Biology

Question 1

What are the two ways in which haematopoietic stem cells can develop?

Answer 1

The ability to self-renew and to differentiate are key characteristics of HSCs necessary for normal haematopoiesis. Self-renewal is the process by which stem cells enter the cell cycle to divide and give rise to more stem cells, this preserving the stem cell pool. Differentiation allows HSCs to develop into more mature cells with progressive lineage commitment.

Question 2

What are clusters of differentiation (CDs)?

Answer 2

As HSCs and progenitors differentiate, they acquire distinct cell surface antigen markers, also known as cluster of differentiation (CDs), while losing the antigens associated with the primitive cell. CDs can be used to differentiate stem cells and diverse progenitors, intermediates and mature blood cells of different lineages. Identifying cell subpopulations via CDs is often done through monoclonal antibodies and flow cytometry with marker panels being readily available commercially. The number of described CDs continues to grow and has exceeded 370 to date. Utilisation of CDs in clinical practice has revolutionised the way we understand, diagnose, classify and treat haematological diseases and is now standard of care to be included in the diagnostic work up.

Question 3

What types of cell are produced from myelopoiesis and what is the progenitor?

Answer 3

Progenitor - Multipotent common myeloid progenitor (CMP) cells

Cells produced - Neutrophils, Eosiinophils, Basophils, Monocytes, Macrocytes

Question 4

What types of cell are produced from lymphopoiesis and what is the progenitor?

Answer 4

Progenitor - Common lymphoid progenitor (CLP)

Cells produced - B-Cell lymphocytes and T-Cell lymphocytes

Question 5

What types of cell are produced from erythropoiesis and what is the progenitor?

Answer 5

Progenitor - megakaryocyte–erythroid progenitor cell (MEP)

Cells produced - erythrocytes (red blood cells)

Question 6

What types of cell are produced from thrombopoiesis and what is the progenitor?

Answer 6

Progenitor - megakaryocyte–erythroid progenitor cell (MEP)

Cells produced - Production of platelets (from megakaryocytes)

Question 7

What are undergoes a process of recombination in order to produce diversity of antibody specificity in B-cells?

Answer 7

• The diversity of antibody specificity is dictated by a process of gene recombination which occurs in B-cell development creating functional antibody heavy and light chain V(D)J transcripts from relatively small sets of Variable (V), Diversity (D) and Joining (J) genes
• Additional diversity is generated by imprecise joining at the V(D)J junctions, principally determined by the extent to which random nucleotides are inserted between joining genes

Question 8

In what order does rearrangement of hIg heavy and light chains occur in the B-cell recombination process?

Answer 8

Ig heavy chain rearranges first
•	Rearrangement of Ig heavy:
o	D (Diversity) joins with J (Joining)
o	V (Variable) joins with DJ (rearranged)
o	Once a viable rearrangement is achieved, the other IGH allele is excluded from rearranging

Ig light chain rearrangement proceeds heavy chain
o Initial attempts occur at the Ig kappa locus
o If a functional IGK rearrangement not achieved Ig lambda locus (IGL) undergoes recombination

Question 9

What occurs following rearrangement and the production of a functional IgH-IgL protein complex (B-cell receptor)?

Answer 9

Cells leave the bone marrow to the lymph node germinal centre (GC) where maturation occurs following encounter of a matched antigen

Question 10

What transformations occur during B-cell maturation n the germinal centre?

Answer 10

• During maturation somatic hypermutation and class switch recombination occurs
• Somatic hypermutation occurs through introducing point mutations in the rearranged genes producing high affinity antibodies that recognise and optimally bind to foreign antigens
• After initial contact with antigen, low affinity IgM is produced and class switch recombination is the mechanism which changes the isotype to IgG, IgA or IgE generating specific antibodies with different functional characteristics.

Question 11

What is chromatin?

Answer 11

The complex of histones and DNA is called Chromatin.

Question 12

What is the function of histone protein in chromosomal structures?

Answer 12

Histones provide structure by wrapping themselves around long thin strips of DNA. Histones also identify and activate the inactive genes involved in cellular mechanisms

Question 13

What are the two sub classifications of chromatin and how are they different?

Answer 13

Chromatins can be further classified as euchromatin and heterochromatin bases on the level of condensation and compaction. Euchromatin is a less compact structure described as ‘beads on a string’ where beads refer to the nucleosomes and string to the DNA. Heterochromatin is more compact and can be further categorised as constitutive heterochromatin and facultative chromatin.

Question 14

What is the difference between constitutive heterochromatin and facultative chromatin?

Answer 14

Constitutive heterochromatin stays condensed throughout the cell cycle and does not actively participate whereas facultative heterochromatin can uncoil to form euchromatin. This heterochromatin is more active and responds to cellular changes during the cell cycle as they contain genetic information used during cell division.

Question 15

What is a centromere?

Answer 15

Small constriction that is considered permanent in the structure of a chromosome. Another name for the centromere is a primary constriction which divides the structure of the chromosome into two arms; the short arm is call a p arm, and the long arm is called a q arm. Each centromeres position is constant in a particular class of chromosomes and thus helps in identification. Furthermore, during the cell cycle, the chromosomes are attached to spindle fibres with the help of centromeres.

Question 16

What are telomeres?

Answer 16

Telomeres – The repeated DNA sequences at the end of the chromosomes are called telomeres. They give the ends of the chromosomes a particular polarity which prevents the other chromosomes fusing with it. Telomere have a role in the life expectancy of an individual and shorten with age. The shorter the telomeres, the shorter the lifespan of that individual.

Question 17

What are the two types of translocations and what are the mechanisms?

Answer 17

Robertsonian - Entire chromosome has attached to another at the centromere resulting in for example a 11p and 12q joined at the centromere and 11q and 12q joined at a centromere
Reciprocal - Segments from two different chromosomes are exchanged

Question 18

What is an isochromosome?

Answer 18

An isochromosome is a mirror image copy of a chromosome segment including the centromere

Question 19

What mechanism leads to the occurrence of chromosome abnormalities in cancer?

Answer 19

Chromosome instability

Question 20

How do TCR rearrangements occur in T-lymphocytes?

Answer 20

In the case of T cells, two types of receptors are known; TCRαβ and TCRγδ. The γδ chain rearranges earlier than the αβ chain during development.

First the TCR D chains (TRD) genes rearrange, then the TCR G chains (TRG), potentially resulting in TCRγδ expression

This is followed by a further TCR B chains rearrangement (TRB) and TCRD deletion with subsequent TCR A chains (TRA) rearrangement, potentially followed by TCRαβ expression

Question 21

What TCR rearrangements have been seen in almost all lymphoprliferative disorders?

Answer 21

Rearrangements of TCR B and/or TCR G

Question 22

What is the function of the RAS-REF-MEK-ERK pathway (Ras/Raf/MAPK)?

Answer 22

This is a signal transduction pathway which activates cell growth, division and differentiation.

Question 23

What are the most frequently mutated genes in the RAS-REF-MEK-ERK pathway?

Answer 23

EGFR / PDGFRA - KRAS / NRAS - BRAF

Question 24

What are the two types of EGFR inhibitors and how do they operate?

Answer 24

Tyrosine Kinase Inhibitors (TKIs) - Inhibit the pocket of EGFR and interfere with extracellular ligand binding
Anti EGFR monoclonal antibodies (mAbs)- Act extracellularly and prevent dimerisation, autophosphorylation and signal transduction.

Question 25

How do BRAF inhibitors work?

Answer 25

In BRAF-mutated cancers, the BRAF kinase becomes hyperactivated resulting in cell proliferation and survival. BRAF inhibitors selectively target BRAF kinase and thus interferes with the mitogen activated protein kinase (MAPK) signalling pathway that regulates the proliferation and survival of cells.

Question 26

How do MEK inhibitors work?

Answer 26

MEK inhibitors bind to and inhibit MEK-dependent cell signaling. This inhibition leads to cell death and inhibition of tumour growth. These are allosteric binding inhibitors or MEK that inhibit MEK1 alone or bother MEK2 and MEK2. Targeting MEK is a means of preventing reactivation of the MAPK pathway in the presence of BRAF and RAF mutations. MEK inhibitors are often used in combination with BRAF inhibitors to prevent pathway reactivation

Question 27

What are the ways in which variants can arise during DNA replication and/or recombination?

Answer 27

1) DNA damage
2) Deficiencies in DNA replication
3) Defects in DNA repair

Question 28

What are the five major pathways of DNA repair?

Answer 28

Base excision repair (BER)
Nucleotide Excision repair (NER)
Mismatch repair (MMR)
Homologous recombination repair (HRR0
Non homologous end joining (NHEJ)

Question 29

What area of the immune system are B and T cells part of?

Answer 29

The adaptive immune system

Question 30

How do B cells participate in the adaptive immune response?

Answer 30

B cell receptors recognise soluble antigens based on its unique antigen binding site. Once linked to a specific antigen, the cell undergoes growth, division and further differentiation into a plasma cell. This leads to the proliferation of a pool of plasma cells from the same clone which collectively secretes a large amount of highly specific antibodies. Antibodies have several effector mechanisms including neutralisation of antigen aggulutination of microbes and activation of complement cascade.

Question 31

How do T cells participate in the adaptive immune response?

Answer 31

T cells recognise antigens in the form of small peptides presented by antigen-presenting cells on the cell surface. Once a T cell is engaged with an antigen presenting cell, it can differentiate into one of many CD4+ effector cells depending on the microenviroment. One path is to differentiate into T helper cells which release cytokines to help activate B cells, NK cells and lymphocytes, Different types of T helper cells have different roles depending on the pathogen and type of immune response.

Question 32

What are monoclonal antibodies?

Answer 32

Monoclonal antibodies are laboratory-produced molecules engineered to serve as substitute antibodies that can restore, enhance, modify or mimic the immune system’s attack on cells that aren’t wanted, such as cancer cells.

Answer 33

Tumour infiltrating lymphocytes can express immune checkpoint molecules such as PD-1 among others. PD-1 inhibition appears to have clinical utility in a variety of cancer and shows durable responses in a proportion of patients, many who have failed other therapies.

Question 34

What are the three mechanisms which regulate epigenetic modification?

Answer 34

DNA methylation
Histone modification
Non-coding RNA

Question 35

How are genes methylated?

Answer 35

• Genes are methylated through a biochemical process in which a methyl group (CH3) is added to the C5 position of cytosine to form 5-methylcytosone (5MeC)
• This is almost entirely restricted to cytosines of CpG dinucleotides – resulting in two methylated cytosines diagonal to each other on opposing DNA strands.
• In mammals, around 70% of all CpG dinucleotides are methylated.
• This is carried out by DNA methyltransferase (DNMT) enzymes

Question 36

How does histone modification occur?

Answer 36

• Histones are proteins that are primary components of chromatin – the complex of DNA and proteins that make up chromosomes
• The N-terminus of histone molecules protrude from the body of nucleosomes
• Chemical modifications of amino acids in these are major determinants of chromatin conformation and consequently influence DNA transcription
• In its more relaxed form, it is active and the associated DNA can be transcribed.
• If chromatin is condensed, DNA transcription does not occur
• Histones can be modified by acetylation/deacetylation or methylation/demethylation

Question 37

How does non-coding RNA impact epigenetic modification?

Answer 37

• Non-coding RNA comes in 2 classes; long and short

• Short: e.g. microRNA
o microRNAs are small strands of RNA around 22 nucleotides long, interfere with gene expression at the level of translation
o They form active ribonuclear complexes with cytoplasmic proteins resulting in RNAse activity

•	Long non coding RNAs 
o	Represent another class of epigenetic markers 
o	Transcripts are around 200 bp long and are thought to form ribonucleoprotein complexes that interact with chromatin, regulating histone modifications and structural transformations