LABEL AND UNDERSTAND SLIDE 27

34
Q

EPIGENOME CHANGES UNDERLIE CELL-SPECIFICATION …

A

SLIDE 28

35
Q

Understanding Mammalian X chromosome inactivation…3

A

1 * EARLY EMBRYO HAS has BOTH X’s ACTIVE.

2 * METHYLATION PATTERNS are HERITABLE through MITOSIS, but RESET DURING Oogenesis

3 * FULLY DEVELOPED FEMALE IS A ‘MOSAIC’ OF DIFFERENT CLONES
(eg calico cat eg human -ANHYDROTIC DYSPLASIA MOCAICISM).

36
Q

Look at Mammalian X chromosome inactivation… SLIDE 29 FLOW CHART 4

A
  1. Sperm + egg
  2. Early zygote
  3. Late blastocyst: RANDOM INACTIVATION OF MATERNAL OR PATERNAL ‘X’ IN DIFFERENT CELLS, RESULTING IN MOSAICISM
  4. EXAMPLE OF PATERNAL X -INACTIVATES: all descendant cells have paternal X-inactivated (STABLE INACTIVATION)

+

EXAMPLE OF MATERNAL X-INACTIVATED (STABLE INACTIVATION)

37
Q

Mechanism of X Inactivation: 3

A

1 * At ~1000 cell stage, cell chooses one X to remain ON.

2 * Other X is inactivated via XIST (X Inactivation- Specific Transcript) – an X chromosome-encoded
lncRNA

3 * XIST coats the X chr LEADING TO HETEROCHROMATIN SPREADING (SILENCING) AND METHYLATION

38
Q

Mechanism of X Inactivation… LOOK AT DIAGRAM OF HOW

A

SLIDE 30

39
Q

X inactivation… WHEN AND WHERE DOES IT OCCUR?

DOSAGE COMPENSATION?

A
  1. Occurs EARLY IN THE DEVELOPMENT of most FEMALE MAMMALS—–> HUMAN FEMALES ARE ‘FUNCTIONALLY MOSAIC’
  2. MOST OF THE GENES ON ONE X-CHROMOSOME ARE INACTIVATED (BY METHYLATION) IN EVERY CELL (E.G whole chromosome inactivation).
  3. OVERALL TRANSCRIPTION DOSAGE OF Chr X genes is EQUAL IN MALES AND FEMALES (“DOSAGE COMPENSATION”)
  4. TAKES PLACE EARLY IN DEVELOPMENT, ~64 – 128 CELL (BLASTOCYST) STAGE IN MOUSE ZYGOTE.
40
Q

Mammalian X chromosome inactivation 6

… EXAMPLE…
Calico / Tortoiseshell cat

A

1 * Early female embryo has both X’s ACTIVE.

2 * RANDOM INACTIVATION OF MATERNAL OR PATERNAL X IN DIFFERENT CELLS.

  1. PROCESS:
    - Zygote
    - Ball of cells with random X inactivated
    - Mixture of X-inactivated cells in the embryo
  2. Cell division
    - active X chromosome, inactive X chromosome (Barr bodies)
    x2
  3. One X chromosome is inactivated in each cell. Which one is by chance.
  4. Coats of tortoiseshell cats have patches of orange and black
40
Q

What is X inactivation? Example?

A
  1. SILENCING OF ONE X CHROMOSOME IS RANDOM (EACH CELL HAS INDEPENDENT CHOICE)
    – All descendants of that cell keep the same pattern
  2. Tortoiseshell cats – X-linked coatcolour gene
    - Each patch of differently coloured fur represents the progeny of one embryonic cell (same inactive X chromosome).
41
Q

What is Skewed X inactivation = 3

1.X activation sometimes
2. Proposed that the abnormal or mutation-carrying X chromosome:

3.. RELEVANCE TO HUMAN DISEASE:

A
  1. X activation sometimes is non-random (skewed)
  2. Proposed that the abnormal or mutation-carrying X chromosome:
    - is preferentially inactivated, and/or
    - has some growth/proliferation disadvantage, so cells in which it is active are fewer in number in adult females
  3. RELEVANCE TO HUMAN DISEASE:
    - can provide evidence that an X-linked disorder might be occurring in a family
    - can explain phenotypic variability in females “carrying” X-linked disorders
42
Q

Understanding Skewed X inactivation… process and diagram

A

a. random
b skewed

slide 35

43
Q

UNDERSTANDING TYHE AGOUTI MOUSE MODEL…

SOURCE?
GENE?
METHYLATED?
UNMETHYLATED?

A
  1. DIET is a MAJOR SOURCE of METHYL DONORS
  2. Mice have an ‘Agouti’ GENE THAT CONTROLS FUR, COLOUR, OBESITY AND SUSCEPTIBILITY TO DIABETES AND CANCER

3.When the Agouti gene is METHYLATED (OFF), mice
are BROWN AND HEALTHY

  1. when the gene is UNMETHYLATED (ON), mice are YELLOW AND UNHEALTHY
44
Q

Epigenome remodeling
during embryogenesis

A

SLIDE 37.. REPEAT

45
Q

UNDERSTANDING EMBRYONIC DEVELOPMENT.. IN ‘Agouti gene’ = 3

A

1 * Mutation in Agouti gene causes obesity & yellow coat colour

2 * Agouti encodes a PARACRINE SIGNALLING MOLECULE THAT PROMOTES FOLLICULAR MELANOCYTES TO PRODUCE YELLOW PHAEOMELANIN RATHER THAN BLACK EUMELANIN PIGMENT.

3 * The MUTANT A(vy ) ALLELE is the RESULT OF TH E INSERTION OF A MURINE ‘IAP’ TRANSPOSABLE ELEMENT ABOUT ‘100kb’ UPSTREAM OF THE TRANSCRIPTIONAL START SITE OF THE AGOUTI GENE

46
Q

UNDERSTANDING EMBRYONIC DEVELOPMENT.. IN ‘Agouti gene

The mutant Avy allele is the result of the insertion of a murine IAP transposable element about 100 kb upstream of the transcriptional start site of the agouti gene1

A

DRAW AND LABEL THE FIGURE …SLIDE 38

47
Q

Dietary supplementation of female mice during pregnancy

A

DRAW AND LABEL SLIDE 39

48
Q

What is Genomic imprinting?

A
  1. Parent-specific expression or repression of genes
    or chromosomes in offspring.
  2. So… even though two copies of a given gene are inherited (one from each parent) only the maternal or paternal allele is expressed.
  3. The non-expressed allele is said to be “imprinted.”
49
Q

Genomic imprinting leads to allele-specific expression
depending on the parent of origin of the allele… process 11

A
  1. Zygote
  2. maintenance
  3. blastocyte
  4. maintenance
  5. Embryo..
  6. reading
  7. Erasure
  8. Primodial germ cells
  9. establishment
    - IC2 or IC1
  10. MATURE GAMETES
  11. ZYGOTE

CLYCLICAL

50
Q

DRAW AND LABEL THE CYCLICAL PROCESS OF:

Genomic imprinting leads to allele-specific expression
depending on the parent of origin of the allele.

A

SLIDE 42

51
Q

Understanding IMPRINTING AND DISEASE…4

A

1 * >70 imprinted genes identified in human genome.

2 * DEREGULATION of IMPRINTED GENES has been oOBSERVED IN HUMAN DISEASES.

    • These diseases are CHARACTERISED BY ‘NON-MENDELIAN’ INHERITANCE PATTERNS THAT EXHIBIT PATERNAL-ORIGIN EFFECTS

4 * SYMPTOMS suggest A ROLE OF IMPRINTED GENES IN GROWTH REGULATION DURING EMBRYONIC AND POST-NATAL DEVELOPMENT, BRAIN FUNCTION AND BEHAVIOUR.

52
Q

LOOK AT SLIDE 45 AND UNDERSTAND…MOUSE IMPRINTED GENES, REGIONS AND PHENOTYPES…

A

SLIDE 45

53
Q

Diseases associated with genomic imprinting: 6

A
  1. Beckwith–Wiedemann syndrome
    • Prader–Willi syndrome
    • Angelman syndrome
    • Wilms Tumor
    • Fragile X syndrome
    • Myotonic dystrophy (congenital)
54
Q

Cancers Associated With Loss of Imprinting:

A

1:Embryonal tumors of childhood
- Wilms’ tumor*
- Hepatoblastoma*
- Rhabdomysarcoma*
- Ewing’s sarcoma*

2: Adult malignancies
- Uterine*
- cervical*
- colorectal*
- esophageal*
- prostate*
-testicular*
- lung*
- breast*
- ovarian
-choriocarinoma
- bladder
-liver
- leukemia

LOI of IGF2 is one of the most common molecular alterations in human cancers *

55
Q

Cancers Associated With Loss of Imprinting:

Embryonal tumors of childhood: 4

A
  • Wilms’ tumor*
  • Hepatoblastoma*
  • Rhabdomysarcoma*
  • Ewing’s sarcoma*

LOI of IGF2 is one of the most common molecular alterations in human cancers *

56
Q

Cancers Associated With Loss of Imprinting:

Adult malignancies: 13

A
  • Uterine*
  • cervical*
  • colorectal*
  • esophageal*
  • prostate*
    -testicular*
  • lung*
  • breast*
  • ovarian
    -choriocarinoma
  • bladder
    -liver
  • leukemia

LOI of IGF2 is one of the most common molecular alterations in human cancers *

57
Q

Angelman and Prader-Willi syndromes both have WHAT IN COMMON?

A

Both involve imprinting defects at 15q11-q13

58
Q

UNDERSTANDING ANGELMAN SYNDROME…

A

1 * severe mental retardation, microcephaly, lack of speech, frequent laughter.

2 * deletion of maternal 15q11-q13 (loss or inactivation of maternal UBE3A gene)

59
Q

UNDERSTANDING PRADER-WILLI SYNDROMES…2

A

1 * mild mental retardation, obesity, short stature.

2 * deletion of paternal 15q11-q13 (deficient snoRNAs expression of paternal allele.)

60
Q

Imprinted 15q11-q13 region

A

LOOK AT DIAGRAMS
DRAW LABEL AND UNDERSTAND

SLIDES 49 AND 50

61
Q

Genome organisation, function and imprinting in Prader-Willi ans Angleman syndromes..

A
  1. PWS, hypotonia, respiratory and failure thrive in the postnatal period, with hyperphagia in early childhood, resulting in OBESITY.
    - Short stature, small ands and feet, hypogonadism, mild-moderate mental redardation, temper tantrums and obsessive-compulsive disorder.
  2. AS, developmental delay, severe mental retardadtion, with lack of speech, movement ataxia, hyperactivity, Seizures, aggressive behaviour and excessive inappropriate laughter.
62
Q

Epimutations in Prader-Willi and Angleman syndromes: a molecular study of 136 patients with an imprinting defect.

FINDINGS

A
  1. Majority of patients show no IC mutation
    - Distribution of mutation status among patients with AS and PWS
  2. Inheritance is completely maternal (AS) r completely paternal (PWS)
    - Grandparental origin of the chromosome carrying the imprinting defect
63
Q

Beckwith–Wiedemann syndrome..FEATURES.. CAUSES..

A

Features: embryonic and placental overgrowth, predisposition to childhood
tumors (big baby or big lamb syndrome).

  1. Cause: genetic and epigenetic changes in a region of about 1 megabase on chromosome 11p, encompassing 15 genes, the majority of them being imprinted.
    • IGF2 and CDKN1C are key genes
    • IGF2 (insulin-like growth factor) Normally this gene is only expressed by the
      paternal chromosome.
    • CDKN1C, on the other hand, is 700kb away, and is
      maternally expressed.
    • Increased expression of IGF2, and suppression of CDKN1C are believed to be
      the major cause of the disease.
64
Q

Normal Control in the 11p15 imprinted locus… LOOK AT SLIDE 54

A

SLIDE 54

65
Q

Conclusions = 5

A

1 * Epigenetic regulation allows changes in gene expression in
response to environmental changes

2 * Allows reversion to be made in response to changes
(because DNA sequence is not changed)

3 * Epigenetic regulation provides a NON-PERMANENT link
between the environment and the genome (eg diet, nurture).

4 * Epigenetic remodeling is crucial for normal development.

5 * Epigenetic de-regulation can drive human disease.

1PATH3305 Medical Genetics (20 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions