GOA old exams

Question 1

1. Name two factors that reduce life-expectancy.

Answer 1

1. Shortened telomeres (GOT lecture 4)
   
   1. Either through mutated telomerase, or haplo-insufficiency, shortened telomeres are a good sign of shortened life expectancy
   2. Can be inherited, even if you don’t inherit the mutated telomerase.
2. Environmental hazards (food availability, diseases, tigers etc.)

Shortened telomere length with successive generations can result in earlier onset and increased severity of a disease – this is known as genetic anticipation

Question 2

Explain antagonistic pleiotrpy. Give an example

Answer 2

1. Pleitropic alleles (ones that can influence two or more seemingly unrelated phenotypes) with positive effects at an early age could be favored by selection, even if they have negative effects at a later age.
2. This would mean that pleotropic alleles with negative effects (even very minor ones) early on would be selected against, even if they have positive effects later in life.
3. Even small beneficial effects early in life will outweigh a deleterious effect late in life, even if this results in senescence and death.
4. Huntington’s disease is an example of this,
   
   1. Usually onset at around age 30
   2. Dominant allele
   3. No negative selection against this late onset disease

People are likely to have reproduced before even manifests at all

Question 3

What is SIPS? Name two factors that induce it?

Answer 3

1. SIPS is Stress Induced Premature Senescence, or Stress Induced Premature Senescence-like phenotype (but only need the first one)
2. The phenomenon wherein cellular senescence is induced prematurely due to extrinsic stress (such as DNA-damage caused by radiation, or oxidative stress caused by high environmental O2 concentration.)
3. Two good examples would be:
   
   1. DNA damage caused by gamma-rays or UVB
   2. Oxidative stress caused by H2O2 or high environmental O2
   3. Bonus: cell cycle arrest and replication associated DNA damage caused by Hydroxyurea.
4. Some factors that induce it (more than 2, but just choose some to memorize):
   
   1. Oxidative stress
      
      1. High O2
      2. H2O2
      3. Tert-butylhydroperoxide
      4. Homocysteine (I think this is oxidative stress)
   2. Anti-neoplastice Drugs – DNA damage
      
      1. Mitomycin-C
      2. Cis-platin
      3. Bleomycin
   3. Hydroxyurea
      
      1. Cell cycle arrest
      2. Replication associated DNA damage
      3. Stops ribonucleotide reductase from converting NTPs to dNTPs
      4. Inactivates ribonucleotide reductase
   4. Gamma-rays and UVB
      
      1. Radiation induced DNA damage
   5. Too much growth factor
      
      1. Stimulation with cytokines (TGF-B1)
   6. Overexpression of oncogenes
      
      1. Normally stimulate cell cycle, but overexpression has been seen to induce senescence
      2. Raf-1
      3. Ras
      4. E2F1

Question 4

C. elegans go into a stage with extended life span. What is this stage? How is it induced? explain briefly

Answer 4

1. This stage is called the Dauer larval state, in this state, the larva is:
   
   1. Thin
   2. Can move
   3. Do not feed (plugged mouth)
   4. Remain viable for around 3 months
   5. Do not age
   6. Show increased stress resistance
2. Induced by environmental stresses like:
   
   1. Overcrowding
   2. Heat
   3. No food, no water
3. Will stay in this state until environmental conditions change, upon which it wil continue into L4 stage of C. elegans life cycle.

Question 5

1. Name and explain two reasons why ageing is not a genetically programmed process?

Answer 5

1. Aging could not have evolved as a death mechanism, since would not increase Darwinnian fitness. Because logically, longer lived individuals would create more offspring and thus be selected for.
2. no evolutionary pressure to develop an aging process, since under real life conditions extrinsic mortality before reaching old age much more common than intrinsic mortality and many more individuals die before reaching old age.

Question 6

What is dietary restriction? How does it influence life span in model organisms?

Answer 6

1. Caloric restriction is a situation in which the caloric uptake of an organism is reduced to 30-40% of normal – nearly starving.
   
   1. In mice has been known to extend life span by 40 – 60%
   2. Also show improved lifespan during aging
   3. Disadvantages are:
      
      1. Reduced body weight and muscle
      2. Sensitivity to cold
      3. Increased sensitivity to bacterial infection
      4. Reduced wound healing
      5. Reduced fertility
2. In some model organisms it can increase lifespan by quite a bit
3. Exact way it works not known, but seems to have something to do with insulin/IGF-1 receptor
4. Also potentially TOR kinase, which has an effect on lifespan

Note: Might need to go over this answer again

Question 7

How is LaminA processed. How is this different in HGPS patients?

Answer 7

1. In WT cells, LaminA is processed post translationally as follows
2. Farnesylation of the CAAX
3. Farnesyl group added to cysteine by protein farnesyltransferase
4. The last 3 amino acids are clipped off
5. Done by ZMPSTE24 and/or RCE1
6. Carboxyl-methylation of the farnesyl-cysteine
7. Done by ICNMT
8. This is a prenyl-protein-specific methyltransferase of the ER
9. Clipping off of the C-terminal 15 amino acids of the protein
10. Includes the farnesylcysteine methyl ester
11. Done by ZMPSTE24
12. In HGPS patients
13. C => T substitution at codon 608 in LMNA gene
14. Causes improper splicing, deleting 50 amino acids from LaminA protein
15. These amino acids contain the protease site for ZMPSTE24 which clips off the farnesyl-cysteine-methyl-ester at the c-terminal
16. This causes an accumulation of farnesylated and methylated prelaminA at nuclear membrane
17. Normally only B-type lamins would be there, but now there are A-type lamins there as well.
18. A similar effect is seen if ZMPSTE24 protease is inactivated
19. So in summary, it is not the deletion of the amino acids directly that causes incorrect localization, but the permanent farnesylation caused by the deletion of the protease site.

Question 8

Rapamycin prevents cell proliferation. What is its target. How does it prevent cell proliferation?

Answer 8

1. Rapamycin forms complex with FKBP12
2. This complex binds directly to mTORC1 complex, inhibiting it
3. Inhibition of mTORC1 causes the cell to stop proliferating

Note: Probably a good enough answer, but maybe more needed, don’t know how much detail they want

Question 9

What is a stem cell? What is the difference between pluripotent and totipotent stem cells?

Answer 9

1. A stem cell is a cell with the ability of self-renewal
2. Has the ability to give rise to a cell that is more mature
3. Totipotent stem cells:
4. Also called omnipotent
5. Can differentiate into embryonic, non embryonic types
6. Fertilized egg is totipotent, as well as a few divisions from fertilized egg
7. Can give rise to a viable organism
8. Can differentiate into all cell types
9. Pluripotent
10. Can differentiate into nearly all cells
11. All cells derived from any of the 3 germ layers
12. Can’t make a viable organism

Question 10

Describe an experiment in detail that demonstrated the limited replicative potential of primary human cells

Answer 10

1. Hayflick experiment
2. Showed the finite lifetime of cultured human cells
3. Showed that normal human fibroblasts will double a finite number of times.
4. Proof for experiment:
5. Mixed equal number of human male fibroblasts with female fibroblasts
6. The male fibroblasts were grown for 40 population doublings prior to mixing
7. Female fibroblasts were grown for 10 population doublings prior to mixing
8. Unmixed cell populations were grown as controls

Question 11

Give a basic (minimal) definition of ageing that is applicable to all (ageing organisms. Name three “markers” to describe ageing by this definition. (II)

Answer 11

• progressive time-dependent functional deterioration of physiological integrity leading to impaired function and increased vulnerability to death
• • three markers: increase in mortality, decline in fertility, decline in physical performance

Question 12

Analyzing ageing, what model organisms are usually used? What are the advantages (name three) and the disadvantages of these model organisms

Answer 12

• classical model organisms: Drosophila, C. elegans, mice, yeast, human cell culture
• • advantages: easy handling, short reproductive life cycle, well charcterized/defined homogenous backgrounds, very controlled environment
• • disadvantages: simple organisms with different life cycles to humans, laboratory strains have been selected for rapid and robust growth over years not extended life span, research may not apply to the wild (very different conditions)

Question 13

What are the advantages and disadvantages of model organisms in ageing research (name at least 3 of each)? Name three model organisms.

Answer 13

advantages: short replicative life span, easy handling,

Question 14

Analyzing ageing processes what are the disadvantages of using mutant laboratory strains with a) shortened or b) lengthened lifespan?

Answer 14

• a) phenotypes with shortened lifespans can have other causes besides ageing
• b) phenotypes that lead to lengthened lifespan are statistically difficult to analyse (censured events)

Question 15

1. Looking for genes involved in aging processes why does it make more sense to analyze mutants with a lengthened lifespan instead of a shortened life span? (II)

Answer 15

1. phenotypes that lead to a shortened life span can have lots of other causes besides ageing, while phenotypes that lead to a lengthened lifespan need to be involved in ageing

Question 16

What basic phenotypes should you analyze in ageing research? Give specific requirements for these phenotypes

Answer 16

1. occurs during physiological ageing
2. aggravation should speed up ageing
3. experimental improvement should slow down ageing

Question 17

Define the terms „life span“ and „life expectancy“. (II)

Answer 17

1. lifespan = maximum number of years an individual of a specific species can live (genetically determined
2. life expectancy = average number of years an organism can expect to live under given conditions (with real world risks)

Question 18

1. Why can human life expectancy be influenced positively and negatively but human life span can not ?

Answer 18

1. life expectancy can be influenced by reducing real world risks
2. life span is genetically determines and genetic modification of humans would be unethical → to some degree life span can be influences by modulating molecular mechanisms of ageing e.g. through caloric restriction

Question 19

1. a) Give a brief definition for the terms “life span” and “life expectancy”. b) How can we influence (human) life span? c) How can we influence (human) life expectancy?

Answer 19

1. life expectancy = the average number of years an organism can expect to live under the given conditions (with real world risks)
2. life span = the maximum number of years an organism can live (genetically determined)
3. human life expectancy can be influenced through reducing real world risks, life span is genetically determines but can be slightly influenced

Question 20

1. What is the statement of the „Mutation accumulation hypothesis“? What is the statement of the „Antagonistic pleiotropy hypothesis“? Give supporting experimental evidence (one example) for each hypothesis.

Answer 20

1. mutation accumulation hypothesis: mutations with late acting effects can not be selected for by natural selection since their effects become evident after the reproduction → they accumulate in the population → chorea huntington as an example
2. antagonistic pleiotropy hypothesis: mutations that have early benefits are positively selected for even if they have very severe detrimental effects later in life because these effects fall into the selection shadow and can’t be selected for → replicative senescence: prevents tumor development (positive effect), but later leads to ageing through loss of tissue homeostasis

Question 21

1. What is the “basic concept” used by the evolutionary theory of ageing? How can this “basic concept” explain the evolution of ageing? What is a “pleiotropic” acting allele?

Answer 21

1. basic concept: natural selection can only select for mutations that have early effects (fall into the reproductive lifespan), later effects fall into the selection shadow and are no longer selected for → selection pressure decreases with increasing age
2. alleles that have negative later effects are not negatively selected against and they can accumulate in the population, sometimes they are even positively selected for if it is a pleotropic acting allele that has beneficial early effects but detrimental later effects
3. pleiotropic allele: an allele that influences more than one seemingly unrelated phenotypic traits

Question 22

1. a) Explain the basic concept of the “evolutionary theory of aging”. b) Explain the “antagonistic pleiotropy hypothesis of aging”. c) Give an example for an antagonistic pleiotropic acting gene and explain how its action is antagonistic pleiotropic (III)

Answer 22

1. antagonistic pleiotropic acting gene = p53 → early in life for cancer preventation, late in life induction of cellular senescence

Question 23

1. Give two examples of theoretical and/or experimental evidence proving/supporting the „Evolutionary theory of ageing“.

Answer 23

1. direct proof: experimentally altering the reproduction time of drosophila
   
   • one time by taking only late produced eggs → high juvenile mortality → longer life span
   • introducing high adult mortality = earlier reproduction → shorter life span
2. opossum experiment

Question 24

1. Explain the basic concept of the „evolutionary theory of ageing“. Give two observations to support this concept (directly or indirectly). Describe an experiment to prove this concept.

Answer 24

1. indirect proof: extreme differences in life span of different species
2. direct proof: experimentally altering the reproduction time of drosophila
   
   • one time by taking only late produced eggs → high juvenile mortality → longer life span
   • introducing high adult mortality = earlier reproduction → shorter life span

Question 25

1. You are looking for a new ageing factor influencing replicative life span in yeast. What criteria should such an ageing factor fulfill?

Answer 25

1. should manifest during physiological ageing
2. experimental aggravation of the factor should fasten ageing process
3. experimental improvement of the factor should slow down ageing process

Question 26

1. Drosophila flies are usually bred by using eggs from 2-week-old females to found the next generation. How does a gradual increase of the eggs “donating” females’ age influence the lifespan of the strain? How can you explain this result?

Answer 26

1. higher juvenile mortality → leads to pressure for lengthened life span and longer reproduction periods

Question 27

1. You are growing four cell cultures: a) bacteria, b) yeast, c) primary human cells, d) human cancer cells. How long do these cultures grow given that the culture conditions are permanently ideal?

Answer 27

1. human cells have a finite growth time since they reach the hay flick limit (50-100 cell divisions)
   
   • mouse primary cells approx. 20 cell divisions
   • reaching the hay flick limits depends on the age of the donor cells
2. all other cultures can be maintained indefinitely

Question 28

Define the term Hayflick limit. (IIIII)

Answer 28

1. number of culture doublings in vitro after which cells cease proliferation and are irreversibly growth-arrested but still “alive” (senescent)

Question 29

1. Which human cell types do not show a Hayflick limit and why not?

Answer 29

1. stem cells, germ line cells (tumor cells) → cells have an active telomerase that elongates the telomeres preventing replicative senescence induced through telomere attrition (cancer cells have either reactivated telomerase or ALT pathway)

Question 30

1. Give two observations leading to the concept of a cell division counter in eukaryotic primary cells.

Answer 30

1. primary cells display a limited proliferation capacity and the cells stop proliferating after reaching a certain number of cell doublings
2. the number of doublings an in vitro cell culture can perform depends on the age of the donor (hayflick limit decreases with increasing donor age)
3. premature ageing syndromes cause a significant decrease in the proliferative capacity of cells in vitro

Question 31

1. What is the end replication problem and how does telomerase solve it? (IIIIII)

Answer 31

1. with each replication cycle the chromosome ends are shortened due to two factors:
   
   • 3’ overhang on the leading strand is lost (plus regeneration of the 3’ overhang through exonuclease after each replication cycle on the 5’ strand)
   • loss of the regions bound by RNA primers at the 5’ end since they cannot by replaced by the DNA polymerase (needs a 3’ OH to bind to)
2. telomerase solves this problem by lengthening the 3’ end of the telomeric DNA using its own RNA template (5’ end can then be synthesized by the DNA polymerase)

Question 32

1. What is the function of telomeres? Why is this function essential? What happens if this function fails?

Answer 32

1. telomeres need to protect the chromosome ends from recognition as a DNA double strand break → essential for genomic stability
2. function fails = (chromosome breakage fusion bridge cycle): chromosome end to end fusion → breakage in cell cycle (when chromosomes are pulled to different poles) → renewed fusion = genomic instability, cell cycle arrest

Question 33

1. a) What is the function of telomeres? b) What factors are essential to establish this function? c) What happens in cells with disfunctional telomeres (basic outcomes)?

Answer 33

1. function of telomeres: to protect the chromosome ends from recognition as a DNA-doubel strand break
2. TRF1, TRF2, POT1 → formation of t-loop + inhibition of recognition as DNA double strand break
3. function fails = (chromosome breakage fusion bridge cycle): chromosome end to end fusion → breakage in cell cycle (when chromosomes are pulled to different poles) → renewed fusion = genomic instability, cell cycle arrest

Question 34

1. Name two essential components of the telomerase enzyme. What is the enzymatic activity of a telomerase? (II)

Answer 34

1. protein (TERT) and RNA (TR), enzymatic activity = reverse transcriptase

Question 35

1. Describe the mechanism of telomere elongation by telomerases.

Answer 35

1. through telomere repeats the telomere template is partially complementary → telomerase then elongates the 3’ end using its own RNA template as the complimentary strand → new cycle
2. the 5’ end can be filled by the DNA polymerase using the regular mechanism

Question 36

1. Explain the T-loop model of mammalian telomeres. What is essential for the formation of the T-loop (no special names necessary)?

Answer 36

1. repetitive nature of the telomere DNA → 3’ overhang can invade the ds telomere region displacing itself

Question 37

What is the shelterin complex? Name three components and their activities and function.

Answer 37

1. TRF1/TRF2: bind the ds telomeric repeats
2. POT1: bind the ss telomeric repeats
3. function: regulate telomere length and inhibit of ATM to prevent DNA damage response

Question 38

1. How do telomeres differ from DNA ends generated by double strand breaks? What is the function of this special structure? (II)

Answer 38

1. binding of telomeres through the shelterin complex and formation of t-loop
2. no recognition through DNA double strand break repair machinery → prevent nonhomologous end joining and homologies recombination

Question 39

1. Give three characteristics of telomere DNA. (II)

Answer 39

1. GT reach telomeric repeats, 3’ ss overhang, bound by specific factors (e.g. shelterin complex) formation of telosome

Question 40

1. TERC+/- heterozygous mice develop disease symptoms. Why?

Answer 40

1. heterozygous mice do not have enough functional telomerase (haploinsufficiency expression of one allele is not enough) which leads to progressive teller shortening

Question 41

1. What does the term “genetic anticipation” mean in hereditary syndromes caused by a mutant hTERT allele? What is the cause for this effect?

Answer 41

1. genetic anticipation means an earlier onset and an increased severity of the diseases with each successive generation → caused by the inheritance of telomere length with each successive generation

Question 42

1. Why is telomerase inactive in somatic cells? Which (mammalian) cell types show telomerase activity? (III)

Answer 42

1. prevented uncontrolled cell proliferation and the development of cancer
2. germ line cells and stem cells (tumor cells in pathological situations)

Question 43

1. How does the loss of telomerase activity influence the ageing process in mice?

Answer 43

1. senescence → leads to loss of tissue homeostasis and regenerative potential through depletion of stem cells
2. genomic instability (loss of tissue function), apoptosis

Question 44

1. how does replicative senescence follow the antagonistic pleiotropy hypothesis of ageing?

Answer 44

1. tumor suppression in early age and contributes to ageing phenotype in later cells (induction of senescence, stem cell depletion)

Question 45

Name four primary signals that induce cellular senescence

Answer 45

1. ROS
2. DNA damage
3. telomere attrition
4. oncogene expression

Question 46

1. How is p53 involved regarding lengthening and shortening of the life span?

Answer 46

1. lengthening of the lifespan = tumor suppression
2. shortening of the life span = induction of senescence/apoptosis

Question 47

1. What effect(s) does p53 activation have on cells, tissue and organism?

Answer 47

1. cells: apoptosis, senescence, replicative arrest
2. tissue: tissue atrophy, repair
3. organism: ageing

Question 48

1. Give experimental evidence that p53 acts like an antagonistic pleiotropic gene.

Answer 48

1. p53 KO mice → increased cancer formation
2. expression of constitutively active p53 → shortened life span/premature ageing

Question 49

1. Give three important phenotypical markers for cellular senescence.

Answer 49

1. irreversible arrest of cell division (cannot be stimulated by physiological mitogens)
2. selected changes in morphology (increased size, cells flatten out, borders tend to vanish, increased lysosomal biogenesis)
3. chromatin changes
4. biochemical changes (expression of p53, activation of Rb)
5. derangement in differentiated function

Question 50

A) What does the abbreviation SIPS mean? b) What is the difference between extrinsic and intrinsic senescence? c) Give four factors that induces for cellular senescence (IIII)

Answer 50

1. stress-induced premature senescence
2. extrinsic = stress induced premature senescence , intrinsic = replicative senescence (telomere shortening)
3. DNA damage, ROS, oncogene expression, telomere attrition

Question 51

1. Define “SIPS”. How is “SIPS” activated? (three examples)

Answer 51

1. DNA damage (e.g. through radiation), oxidative stress, oncogene expression

Question 52

1. Why are only a few cells malignantly transformed even if the key players fail to induce senescence?

Answer 52

1. only a few cells are malignantly transformed since only a few manage to stabilize telomeres to escape genomic instability

Question 53

1. Why are only a few cells malignantly transformed even if p53 fails to induce senescence? What is meant by “first barrier” and “second barrier” against cancer formation? (IIII)

Answer 53

1. first barrier = cellular senescence
2. second barrier = genomic instability resulting from too short telomeres that kills cells

Question 54

1. What is the first barrier against cancer formation? What happens if the first barrier against cancer formation fails?

Answer 54

1. activation of senescence
2. if they circumvent senescence the cells enter a cellular crisis due to genetic instability which usually kills them

Question 55

1. Why poses cancer a major challenge to the longevity of organisms with renewable tissues? (II)

Answer 55

1. you need to balance tissue renewing with cancer prevention → if you have enough tissue renewal to ensure longevity you enable cancer formation, if you suppress cancer formation you limit tissue renewability and thus negatively effect organism longevity

Question 56

1. What defines a stem cell? What is the special feature of a totipotent stem cell in comparison to other stem cell types?

Answer 56

1. self-renewal and potential to give rise to more differentiated (more mature cells)
2. totipotent stem cells can give rise to an entire new organism (i.e. also to the extra embryonic tissues like placenta)

Question 57

1. How do stem cell properties change during life?

Answer 57

1. with increasing age stem cells express higher levels of tumor suppressors (mediated through heterochronic genes, let-7 polycomb) which inhibit their proliferative potential
2. young = rapid proliferation to support growth → young adult hood: slowed division, in quiescent stage (proliferation can be reactivated) → old: high expression of tumor suppressors prevents proliferation, decreased proliferative ability

Question 58

1. What is the role of tumor suppressor genes in stem cells and how do they contribute in general to stem cell ageing?

Answer 58

1. tumor suppressor genes are important to prevent the development of cancer → also inhibit stem cell function (prevent their proliferation)

Question 59

The daf-2 pathway regulates dauer formation and life span in C. elegans. What happens phenotypically and on a molecular level if

1. daf-2 activity is up-regulated (II)
2. delete daf-2 (II)
3. daf-18 activity is up-regulated (III)
4. daf-18 activity is down-regulated (II)
5. age-1 is deleted (II)
6. the phosphoinositide-3-phosphate generating kinase is down regulated
7. the phosphoinositide-3-phosphate generating kinase is upregulated
8. upregulate p85/age-1 activity (II)
9. olfactory and chemosensory neurons are removed
10. inactivate unc-64 and unc-31 (II)

Answer 59

1. daf-2 activity is up-regulated (II) → daf-16 is inactivated → no dauer formation
2. delete daf-2 (II) → daf-16 is active → dauer formation
3. daf-18 activity is up-regulated (III) → daf-16 is more active → dauer formation
4. daf-18 activity is down-regulated (II) → daf-16 is less active → no dauer formation
5. age-1 is deleted (II) → daf-16 is active → dauer formation
6. the phosphoinositide-3-phosphate generating kinase is down regulated → daf-16 is active → dauer formation
7. the phosphoinositide-3-phosphate generating kinase is upregulated → daf-16 is inactive → no dauer formation
8. upregulate p85/age-1 activity (II)
9. olfactory and chemosensory neurons are removed → no stimulation of daf-2 pathway → active daf-2 → dauer formation
10. inactivate unc-64 and unc-31 (II) → no stimulation of daf-2 pathway → active daf-2 → dauer formation

Question 60

1. Removal of the olfactory and chemosensory neurons in C. elegans results in life span extension. Explain the underlying mechanism. (II)

Answer 60

1. Insulin is no longer released in the presence of nutrients → daf-2 pathway is not activated which leads to activation of daf-16 → dauer formation

Question 61

1. If you generate a daf-2 genetic mosaic will you get a phenotypically mosaic worm or an uniform phenotype? What does the outcome say about the daf-2 function? (II)

Answer 61

1. you get a uniform phenotype → daf-2 works in a non- cell autonomous fashion (systemic effect) → a few cells that have daf-2 pathway regulate the phenotype development in the entire organism

Question 62

1. Does the daf-2 pathway act cell-autonomous or cell-nonautonomous? How was this shown?

Answer 62

1. cell-nonautonomous → generation of mosaic flies where some cells express daf-2 but the phenotype is uniform (some cells exhibit a different phenotype than their genotype)

Question 63

1. Name two cellular signals that lead to mFOXO transcription factor activation by phosphorylation.

Answer 63

1. low ATP to AMP ratio (Low energy load) → AMPK
2. ROS (oxidative stress) → JNK

Question 64

1. What is “caloric restriction” (CR)? What happens if you apply CR to rodents? Which pathways seem to be involved in CR control? (II)

Answer 64

1. caloric restriction: the reduction of the uptake of nutrients to the bare minimum (Barely not starving) where still all necessary vitamins are taken up longer lifespan,
2. longer health span, infertility, cold-sensitivity
3. Insulin receptor signaling → FOXO (upregulated) and mTORC (downregulated)

Question 65

1. Name two pathways that are involved in regulating the response to Caloric Restriction.

Answer 65

1. nutrient sensing and IGF-signaling

Question 66

1. What is the function of the TOR kinase? What is caloric restriction (CR) and how does CR influence TOR activity?

Answer 66

1. decreased uptake of nutrients
2. activation of protein translation, cell cycle progression, inhibition of autophagy, (general growth and proliferation status)
3. nutrient sensing under caloric inhibition down-regulated (less amino-acid export from the lysosome) less insulin signaling → TOR not activated → downregulated protein translation

Question 67

1. What is the major function of the TOR kinase? How does TOR activity influence lifespan? (II)

Answer 67

1. mTOR up: S6 kinase up = inhibition of 4e BP (translation inhibitor) = protein translation up, respiration rate goes down
2. mTOR down: S6 kinase not activated = active 4E BP (translation inhibitor) = protein translation down, respiration rate goes up

Question 68

1. Name two biological processes regulated by the mTOR complexes that are probably relevant for life span extension by caloric restriction. Very briefly explain how mTOR complexes regulate these processes.

Answer 68

1. protein translation
2. mitochondrial respiration

Question 69

1. Explain briefly the posttranscriptional processing of Lamin A (how is mature Lamin A generated from its precursor prelamin A) and how this process is disturbed by the HGPS mutation. (II)

Answer 69

1. farnesylation of cysteine in c-terminal CAAX domain (membrane association) by farnesyltransferase 2. cleavage of -AAX from the c-terminal domain through Zmpste24 /Rce1 3. methylation of farnesylated cysteine residue (ICNMT) 4. cleavage of c-terminal 15 aa including the farnesylated cysteine (no longer membrane associated)
2. HGPS → spontaneous mutations activate a cryptic splice site → 50 aa sequence in protein deleted that contains the recognition sequence for the second cleavage by Zmpste24 → the protein (called progerin) remains farnesylated (and thus associated with the membrane)

Question 70

1. Name three functions of the nuclear lamina. How are these functions disturbed in HGPS patients and how does this contribute to the premature aging phenotype. (II)

Answer 70

1. lamin proteins are associated directly and indirectly with chromatin connecting the nuclear structure with the cytoplasm (LINC complex) → structure of the nuclear envelope/ mechanical stability and chromatin organization and telomere regulation
2. nuclear envelope/mechanical stability is weakened in HGPS → cell rupture/cell death
3. altered gene expression → DNA damage response is impaired → p53 activation and senescence
4. DNA replication fork formation impaired → DNA double strand breaks

Question 71

1. Do bacteria age? Briefly describe two experiments that support your answer. (IIII)

Answer 71

1. Bacteria can be cultivated indefinitely in vitro
   
   • Have circular genomes, making telomere attrition not a problem
   • Asymmetrical cell division to produce rejuvenated daughter cell
2. Could take one culture, grow it for a certain number of divisions, then put on same medium as another culture that has just started growing and see if either stops dividing first. Hypothetically, they should both grow indefinitely, and neither should stop first, but if not, the one that started earlier should stop first.
3. Could also look at “fertility” in older colonies of bacteria vs. younger ones.
   
   • Compare the rate of doubling over time.
   • If there is not aging, rate of doubling should not decrease over time.