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Biosci 353 > Exam, > Flashcards

Exam, Flashcards

1
Q

What does proteostasis ensure

A

correct folding, concentration and location

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2
Q

Aspects of proteostasis

A
  • protein synthesis
  • protein QC maintenance
  • protein degradation
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3
Q

Protein synthesis

A
  • Co or post translational folding
  • correct concentration
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4
Q

Protein QC maintenance

A
  • unfolding and refolding
  • correct location
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5
Q

Protein degradation

A
  • correct concentration
  • destroy aggregates
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6
Q

Cellular stressors

A
  • temperature
  • chemical
  • oxidative
  • osmotic
  • mutations
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7
Q

How does heat affect protein folding

A

breaking of VDW and H bonds leading to denaturation but can also lead to bond reformation

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8
Q

2 types of proteins

A
  • stable
  • unstable
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9
Q

stable proteins

A
  • common
  • low propensity to aggregate
  • less sensitive to stressors
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10
Q

unstable proteins

A
  • have intrinsically disordered regions for flexibility
  • sensitive to misfolding
  • sensitive to temperature due to loss of a small set of crucial effectors and regulators of biological processes
  • biosensor for stress
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11
Q

Concentration affecting protein folding

A
  • correct folding is impaired by high protein concentration due to overloading chaperones so aggregates occur
  • possible reason for trisomy 21 leading to increased alzheimers as APP gene is found on chr21
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12
Q

How is protein misfolding toxic

A
  • loss of normal biological function
  • gain of toxic functionG
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13
Q

Gain of toxic function

A
  • clog up intracellular transport/degradation
  • induce inflammation
  • sequester other proteins
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14
Q

How do cells prevent/cure misfolded proteins

A
  • halt transcription/translation
  • degradation
  • increase chaperones for folding and refolding
  • cell death
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15
Q

When do chaperones function

A
  • during synthesis = prevent peptide folding before domain is complete
  • during folding = help partially folded intermediates to cross energy barriers
  • during misfolding = unfold and refold proteins
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16
Q

Intracellular protein clearance pathways

A
  • proteasome
  • autophagy
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17
Q

The discovery of the heat shock response

A
  • found in drosophila salivary gland chromosomes
  • observed puffing induced by heat shock or chemical stressors
  • puffing was rapid, reversible and positively stains for RNA
  • heat shock caused a change in the types of protein expressed thus RNA
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18
Q

heat shock response

A
  • induced at 15-15 degrees above optimum growth temperature
  • increased transcription/translation of heat shock proteins
  • aids survival of stimulating stress
  • primes for survival of subsequent stress
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19
Q

how does priming by the heatshock response help survival

A
  • switches cells to making heat shock proteins
  • prepares cells to combat following stress
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20
Q

Hit5 seedlings - heat shock response

A
  • mutant Hit5 seedlings can survive a 44 degrees heat shock without priming
  • mutation increases basal levels of heat shock protein mRNA
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21
Q

Transciptional changes allowing HSPs to become a major protein product

A
  • repress transcription of most mRNAs
  • increased transcription of specific mRNAs
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22
Q

What does the duration of gene expression changes correlate with

A

severity of stress and may also be stress specific

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23
Q

Repression of transcription via SINES

A
  • act like transcription factors
  • transcribed by RNA Pol III during stress
  • bind and inhibit RNA Pol II transcription
  • when SINE RNA binds RNA Pol II it keeps the pre-initiation complex (PIC) in the closed conformation
  • PIC cannot access DNA for transcription
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24
Q

Increased transcription via HSF1

A
  • sensor of protein misfolding which is normally kept as a monomer but in some species it does have basal activity
  • HSF1 is a monomer which is bound to Hsp70,40,90
  • stress causes Hsp90 to bind misfolded proteins thus will unbind HSF1
  • HSF1 can now form a homotrimer
  • homotrimer binds to promoters containing heat shock transcription elements in heat responsive target genes
  • binding to promoters allows RNA pol II to dissociate from NELF and DSIF
  • HSF1 recruits P-TEFB to phosphorylate promoters causing NELF to be released
  • RNA Pol II is now free and can transcribe heat shock proteins
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25
Q

HSPs chaperone protein folding mechanism

A
  • open position with ATP bound
  • misfolded protein is loaded
  • ATP to ADP causing lid to close
  • closing of lid protects hydrophobic components allowing for remodelling
  • ADP to ATP
  • protein released
  • can be constitutive or inducible
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26
Q

Stress granule assembly order

A

cores then shell

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27
Q

Stress granule disassembly order

A

shell then core

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28
Q

3 steps of stress recovery

A
  • dephosphorylation of pEIF2a
  • SG disassembly
  • resumption of translation
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29
Q

Dephosphorylation of pEIF2a

A
  • depletion of ternary complex leads to the translation of ATF4
  • ATF4 helps chaperones to transcribe and translate CHOP
  • CHOP induces Gadd34
  • Gadd34/CreP binds PP1 which inhibits phosphorylation of EIF2a
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30
Q

SG disassembly

A
  • mRNAs are released to polysomes for translation
  • done through surface exchange of loosely interacting proteins on shell with polysomes and remodelling via disaggregase chaperones
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31
Q

SG disassembly - disaggregase chaperones

A
  • AAA ATPases
  • homohexamer of 6 B6P monomers
  • central hydrophobic channel for proteins to thread through
  • unfolds UB substrates and unpicks protein complexes in the presence of ATP
  • KO leads to abnormal presence of stress granules
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32
Q

Resumption of translation

A
  • increase in ternary complexes
  • polysome formation
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33
Q

ALS mutations in SG proteins

A
  • can recruit more SG proteins to SG
  • decreased recovery when TIA1 mutants
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34
Q

CHOP as a transcriptional activator and repressor

A

indices DDIT3 which activates pro-apoptotic genes and represses anti-apoptotic genes

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35
Q

Pro-apoptotic genes

A
  • trail receptor (extrinsic)
  • Bim (intrinsic)
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36
Q

Anti-apoptotic genes

A
  • Bcl-2
  • Bcl-XL
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37
Q

Why do cells die

A
  • CHOP induced
  • defence
  • development
  • homeostasis
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38
Q

How is cell death classified

A
  • morphology
  • biochemistry
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39
Q

Apoptosis morphology

A
  • cells shrink
  • organelles remain intact
  • condensed chromatin
  • controlled DNA fragmentation = ladders
  • apoptotic bodies engulfed by phagocytes
  • cell blebbing
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40
Q

Necrosis morphology

A
  • cell and organelle swelling
  • moderate chromatin condensation
  • random DNA fragmentation = smear
  • cell lysis
  • inflammatory products produced
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41
Q

Autophagic morphology

A
  • accumulatio of double membraned vacuoles
  • lack of chromatin condensation
  • little or no phagocytotic uptake
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42
Q

Accidental cell death (biochem classification)

A
  • cell murder
  • always necrotic (not all necrosis is ACD)
  • harsh injury that cell cannot recover from
  • no adaptive response and no signalling regulation
  • release of inflammatory molecules = DAMPs/alarmins
  • DAMPs/alarmins then activate regulated cell death in nearby cells
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43
Q

Regulated cell death (biochem classification)

A
  • cell suicide
  • can be not stress driven (development) or stress driven
  • defined signalling pathways
  • morphology is apoptotic or necrotic
  • stress driven ways to die = apoptosis, necroptosis, autophagy
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44
Q

Extrinsic apoptosis

A
  • Ligand causes receptor (fas etc) to form a homotrimer
  • formation of homotrimer brings together the FAS associated death domains
  • leads to the recruitment of procaspase 8 which is cleaved leading to active caspase 8
  • caspase 8 cleaves procaspase 3 and Bid
  • formation of tBid can upregulate pro-apoptotic genes (link to intrinsic)
  • Caspase 3 dismantles cell via cleaving cytoskeleton and DNA = apoptotic body forms
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45
Q

Intrinsic apoptosis

A
  • not activated by an extracellular ligand but instead cell senses stress
  • increased pro-apoptotic genes and decrease in anti-apoptotic genes via BH3 only proteins activating Bax/Bak
  • pro-apoptotic molecules can homo oligomerise to form a pore/channel in the mitochondrial OM
  • release of cyt c, APAF1 and procaspase 9 to form a apoptosome
  • apoptosome cleaves procaspase 3 = apoptosis
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46
Q

What is necroptosis

A

an alternative mode of RCD mimicking features of apoptosis and necrosis

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47
Q

When is necroptosis common

A

when caspase 8 is inhibited and FADD is depleted as the apoptotic pathway cannot be activated

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48
Q

How is necroptosis activated

A

same way as extrinisic apoptosis

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49
Q

What does necroptosis require

A

death domain containing kinases such as RIPK3

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50
Q

Necroptosis morphology

A

same as necrosis

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51
Q

RIPK1 as a checkpoint

A
  • can activate all 3 pathways = apoptosis, necroptosis, survival
  • activated by TNF
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52
Q

RIPK1 activating cell survival - acting as a scaffold

A
  • phosphorylation at Ser25 which is promoted by Ub of RIPK1
  • acts as a scaffold where it recruits molecules required for cell survival
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53
Q

RIPK1 in cell death - kinase

A
  • dephosphorylated at Ser25
  • if caspase 8 and FADD are present then will go to apoptosis
  • if both are absent then RIPK3 will be activated for necroptosis
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54
Q

RIPK3 activation by

A
  • Death domain receptors (FAS) via RIPK1
  • Toll-like receptors via TICAM1
  • viral nucleic acids via DAI
  • adhesion receptors
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55
Q

MLKL conformational changes

A
  • MLKL phosphorylated by RIPK3 at its C terminus
  • extension of MLKL 4 helix barrel
  • conformational change allows IP6 to bind (-)
  • increased affinity for membrane (+)
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56
Q

MLKL possible mechanisms in membrane

A
  • activates TRPM7 or Na channels = influx = depolarisation = water = bursting
  • permeabilises the membrane
  • pore creation for ion transport = ion in = water = bursting
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57
Q

3 assays for cell death

A
  • TUNEL
  • Live/dead
  • apoptotic/dead
58
Q

TUNEL assay

A
  • tissue or fixed cells
  • imaging
  • enzymatic addition of labelled dUTP which labels a 3’OH
  • Detects DNA breaks formed from DNA fragmentation
  • cannot distinguish between necrosis or apoptosis as both involve DNA fragmentation
  • may also detect basal DSDNA breaks and cell turnover
59
Q

Live/Dead assay

A
  • cell cultures
  • imaging or flow
  • Uses 2 fluorophores
  • Live = Calcein-AM cleaved by enzymes in live cells
  • Dead = Ethidium homodimer/propidium iodine
  • discriminates between necrotic or late apoptotic but would also show necroptosis
60
Q

Apoptotic/dead assay

A
  • cell cultures
  • imaging or flow
  • Uses 2 fluorophores
  • Live = Annexin V binds to phosphatidylserine
  • Dead = propidium iodine
  • discriminates between necrotic or late apoptotic
  • doesnt consider necroptosis
61
Q

Cell death in cancer

A
  • have dysregulated cell divison/death
  • therapies attempt to decrease cell division or induce cell death
62
Q

Primary cell cultures

A
  • derived from normal animal tissue
  • limited ability to proliferate
63
Q

Transformed cells

A
  • can be derived from cancer tissues
  • differentiated primary cells transformed via oncogenic viruses
  • immortal and divide continuously
64
Q

Optimum culture growth conditions

A
  • 37 degrees
  • 5% CO2
  • normoxia or hypoxia
  • humidified
  • growth media
  • serum
  • additives such as growth factors
65
Q

Growth media components

A
  • salts
  • glucose
  • vitamins
  • amino acids
  • buffer
66
Q

Serum

A
  • produced from the blood of a fetal animal
  • low antibodies
  • contains many growth factors
67
Q

Factors influencing a cells fate and identity

A
  • specified changes in gene and protein expression leading to a fully differentiated cell from embryonic stem cells
  • intrinsic factors = genetic program
  • environmental factors = growth media
68
Q

Methods to determine cell identity

A
  • morphology
  • protein expression
  • gene expression
69
Q

Methods to assess morphology

A
  • bright field = stained
  • phase contrast = live cells
  • fluorescence
  • SEM
  • TEM
70
Q

Methods to assess protein expression

A
  • immunohistochemical analysis
  • microscopy
  • flow cytometry
71
Q

Single cell RNA sequencing for gene expression analysis

A
  • isolates cells from tissue
  • add barcoded beads
  • suspended single cells
  • amplify and sequence
  • identifies the type and number of mRNA in each cell
  • can lead to the identification of new and rare cell populations
72
Q

Human cell atlas uses

A
  • look at development = retina
  • evolution = primate species brain dev
  • find rare and new cell types = trachea
  • study disease states = covid-19
  • validate disease models = breast cancer
  • aging = skeletal muscle changes with age
73
Q

Somatic cell nuclear transfer

A
  • e-nucleate egg
  • add nucleus of terminally differentiated cells
  • differentiate to inner cell mass stage
  • isolate ESC
  • add ESC to a tetraploid blastocyst where it contributes to developing embryo
  • embryo develops and clone is formed
74
Q

uses of cell fate manipulation

A
  • growing cell types which arent accessible
  • regenerative medicine
    -study aging cells
75
Q

methods to make iPSC

A
  • transfection using DNA, RNA or protein
  • viral vectors
  • gene editing
  • chemical induction
76
Q

considerations when selecting a viral vector

A
  • tropism
  • integration
  • lytic
  • size
77
Q

Yamanaka and Takahashi factors discovery

A
  • used a dispensable gene = Fbx15
  • inserted reporter Bgal-NeoR into Fbx15 locus
  • transduced MEF with pluripotency factors
  • selected for NeoR to find pluripotent cells
  • found 4 factors = Oct4, Klf4, Sox2 and c-myc
  • problem where it didnt contribute to developing embryo so redid experiment with nanog instead of Fbx15
  • inserted GFP-puromycin-resistance cassette into 5’UTR of Nanog = generated chimaeric mice
78
Q

How to confirm pluripotency

A
  • morphology
  • cell surface markers
  • differentiation into all germ layers
  • could terminally differentiate
  • gene expression of factors
79
Q

Similarities between iPSC and ESC

A
  • morphology
  • gene expression
  • protein expression
  • proliferative rate
  • telomerase activity
  • epigenome
  • chimeric mice
  • germline transmission
80
Q

Differences between iPSC from ESC

A
  • iPSC has integration of RV
  • iPSC has some epigenomic memory retained from starting cell type
81
Q

iPSC applications

A
  • modelling aging diseases such as alzheimers (inserting mutations for familial and sporadic)
  • modeling complex orders such as bipolar
  • potential to make germ cells which can generate offspring
82
Q

General steps for human iPSC based disease modeling

A
  • extract somatic cells from patient
  • reprogram cells into iPSCs
  • create an isogenic control with Cripsr-Cas9
  • differentiate into desired cell types
  • characterise disease phenotypes and identify molecular mechanisms
83
Q

iPSC derived cardiomyocytes - in vitro example

A
  • hard to obtain primary cardiomyocytes so we can use iPSC to help
  • human peripheral blood mononuclear cells were reprogrammed into iPSCs via OKSM episomal factors
  • iPSCs differentiated into cardiomyocytes via extrinsic factors
  • infected them with SARS-COV-2
  • saw that it could enter and replicate via ACE2
  • saw cells stopped beating in 3 days and apoptosis was induced
  • developed an ACE2 antibody to treat these cells
84
Q

Reversing aging for heart repair example - in vivo

A
  • transient OKSM reprogramming in mice
  • OKSM in genome was under a doxycycline inducible cardiac specific promoter
  • restricted oxygen to mimic a heart attach
  • saw rejuvination where reprogrammed cells differentiated into immature cardiomyocytes which could proliferate and repair the heart
  • detected via cell surface markers
  • very specific on/off switch with factors as could lead to tumour growth and death if left on too long
85
Q

Understanding Alzheimers disease using iPSC - in vitro

A
  • RV-OKSM hiPSC with a PSEN-1 mutation
  • no normal control but had iPSCs from a patient with sporadic AD
  • differentiated into cortical neurones
  • mutant secreted a Tau peptide (eTau) into the media
  • eTau media was then put on healthy cells and saw an increase in AB42, decreased a-secretase APP cleavage and soluble APPa
  • also caused neuronal hyperactivity
86
Q

AD drug screening using iPSC

A
  • RV OKSM iPSC from renal tubular cells
  • differentiated into normal cortical neurones
  • exposed to AB42 and screened for neuroprotective agents
  • found that CDK inhibitors appeared protective
87
Q

Methods to avoid using RV

A
  • non-integrating vectors
  • chemical reprogramming
88
Q

Non-integrating vectors

A
  • RNA is modified to be stable and non-immunogenic
  • inducible expression
  • ex = episomal vectors
  • alternative to RV which may lead to cancers and mutations in cells
89
Q

Chemical reprogramming

A
  • uses no viral vectors
  • use of small molecules instead of OKSM RV
  • low efficiency to protocol needs to be improved
  • found via scRNA-seq that cells take a different gene expression route with this reprogramming
90
Q

Direct reprogramming

A
  • avoids using oncogenes which promote tumour growth when transplanted
  • goes straight from differentiated cell to differentiated cell = no pluripotent cells
91
Q

Benefits of direct programming

A
  • faster
  • more efficient
  • less costly
  • doesnt rejuvinate cells = good for age-related disorders
  • avoids tumour growth
92
Q

Neuron marker

A

TUJ1

93
Q

Problems with 2D cultures

A
  • can be hard to get cells to stick on plastic
  • feeder layers of other cells may be needed to secrete supportive factors
  • secrete GF and ECM components - not ideal when looking to transplant
  • doesnt fully represent the in vivo environment
94
Q

Benefits of 3D in vitro cultures (organoids)

A
  • cells organise themselves into complex structures of multiple cell types
  • can themselves secrete growth and supporting factors so dont need a feeder layer
  • can form mini organs
  • more physiologically relevant than 2D cell culture is
95
Q

Potential organoid uses

A
  • study development (Avoid 14 day rule)
  • study disease
  • evolution
  • personalised drug screening
  • transplants
96
Q

Organoid production

A
  • starting cell types = ESC,ASC and iPSC
  • need a defined media of chemical factors
  • series of steps that reflect the cells signalling environment within a developing embryo
  • initiate and maintain normal temporal gene expression to produce correct cell identity
  • often need an ECM component
97
Q

2 types of brain organoids

A
  • cerebral (Lancaster protocol)
  • cortical (velasco protocol)
98
Q

Lancaster protocol

A
  • Cerebral organoids
  • self organised from hESC or LV-OKSM iPSC
  • differentiate themselves in culture = follow own developmental profile
  • have forebrain, midbrain and hindbrain
  • cells plated in a suspension media
  • neural induction media
  • differentiation media and matrigel
  • spinning bioreactor
  • problem = each brain produced is different
99
Q

Velasco protocol

A
  • Cortical organoids
  • defined media with chemical signals to direct differentiation into cortex
  • little variation
100
Q

Multi cell organoids

A

good for when studying physiology and for organ replacement

101
Q

Single cell organoids use

A
  • drug efficacy and toxicology
  • genetics
102
Q

Organoid limitations

A
  • size
  • lack of vasculature = lack of nutrients
  • not complex enough, cant study aging
  • variable
  • ethical concerns
103
Q

Gut assembloid

A
  • Tabeke et al 2021 took iPSC derived gut spheroids differentiated into anterior and posterior
  • fused these spheroids together in matrigel
  • spheroids signalled to one another leading to the development of a pancreas, liver and bile duct
  • allowed for the modelling of a gut disease caused by HES1 mutant
  • growth was confirmed via a PROX1-tdTomato reporter system
104
Q

Assembloids

A

3D structures that incorporate 2 or more organoids/spheroids that are fused together to form a multi regional or multi-lineage assembloid

105
Q

Brain assembloid

A
  • Sergiu P. Pascas group
  • fused a cortical organoid with a striatal organoid
  • also fused a cortical-spiral-cord-muscle assmbloid
  • saw that stimulation of neurones triggered muscle contraction
106
Q

Somatosensory assembloid

A
  • 4 part organoid
  • combination of cortical, diencephalic, spinal and sensory
  • sensory pathways
  • responsive to chemical signals
  • used it to model SCN9A mutations which lead to no pain = due to abnormal signalling and inefficient signals reaching the brain
107
Q

Organ on a chip models

A
  • allows for vasculature
  • flow system with cells which will form endothelial networks
  • blood vessels organoids form throughout organoids allow for increased growth, maturation and function
108
Q

Venom secretory organoid

A
  • solves problem of the difficulting in developing antivenoms
  • AdSc derived
  • venom could be harvest from media
109
Q

Problems with venom secretory organoids

A
  • different development
  • cold blooded animals so have to be cultured at a different temperature
  • growth media formulation
110
Q

SARS-COV2 organoid modelling

A
  • Lancaster developed a choroid plexus organoid which secretes CSF
  • covid infected and broke down choroid plexus cells allowing for CSF fluid containing SARS and inflammatory molecules to pass the blood-brain barrier
  • intestinal organoid found that SARS can be passed through fecal matter
  • proved Hydroxychloroquine didnt target endocytosis of virus = not a good treatment
111
Q

Zika virus infection mechanism using organoids

A
  • cannot be modelled in a mouse due to their different brains
  • cortical organoids generated and used to determine pathology
  • found it preferentially effected neural progenitor cells
  • screen for drugs
112
Q

Autism modelling with organoids

A
  • CHD8 is a common mutant in ASD
  • mutant iPSC generated using CRISPR from human skin and differentiated into cerebral organoids
  • used a isogenic control
  • saw alterations in inhibitory neuronal differentiation
113
Q

Ex vivo

A

cells cultured in the lab before transplant back into patient

114
Q

Autologous cell therapy

A

cells used are derived from the patient

115
Q

Autologous cell therapy advantage

A

wont be rejected so no immunosuppression needed

116
Q

Allogenic cell therapy

A

human origin but from an individual distinct from patient (cell banks)

117
Q

Allogenic disadvantages

A

will need to be on immunosuppressants

118
Q

Allogenic advantages

A
  • good if patient has lost cells
  • decreased culture process required
119
Q

Xenogenic cell therapy

A

non human origin

120
Q

Xenogenic benefit

A

more accessible and cheaper

121
Q

In vivo

A

cells modified within a living animal or human

122
Q

Good manufacturing process

A
  • a set or principles and procedures that when followed helps ensure that therapeutic goods are of high quality, safe and potent
  • regulations differ by country
123
Q

Quality control for cells

A
  • no contaminants
  • cells produced are the same every time
  • contains no pluripotent cells in order to avoid tumors
  • sterility
  • use of animal free media
124
Q

3 different CRT to cure blindness

A
  • ASC
  • iPSC derived ocular organoid
  • autologous iPSC derived retinal pigment epithelial cells
125
Q

ASC blindness CRT

A
  • autologous
  • culture of adult corneal stem cells extacted via biopsy
  • grown into a sheet of cells on a sample of amniotic membrane
  • grown on a MEF feeder layer = possible contamination
126
Q

iPSC derived ocular organoid to treat blindness

A
  • self patterened
  • mimics whole eye development
  • corneal epithelial zone cells grown into a sheet and transplanted into mice showed recovery
  • some recovery in human trials
127
Q

Autologous iPSC derived RPE cells to treat blindness (md)

A
  • looking at age related macular degeneration which causes RPE cell death
  • OKSM reprogramming with episomal vectors from patients skin
  • differentiated into RPE in vitro on MEF feeder layers
  • cells were screened to check RPE identity and ensure all PSCs gone
  • Patient 1 = successfully integrated
  • Patient 2 = mutants formed
128
Q

Considerations with iPSC therapies

A
  • must check for left over PSC
  • check for mutants
  • find alternatives to matrigel and feeder layers
  • must use GMP
129
Q

iPSC derived neurones to treat parkinsons in monkeys

A
  • parkinsons = neurodegeneration of dopamine neurones leading to cognitive and motor symptoms
  • iPSC from monkey fibroblasts via OKSM RV
  • iPSC differentiated into dopamine neurones and transplanted back into monkey
  • cells survived and integrated causing a functional recovery
  • no tumours and no immunosuppression
130
Q

Liver organoids for transplantation

A
  • use HLA matched iPSC which differentiate into hepatic cells, blood vessel cells and mesenchymal cells
  • self assemble into liver bud organoids
  • transplant into liver failure mice showed recovery of hepatic function
131
Q

Cholangiocyte organoids as alternative to liver organoids

A
  • donor livers may not have functioning bile ducts
  • possibility of injecting cholangiocyte organoids into bile ducts to recover them
  • known as ex vivo perfusion models
132
Q

Severe combined immunodeficiency syndrome (CRT RV)

A
  • mutation in the gene coding for IL2RG
  • faulty development of T cells and NK cells in the immune system
  • Ex vivo RV-IL2RG transduction into an adult immune progenitor cells and inject into patient
  • results = persistent proliferation and correction of the disease
  • some developed T cell cancer = RV integration?
133
Q

Epidermolysis Bullosa (CRT)

A
  • mutation in LAMB3 prevents normal epidermal anchoring
  • RV-LAMB3 into unblistered epidermal cells from patient
  • Normal LAMB3 was integrate into the genome of a heterogenous skin cell culture
  • Grown into sheet and grafted onto boy
  • saw over time all his epidermis was regenerated
134
Q

Mesenchymal stem cell sources

A
  • adult bone marrow
  • stromal vascular fraction of adipose tissue
  • placenta
  • umbilical cord lbood
135
Q

Mesenchymal stem cell characteristics

A
  • heterogenous (problem with GMP)
  • wide differentiation potential
  • doesnt have a defined marker (problem)
  • secretes cytokines and growth factors = paracrine provider
  • when injected they home to parts which are inflammed where they will secrete cytokines/GF to stimulate cell repair
  • could help with heart attack scar tissue etc
136
Q

Questions to determine if a therapy is legitimate

A
  • efficacy in humans
  • safe and effective
  • GMP
  • kind of cells used
  • immunosuppressants
  • source of cells
  • what is done with extra cells
  • does it prevent joining of future clinical trials
  • consent
  • delivery method
  • regulations
  • costs
137
Q

eIf2a stress kinases

A
  • HRI
  • PKR
  • PERK
  • GCN2
138
Q

Why HSP can still be made when translation is halted

A
  • HSP undergo cap-independent translation
  • the translation that is halted is cap-dependent
139
Q

Ethical issues with HeLa cells

A
  • unjust enrichment
  • consent
140
Q

How can HSF1 be activated

A
  • intrinsic heat stress
  • HSR1
  • Hsp90

Biosci 353 (4 decks)

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