advanced molecules, membranes and cells Flashcards
1
Q
Embryonic stem cells are…
A
pluripotent stem cells derived from the inner cell mass of the blastocyst of preimplantation embryos, at which time they consist of 50-150 cells
2
Q
Entire embryo will derive from the
A
‘inner cell mass’ (ICM)
3
Q
ICM expresses what marker
A
oct 4 and nanog
4
Q
dissociating a blastocyst and culturing the ICM produces
A
embryonic stem cells
5
Q
embryonic stem cells introduced into adult mice will form
A
teratomas
6
Q
if embryonic stem cells are reintroduced into a blastocyst they will contribute to and form
A
all parts of the embryo forming chimeric mice with ES cells contributing randomly
7
Q
Oct4 is
A
A transcription factor, expressed in ICM cells
8
Q
Oct4-null embryos develop to
A
blastocyst stage but die because the ICM cells are not pluripotent
9
Q
Nanog null ICM cells lose
A
pluripotency and develop as extra-embryonic tissues (parietal endoderm)
10
Q
Leukemia Inhibitory Factor (LIF) role in ES cell manipulation
A
prevents differentiation into mesoderm/endoderm
11
Q
BMP role in ES cell manipulation
A
prevents differentiation into neuroectoderm
12
Q
the important highlights of the maintenance of mouse ES cells pathway
A
Leukemia Inhibitory Factor promotes GP130/LIFR -beta to work on three pathways JAK, Grb2, PI3K which through kinases goes on to influence SOX2 and Nanog which then alter OCT4
13
Q
how may ELECTROPORATION introduce DNA into ES cells
A
Electroporation blows tiny lesions in cell membranes ⟶ DNA enters before or during repair ⟶ somehow makes its way to nucleus.
14
Q
process of using ES cells to produce GM mice
A
DNA electroporation->selection->analyse survivors-> inject into ICM of host blastocyst->implant into pseudo pregnant female
15
Q
some examples of spontaneous mouse mutations
A
small eye mouse
looptail mouse
clubfoot mouse
16
Q
random mutagenesis involves
A
male animal exposed to mutagens such as ethyl nitrosourea (ENU) or ethyl methansuplhonate (EMS)
17
Q
ethyl nitrosourea (ENU) mutagenesis induces
A
point mutations by ethylating DNA base pairs in replicating sperm cells
18
Q
ethyl methansuplhonate (EMS) mutagenesis induces
A
turns G/C base pairs into A/T
19
Q
low doses of EMS/ENU mutagenesis ensure
A
randomly distributed point mutations at low frequency throughout genome
20
Q
the point of a mutagenesis screen is to
A
screen for recessive mutations and breed to produce heterozygous mutations
21
Q
mutagenesis screen benefit
A
Can generate mutations in tissues without a priori assumptions or knowledge about which genes are important
Can generate new alleles of genes that you would never have been able to make deliberately, or would never have thought of
22
Q
mutagenesis screen limitations
A
Uses very large numbers of animals
Wasteful - you only find what you’re looking for
e.g. limb defects, and may miss interesting mutations just by not looking at the right bit of the animal
23
Q
Gene Targeting (Knock-Outs) mean
A
To delete genes (‘knock genes out’) from animals by Homologous Recombination in Embryonic Stem Cells
24
Q
Homologous Recombination Usually occurs during
A
meiosis
25
Homologous Recombination entails
Identical (homologous) sequences on maternal + paternal chromosomes find each other, line up and may ‘cross over
It can occur ‘accidentally’ between identical DNA sequences in any cell at any time ⟶ at low frequency
Can be used to introduce new DNA into cells
26
in practice genetic manipulation using homologous recombination involves
transgene
| flanking DNA identical to sequence and hope they cross over
27
ganciclovir for Homologous Recombination ensures
cells with TK are killed (herpes simplex thymidine kinase gene)
28
where is TK attached on the transgene
outside the flanking sequences so it should be lost after undergoing homologous recombination
29
neomycin for Homologous Recombination ensures
cells without neoR are killed
30
NeoR is attached where on the transgene
next to the gene so it should be present after homologous recombination
31
after positive and negative selection following Integration of Target Vector by Homologous Recombination what should be conducted?
PCR and Southern Blotting
32
following successful targeted ES cells what happens next?
inject them into inner cell mass (ICM) of host blastocysts, implant into uterus of pseudopregnant female
Get chimeric offspring that can be used for breeding
33
Homologous Recombination accounts for what percentage of all integration events
<2%
34
issues with Knockouts
Redundancy can lead to mild or no phenotype (at the level of analysis)
Early embryonic lethality may prevent analysis of later events
Genetic background
The strain of mouse you use may affect the phenotype you see
Genes don’t act in isolation, and there may be many steps between gene dysfunction and animal phenotype
35
Eukaryotic genes need:
1) A promoter that will drive expression in the tissues you require…
2) An open reading frame encoding the gene you want to express…
3) Sequences that ensure correct mRNA processing
36
what signal ensures correct mRNA processing?
polyadenylation signal
37
cDNA refers to
complementary DNA - Contains coding region of gene, with no introns
38
Minimal promoter is where
transcriptional machinery
| binds to
39
DNA regulatory elements refer to
DNA sequences known to be bound by transcription factors that activate or repress transcription
40
methods of introducing transgenes into an animal
direct injection
chemical transfection
electroporation
infection
41
chemical transfection transgene delivery involves
Incubate cells in culture medium containing DNA and a chemical that wraps the DNA up and is either endocytosed or that diffuses through the cell membrane
42
to increase the chance of the transgene being carried by all the cells we introduce it to the
one cell embryo (the fertilised zygote) otherwise you get a mosaic
43
direct injection delivery of a transgene is most effective when you target
male pronucleus after fertilisation of oocyte but before nuclear fusion
44
nuclear processing and free DNA
Repair mechanisms presumably recognise ‘free’ DNA in nucleus and try to do something with it
45
nuclear processing and our transgene
May randomly accidentally introduce breaks in host DNA and ligate transgene
46
When the transgenic animals are made ⟶ what if expression is not as expected, what happened?
Weak promoter / Insufficient regulatory elements
Copy number (too large gets silenced by epigenetics)
Position effects (site of integration - random)
Epigenetic modification
Genetic background (farm animals, hence Dolly)
Very big transgenes (>1 Mb), which put the promoter of the transgene in its normal chromosomal context, usually work best
47
viral GAG gene is for
encodes proteins of nucleoprotein core of virion
48
viral POL gene is for
encodes reverse transcriptase, integrase etc functions
49
viral ENV gene is for
encodes surface protein components of virion
50
after getting the transgene packed up as an infective particle it is then
introduced into ‘helper’ cells
51
why is helper cells needed for viral transgene delivery
Packaging cells contain the genes encoding gag, pol, env, but do not have the packaging signal so the viral genes cannot be included in viruses
Instead, the gag, pol, env proteins package and make viral particles around the transgene DNA
52
are viral transgene delivery used to infect the whole animal?
no, Often used to introduce a gene into bits of the mid-gestation embryo to create a mosaic
53
viruses commonly used for gene viral delivery
Adenoviruses
Adeno-Associated Virus AAV
Lentiviruses
54
issues with adenovirus gene delivery
DNA genome
Gets eliminated by immune system (immune activation)
DNA genome
55
Adeno-Associated Virus AAV benefits
No pathogenicity
Can infect non-dividing cells
Can integrate - usually at AAVS1 site in Human chromosome 19
Popular for gene therapy
56
Lentiviruses is a
Retroviruses
57
benefits of lentiviruses
Very efficient infection, dividing and non-dividing cells
| Stable integration - long term expression of transgene
58
why is long term expression with viral transgene delivery a problem?
Viral sequences are subject to silencing mechanisms
59
any side effects to viral transgene delivery?
recombination leads to infective new viruses
60
solution to a knockout gene mouse dying before the gene can be explored
conditional knockout/tissue specific knockout
61
tissue specific knockout uses
Cre/loxP or flp/frt
62
cre is a
A site-specific Recombinase Enzyme from the P1 phage
63
cre recognises
Recognises a 34 bp DNA sequence loxP
64
cre's role
recognises two loxp segments in DNA, brings them together and then removes the excess lox p segment
65
principles of using cre/loxP conditional knockout
A line of mice in which loxP sites have been inserted around the gene of interest. The gene is said to be ‘floxed’.
and a different line of mice that express Cre recombinase from a tissue-specific promoter (e.g. directs Cre expression to pancreas only.
breed the two together
66
how to created floxed mice?
conditional targeting by homologous recombination in ES cells, inject into blastocysts, create chimeras, and breed
67
what is different about the homologous recombination when using lox P and FRT
post-homologous recombination the neomycin resistance gene can be removed to avoid it interfering with gene expression
68
how do you target cre expression to a tissue of interest?
Create a ‘Designer’ Promoter
| or knock-in cre gene to the correct tissues in ES cells
69
why is an internal ribosome entry site necessary for a typical cre construct
necessary to ensure expression of cre and GFP/LacZ which enable you to verify the transgene delivery was successful
70
a typical cre construct then consists of
promoter, cre, IRES (internal ribosome entry site), GFP or LacZ, intron and SV40
71
how can we temporally control the effect of the cre-lox transgene?
Put the Cre gene on promoters that are responsive to drugs you can give the mice
72
what drugs can cre promoters be vulnerable to
Tetracycline
| Tamoxifen
73
Tetracycline "tet-off system" Induction of Cre expression involves
The Tet-activator protein ⟶ Binds to the tet operon (tetO) to activate transcription of Cre
But not in presence of Tetracycline
74
tet-off tetracycline cre system without tetracycline results in
Tetracycline then withdrawn ⟶ TetA binds tetO ⟶ Cre is transcribed ⟶ floxed gene knocked out
75
tamoxifen induction of cre expression involves
Uses a fusion protein combining activity of Cre and a mutant form of the ligand binding domain of the estrogen receptor (ERTM)
Cre-ERTM does not bind estrogen, but does bind 4-OH-tamoxifen
In absence of Tamoxifen ⟶ Hsp90 grabs Cre-ER and keeps it in cytoplasm ⟶ no recombination in nucleus
76
upon addition of tamoxifen Cre-ERTM is
released from Hsp90, allowing access to the nucleus and Cre-mediated recombination
77
any issues with the cre-loxp system?
Not 100% efficient
Not reversible
Some evidence that high levels of Cre may be toxic to cells in some cases
78
3 mechanisms of genome editing include
zin finger nucleases
TALEN
cas9/CRISPR
79
the zinc finger is
Protein motif that binds ~3 bp of DNA
80
multiple zinc finger motifs in a protein can
give long, >> 3 bp binding specificity to a transcription factor
81
normal physiological function of zing fingers are to
Zn-finger transcription factor binds specific consensus DNA sequences around genes and activates transcription
82
experimental zinc finger involves
the transcriptional regulatory domain is replaced by a DNA nuclease called FOK1
2 ZFN bring 2 FOK1 together and generate a double stranded break in DNA
83
2 ZFN bring 2 FOK1 together and generate a double stranded break in DNA is important because
Ds break will be repaired in the cell, but repair is ERROR-PRONE
Random bases inserted, or get small deletions or insertions as DNA repair enzymes join the two cut ends together
Makes mutations at the cut point with very high efficiency
84
the process by which zinc finger (ZFN) generate mutations is called
non-homologous end joining
85
TALENs stand for
Transcription Activator-Like Effector Nucleases
86
talens in principle work similar to
ZFNs
87
TALE’s are
highly conserved 33-35 bp repeat domains encoded by Xanthomonas spp. proteobacteria
88
TALEs consist of
a nonspecific FokI nuclease domain fused to a customizable DNA-binding domain
89
TALEN method of Non-homologous End Joining Repair is
2 TALENs binding specific sequence of target gene to create double strand break
2 hypervariable amino acids determine which base each TALE repeat binds to
90
Cas9 is
```
An endonuclease (cuts DNA)
From bacterial acquired immunity against viruses
```
91
cas9 is based on
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
92
cas9 is targeted to cleave DNA by
short RNAS:
CRISPR-RNA (crRNA) and transactivating CRISPR-RNA (tracrRNA)
Are complementary to the site to be cut
93
Cas/CRISPR system can cut any piece of genomic DNA that is adjacent to a
‘PAM’ (Protospacer adjacent motif)
94
cas 9 PAM is
NGG
95
the first stage of CRISPR/mas mutagenesis then involves
The first CRISPR crRNA has a region that is complementary to part of the target gene, upstream of the PAM
It wriggles into the DNA duplex and binds by base-pairing
96
once the CRISPR crRNA has bound upstream to PAM then
The transactivating CRISPR tracrRNA has a region that is complementary to the rest of the crRNA
It binds by base-pairing
97
once cas9 binds to the CRISPR crRNA/ tracrRNA complex
cas9 will make a double-stranded break in the target DNA, a variable number of bases from the PAM
Break will be ragged, with single strand overhangs
98
the consequent break by cas9 stimulates
DNA repair enzymes will try to repair the damage
| They will screw it up, creating a mutation
99
cas9 then generates mutations through
non-homologous end joining
100
in the lab crRNA-tracrRNA are fused into
sgRNA
101
Targeted ES cells with Cas9, and sgRNAs against multiple alleles of a gene was found to
With high efficiency, produced ES cell clones with mutations in all 6 alleles of all three genes
to quickly target ES cells with multiple mutations and make mutant mice
102
When Cas9 was injected into 1 cell zygotes what percentage of offspring had the mutations against TET genes?
50-90%
103
a limitation of using cas9 in 1 cell zygotes were
mutations induced are Uncontrolled and Variable
104
how may cas9 Uncontrolled and Variable mutations be overcome?
Catalyse some Homology-Directed Repair (HDR
105
Homology-Directed Repair (HDR) involves
Electroporate or inject cells with Cas9, sgRNA AND a single-stranded DNA oligonucleotide homologous to the area around the breakpoint
106
can Homology-Directed Repair be used alongside TELNS or zinc fingers?
yes
107
Cas9 + HDR can be used to introduce what other form of genetic manipulation into 1 cell zygotes?
loxP sequences
108
any examples of Cas9 + HDR + loxP being used
Floxed the Mecp2 gene
| that is mutated in Rett Syndrome
109
limitations of cas9 are
Off-target mutations of Cas9/CRISPR remain a problem (especially for human therapy).
110
how to clone an animal?
1. Take a cell from the donor animal to be cloned
2. Take the nucleus (pronucleus) out of a recipient unfertilised oocyte
3. Put the nucleus of the donor somatic (e.g skin) cell into the enucleated oocyte
4. ‘Activate’ the oocyte to start its development
5. Transfer it to the uterus of a pseudopregnant female
6. Completes development as normal in utero
111
why does any cell work for cloning?
Every Cell Contains a Complete Genome
112
how can an oocyte be activated to start its development?
- electrical or chemical activation
113
for cloning how can you put the nucleus of the donor somatic (e.g skin) cell into the enucleated oocyte
direct injection or electrofusion
114
any examples of chemical activation for oocyte development?
strontium chloride will stimulate the calcium activation wave
115
issues with cloned animals include
Dolly died young of lung disease
⟶ Viability of cloned embryos very low
⟶ ‘Large offspring syndrome’ common
⟶ Respiratory and circulatory problems common
⟶ Weak immune system
⟶ Liver failure
⟶ Premature ageing (including arthritis))
116
why do issues arise with cloned animals?
⟶ Failure of epigenetic reprogramming (some retention of DNA character of donor cells) – this is a technical not a biological hurdle.
⟶ Accumulation of damage macromolecules – should be diluted out
⟶ Shortened telomeres - yes in sheep and goats, not in all species
⟶ Retention of mutations that happened during life of donor – very likely to happen.
117
can serial cloning for many generations work?
yes for mice!
118
what is the possible pathway for human cloning?
skin sample->inject into host oocyte and make blastocyst clone->harvest ICM and make ES cells->differentiate into necessary cells, tissues etc
119
what is important about this process of human cloning (protocol for dopamine neurone production e.g.)
follows a stepwise process that mimics using external chemical influences actual human physiological differentiation and maturation
120
can these cloned cells, such a dopamine neurones be grafted onto animals?
yes and survive
121
did the grafting of human cloned dopamine neurones onto PD animals yield any improvements?
yes
122
stem cell technology challenges
clinical grade culture facility free of pathogens
preventing spontaneous differentiation
animal product free mediums (risk of virus and immunity)
to minimise tumorigenic potential of transplants (teratoma)
overcome host immunity
produce all cell types necessary
123
Adult somatic cells can be induced to become pluripotent by forced expression of a few key transcription factors such as
c-Myc, Oct4, Sox2, Klf4
124
these induced pluripotent stem cells were verified by demonstrating
Teratoma formation with differentiated cells from all three embryonic layers
forming embryoid bodies (clumps of differentiated cells)
and could produce chimera
125
Oct4, Sox2 (with Nanog) function as .
core transcription factors in maintaining pluripotency
126
C-Myc is an
An oncogene, enhances proliferation
127
C-Myc use in IPSC may be
Associates with histone acetyltransferase complexes responsible for opening up DNA for transcription. Possibly allows Oct4 and Sox2 to bind target loci.
128
Klf4 use in IPSC may be
represses p53 which in turn represses Nanog (may therefore be back door to induction of endogenous Nanog).
129
Tetraploid cells in pregnancy can
can contribute to the placenta but not to the embryo proper.
130
injecting iPS cells into a tetraploid mouse blastocyst and transferring the blastocyst to the uterus of a recipient female to complete pregnancy, Boland et al were able to create
adult mice derived entirely from iPS cells
131
other alternative transcription factors for producing ISPC
OCT4, NANOG, SOX2 and LIN28
132
any proof of therapeutic use of ISPCs?
Treatment of Sickle Cell Anemia mouse model with iPS cells generated from autologous skin.
133
Treatment of Sickle Cell Anemia mouse model with iPS cells generated from autologous skin limitations when considering humans?
Paper ends with a caveat that this is not suitable for humans because of the requirement to use retroviruses (risk of insertion mutagenesis) and oncogenes (c-Myc).
134
when producing differentiated stem cells, in comparison to ES-cells, iPS cells were
dramatically less efficient at forming haemangioblasts, significantly more apoptotic, and with severely limited growth and expansion capacity
135
why were IPS cells limited in their ability to differentiate
Induction of pluripotency requires somatic cells to lose their epigenetic memory
iPS cells retain some of the methylation pattern of the somatic cells from which they were derived, and are more prone to differentiate back into those same sorts of cells than into new cell types
136
what percentage of AD is familial
1%
137
BACe1 codes for
beta secretase
138
an ideal gene delivery system should
transfer genes into both developing and mature animals;
transduce cells with high efficiency;
be cell-type / tissue specific;
mediate high level, long-term expression;
cause limited cytotoxicity;
elicit a small/negligible immune response;
allow sufficient lengths of DNA to be introduced;
avoid over-expression;
allow regulatable expression
139
Novel viral models of Alzheimer’s disease enable studies on
Cellular mechanisms of degeneration
Drug target identification
Initial evaluation of treatments
On demand delivery to many species and preparations,
hence addressing the 3Rs of animal experimentation
140
problems with transgenic mice models when modelling AD and using multiple genes
```
Varied promoters
Varied genetic backgrounds
Technical uncertainties
No suitable controls
Difficult to compare
```
141
PLB mice for understanding AD were produced using
Knock-in (vs pronuclear injection)
142
the benefits of the PLB mice model for AD were
Safe, removable, single-copy, stable genetics
Comparable triple / double / single lines [APP, Tau, PS1 & BACE]
Low / relevant expression levels (model of prodromal dementia)
Ideal to develop sensitive, early & translational biomarker
143
BACE is rate limiting step in production of
b-amyloid
144
rational for the production of PLB 4 hBACE1 knock in model
No hBACE tg mouse available
BACE is rate limiting step in production of b-amyloid
hBACE1 allows improved drug discovery
Model does not rely on rare genetic mutations
145
PLB 4 hBACE1 knock in mice model pathological findings for AD were
reduced full length APP
accumulation of APP cleavage products
severe inflammation in the hippocampus and cortex
146
PLB4 BACE1 knock-in mice: Behavioural characterization
```
reduced:
Activity
Spatial memory
Working memory
Semantic memory
```
Anxiety
Age-dependent
AD-relevant
147
did hBACE 1 mice models demonstrate any systemic effects despite being specific for neuronal tissue?
PLB4 mice are susceptible to diabetes, high fat diet
A glucose tolerance test, determine their fasting glucose level and then see how fast they can cleave glucose from their system. PLB4 much higher glucose level at 5 month
148
BACE1 may be a link between what two pathological conditions
between AD and T2D
149
what hypothalamic gene expression profiles were altered in PLB4 mice
changes related to appetite & energy regulation, a dramatic up-regulation of CHOP (multifunctional & pro-apoptotic transcription factor)
CHOP reflects ER stress
150
how may BACE1 relate to ER stress
factors induce ER stress, this results in a signalling molecule being sent to activate stress response genes and alter translation necessary for plasticity. it is believed inflammation and BACE1 can interact with this pathway increasing it and thus altering plasticity and triggering apoptosis & autophagy
151
factors causing ER stress
Ageing (mitochondria)
Obesity / Metabolic failure
Excitotoxicity
Ca2+ dyshomeostasis
152
symptoms of parkinsons
Tremor
Ridigity
Akinesia
Postural instability
(TRAP)
153
indirect symptoms of parkinsons
Depression
Dementia
Fatigue
Apathy
154
ideal model of PD should include
```
Pathological and clinical features
Dopaminergic and nondopaminergic systems
Central and peripheral nervous system
Motor and non-motor symptoms
Age-dependent onset and progressive nature
```
155
are there any natural models for PD?
no
156
in silico models for PD are useful for
Drug candidates:
Design
Screening
Mathematical models:
Metabolomics
157
in vitro models of PD are useful for
Disease mechanism on cellular level:
Uptake of α-synuclein
Seeding capacity
Screening of drug candidates:
Reaction to molecules on cellular/organ level
158
in vivo non mammalian models for PD are useful for
Genetically easily manipulated:
Understanding basis of pathogenesis
Molecular interactions
Mitochondrial studies
Less ethical issues
Drug discovery
159
what is the limitation of in vivo non-mammalian models
99% of PD is non-hereditary
160
benefit of mammalian models of PD
Validation of in vivo
Complex organism
Behaviour
161
what percentage of DNA is shared between mice and men
97.5%
162
gold standard mice neurotoxic model of PD
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model
163
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model works by
Mitochondrial inhibition in nigrostriatal pathway:
| Oxidative stress -> loss of dopaminergic neurons
164
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model pathology
PD-like motor symptoms
| Lacks formation of Lewy bodies
165
can 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model pathology be injected across the BBB
easily and systemically
166
key drugs developed using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model were
Levodopa
MAO inhibitors
COMT inhibitors
167
an alternative neurotoxic model of PD is
6-hydroxydopamine (6-OHDA) model
168
6-hydroxydopamine (6-OHDA) model is produced by
Injected into the substantia nigra or striatum
169
6-hydroxydopamine (6-OHDA) models specifically
Modelling motor symptoms, not disease etiology
170
6-hydroxydopamine (6-OHDA) model produces
Oxidative stress
Neurodegeneration
Neuroinflammation
Neuronal death by apoptosis
171
PD mouse model transgenes are
LRRK2, SNCA
| PINK1, PRKN, PARK7
172
PD mouse model transgene features
Molecular pathways
No or little neurodegeneration
No Lewy body formation
173
α-synuclein spreads through the brain according to the
Prion-hypothesis
174
Mouse models of PD: Seeding of α-synuclein involve
Injection of human α-synuclein directly into brain which stimulate murine alpha synuclein to propagate and become pathological
175
techniques to study mice behaviour include
```
catwalk
rotarod
elevated plus maze
agora
open field
morris water maze
y maze
```
176
behaviour affected by neurotoxic PD models?
yes, heavily
177
behaviour affected by transgenic PD model
not at all
178
behaviour affected by seeding PD model
yes mild
179
cognition affected by neurotoxic PD model
yes
180
cognition affected by transgenic PD model
not at all
181
cognition affected by seeding PD model
yes
182
social behaviour affected by transgenic PD model?
not at all
183
social behaviour affected by seeding PD model?
yes, mild
184
equation for energy balance
(ES)=(EI)-(EO)
185
ES stands for
Rate of exchange in the body stores
186
EI stands for
Chemical energy from the diet
187
EO stands for
Work and heat
188
(EO) or work and heat energy balance can be divided into
```
Resting Metabolic Rate
(RMR)
Physical Activity
(PA)
Diet-induced Thermogenesis (DiT)
Thermogenesis
```
189
what does diet really reflect?
bioavailability rather than just intake
190
factors of bioavailability include
```
Food addictions
Nutrient density &
quality
Allergies/Intolerances
Digestive Tract Diseases
Hunger/Satiety
```
191
energy expenditure by resting metabolic rate has to consider
body composition (fat, fatre free mass, TBW, bone, organs etc)
192
does base metabolic rate decrease with age?
yes, but plateaus around 20 rather than continuing to decrease
193
what are the two central regulators of energy balance
hindbrain and hypothalamus
194
what hypothalamic nuclei regulates energy balance
arcuate nucleus (ARC)
195
arcuate nucleus (ARC) receives inputs from
grehlin, satiety peptides, leptin and insulin
196
hindbrain nucleus receives inputs from
vagal afferents (stretch, chemo, nutrient)
197
the hind brain nucleus is
nucleus tractus solitarius
198
central regulation of energy balance outputs are
meal timing
meal volume
gastric emptying
energy expenditure
199
benefits of mice models when considering obesity models
1. Mouse obesity and metabolic phenotypes are comparable to humans
2. Phenotype measured with standardized test
3. Easy study of the brain
4. Manipulate their genome
5. Use identical strains when inbreeding
6. Control of environmental factors
7. Accelerated lifespan 1/30
8. Cost-effective and efficient tool
200
3R's of animal research
replacement, reduction, refinement
201
Replacement -
Methods which avoid or
replace the use of
animals
202
Reduction -
Methods which minimise
the number of animals
used per experiment
203
Refinement -
Methods which minimise
animal suffering and
improve welfare
204
what is important to consider about the gross anatomical arrangement of the mouse brain and energy circuit
the energy circuit is close to the centres for decision making, motivation and arousal with several links
205
environmental models for studying obesity use
```
Maternal and early-life diet
Scheduled meal events
Fat/sugar choice diets
Binge-type feeding
Cafeteria/Junk diets
```
206
chemical models for obesity use
Glucocorticoids
Streptozotocin
Antipsychotics
207
chemical models for obesity - Glucocorticoids
Adipocyte differentiation,
lipolysis, proteolysis, gluconeogenesis. Induce
food intake, weight gain, adiposity, glucose
impairment.
208
chemical models for obesity - Streptozotocin
Chemical ablation of pancreatic b-cells.
| Model of diabetes
209
chemical models for obesity - Antipsychotics
Fat deposition, FFAs
210
pros of environmental/chemical models
Suitable for the investigations of non-genetic lifestyle dependent metabolic syndrome in humans
Inexpensive
211
cons of of environmental/chemical models
Sometimes not complete reproducibility
Lack of standardized diets
Delayed onset of metabolic syndrome
212
the leptin receptor when bound by leptin
inhibits appetite
213
a single molecule, have been crucial to understand their role
in the pathophysiology of obesity. ie
leptin
214
3 forms of genetic models for studying obesity
Spontaneous mutations
Artificially-induced mutations
Targeted mutations.
215
Artificially-induced mutations limitations
Not targeted mutation
Need to screen for all genome and study whether it causes a phenotype
Expensive
216
leptin acts through what signalling molecule
STAT3 signalling
| to regulate adiposity
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Mutation in STAT3 (s/s) shows
the same overweight phenotype, but
glycemia and insulin sensitivity is
corrected under food restriction.
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conventional GMO mice model limitations
Knocking-out a gene can create early embryonic death
preventing the study in adults
Since the animal develops and grows without a specific gene,
compensatory mechanisms can appear.
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what alternative GM Advanced technique allows to interrogate genes
in specific tissues, cell type and even at precise times.
Cre/LoxP > Flp/Frt > Dre/Rox systems
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an example of mice obesity study using cre/LoxP
Mouse InsRLoxP
| insulin receptor insR
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Recombinant virus targeting in cre mouse models for obesity study enables
long-term, cell specific and spatio-temporally genetic access to
certain cell populations
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what three areas in regards to neurones can viral particle and cre mouse models allow us to study
neuronal effect, connection effect and mapping -
| with anterograde and retrograde tracing
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chemo genetics requires
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)
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how does DREADDs work
using viral particles and cre mouse models to insert receptors that are only activated by specific drugs to assess function of specific neurones in specific brain regions
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Opsins are
light-gated ion channels or pumps that absorb light at specific
wavelengths. Upon activation by light, these channels and pumps respond by
opening or closing, which conducts the flow of ions into or out of the cell
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Optogenetics enable the ability to
modulate terminal projections to evaluate target regions in real time
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Photometry uses
Calcium imaging enables neuroscientists to visualize the activity of hundreds
of individual neurons simultaneously using fluorescent activity sensors.
Changes in fluorescence indicate fluctuations in intracellular calcium, which is
an indirect indicator of neural activity
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Chemogenetics revealed AGRP neurones in arcuate nucleus (ARC)
activation of AgRP neurons induce food intake and
| body weight gain
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Optogenetics revealed AGRP neurones in arcuate nucleus (ARC)
Activation of AgRP neurons do not induce food intake through the parabrachial nucleus
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photometry revealed AGRP neurones in arcuate nucleus (ARC)
AgRP neurons drive only the initiation of the meal event
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channelrhodopsin is both
fluorescent and an ion channel
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in response to blue light the channelrhodopsin channel
opens, Na+ and Ca2+ ions flow in, the nerve cell depolarizes and fires action potentials
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halorhodopsin is a
light-gated chloride pump found in archae
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halorhodopsin in responds to what colour of light
yellow
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halorhodopsin in response to yellow light
activates halorhodopsin pump, chloride enters, nerves hyperpolarise, silences action potentials
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the light sensitive protein channelrhodopsin is harvested from
algae
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together channelrhodopsin and halorhodopsin allow
multiple-color optical activation, silencing and desynchronization of neural activity.
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Optogenetic modulation occurs at three levels of neuronal activity:
1) light activated signal transduction cascade
2) light mediated intracellular protein-protein interactions
3) light switchable transgene for control of gene expression
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what are ospin receptor chimeras?
G-protein channel receptors combined with rhodopsin
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what is the intracellular loops of rhodopsin replaced with in chimeric receptors
specific adrenergic or serotonin receptors
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chimeric receptors enable manipulation of
alpha 1 adrenargic receptor - IP3, DAG
| beta 2 adrenergic receptor - cAMP
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Optogentics + pharmacology can allow the study of
signalling - protein-protein interactions
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LightOn, uses the light dimerization property of Vivid to produce a
a light oxygen voltage domain containing protein in circadian clock systems, to form a synthetic light-switchable gene-promoter system.
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LightOn thus then can
binds promoters after blue light exposure
rapidly initiate transcription of target transgenes in both mammalian cells and mice
Giving precise spatiotemporal control of genes in a cell type–specific fashion - with light
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Therapeutic uses for optogenetics
gene therapy - install light-sensitive opsins in nociceptor nerve endings mouse skin
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in therapeutic optogenetics blue light causes
pain
247
in therapeutic optogenetics yellow light causes
blocks pain
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Channelrhodopsin can be used to tranfect what to achieve potential therapeutic effect
retinal ganglion cells in humans blinded by retinitis pigmentosa - restore vision
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the connectome project aims to
mapping the entire connections of the human brain
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the connectome project uses
loxP and cre recombinase system
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why can cre/LoxP generate different colours for the brainbow
Cre/LoxP system can be used to introduce different colour tags with some being removed. Furthermore, whilst two forward facing LoxP sections will result in excision, two loxP sections facing one another will cause inversion of the code
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what are the four initial cre-lox P brainbow colours
red, green, yellow, blue
253
how does a rainbow be generated from LoxP however
expression of trimeric combination of different fluorescent proteins and different numbers of copies result in different intensity
254
how many colours are available for the brainbow project
ten
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has the brainbow project produced any revelations?
challenges concept of action potential dependent synapse withdrawal in developing visual system -
See clusters of retinal terminals from >10 uniquely labeled RGCs make complex retinothalamic synapses
Synapses are not withdrawn – lots of convergence persists on thalamic relay cells.
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the goal of the clarity project is to
creates see through brain
travel through brain structures in 3D, dynamically
label lots of molecules in whole brain
trace single cells through a forest of other cells
257
is clarity just limited to the brain
no, other organs can undergo
258
clarity process involves
infuse mouse brain with a hydrogel solution.
set hydrogel mesh binds and supports rest of brain
gel does not bind lipids or fats
they need removed as they are opaque
extract unbound fat - leaves clear view of everything else
proteins embedded in cell membranes and
dendritic spines on neurons - both remained.
Add brainbow labels
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has clarity been used to demonstrate anything?
Mice given cocaine, or electric shocks + Clarity + dyes to highlight cells that fired
shows which networks of neurons corresponded to pleasure, or to fear
both emotions are laid down in the medial prefrontal cortex
but stored along different pathways or axonal projections
And they are connected to different regions in the brain
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the results of the clarity pleasure and fear study
both emotions are laid down in the medial prefrontal cortex
but stored along different pathways or axonal projections
And they are connected to different regions in the brain
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Algebraic Topology seeks to
describe emergent behaviours of neural networks from their underlying structural firing patterns – single cells, groups, networks, synchrony/asynchrony
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according to Algebraic Topology neurones
tend to form families (called "cliques"), where each neuron is connected to each other in the clique
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according to algebraic topology the model brain reacts by
building and then collapsing a multi-dimensional connected tower
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what are some of the controversies regarding algebraic topology
some accuse it of lacking any substantial data, using too much jargon and having grandiose claims
265
algebraic topology grandiose claims include
being able to code for decision making in brains and if correct, one day, be able to stimulate decisions in brains
