Preguntas de clase Flashcards
Assuming that there were no time constraints on replication of the genome of a human cell, what would be the minimum number of origins that would be required?
If there were no time constraints on replication, one origin would be required for each chromosome; thus, a minimum of 46 origins, equal to the number of chromosomes in a human cell, would be needed.
What consequences can you think of that would arise if a eukaryotic chromosome (150 Mb in length) had one of the following features:
* A single replication origin located in the middle of the chromosome (DNA replication proceeds at about 150 nt pairs per second).
* A chromosome with no telomeres
* No centromere
A single replication origin in the middle of the chromosome: Slow Replication, limit severily the cell division
A chromosome with no telomeres: would loose nucleotides during each DNA replication, essential genes would be lost
No centromere: crucial for proper chromosome segregation during cell division. Without a centromere, chromosomes might not be correctly distributed to daughter cells during mitosis and meiosis.
(T/F) Human cells do not contain any circular DNA molecules
False. some mitochondrial DNA molecules are circular
Why is Hoechst used for in vivo imaging ,…. but DAPI rarely used , and PI not used
Hoechst: bind specifically to the minor groove of DNA, resulting in strong fluorescence signals within the cell nuclei. This makes them useful for labeling and visualizing the nuclei in living cells.
DAPI: excessive exposure to UV light during live-cell imaging can lead to cell damage. This phototoxicity can limit the use of DAPI in prolonged in vivo imaging experiments.
Propidium Iodide (PI): is excluded from live cells with intact plasma membranes, and it only enters cells with compromised membrane integrity, such as dead or apoptotic cells. As a result, PI is not suitable for in vivo imaging of live cells.
Can you think of disadvantages of using UV light to visualize DNA/RNA?
DNA and cellular damage: phototoxic, mutations and apoptosis
The Human genome project and the company Celera used 2 different approaches to fully sequence the human genome.
Sequencing of mapped clones (=Bacterial Artificial Chromosomes) vs. shotgun sequencing
Shotgun sequencing is much faster compared to traditional methods but offers very short read length. If you think about the composition of DNA sequences in the human genome, what are putative problems a shotgun approach could face?
Challenges with Repeat Regions: The human genome contains a significant portion of repetitive elements, such as transposons and tandem repeats. Short read lengths in shotgun sequencing may make it difficult to resolve and assemble these repetitive regions accurately, leading to gaps or misassemblies in the genome
Difficulty in Resolving Structural Variations: Structural variations, including insertions, deletions, duplications, and inversions, are prevalent in the human genome. Short reads from shotgun sequencing may not provide enough information to accurately resolve complex structural variations, leading to challenges in reconstructing the true genomic architecture.
Which mutational changes would you predict to be the most deleterious to gene function?
Removal of a single nucleotide near the beginning of the coding sequence is noted as harmful. This is because it is likely to cause a frameshift mutation, altering the reading frame and leading to the production of a completely different protein or a truncated version.
There is a persisting expression of AAV transgene for years in a patient with retinal dystrophy. However, after 5-6 years there is a loss of expression. What are the possible reasons for that?
1) The patient’s immune cells might recognise the AAV vector at some point and eliminate it.
2) Epigenic modifications may silence the transgene / its promotor
3) Cells in the retina have a limited lifespan and eventually are replaced by new cells, lacking the AAV
Integrating viral vectors can lead to genotoxic events e.g., inflammation, random insertion disrupting normal genes, activation of proto-oncogenes, and insertional mutagenesis. Explain possible genotoxicity effects
A (i)
Promotor insertion upstream of our transcription unit -> expression of both transgene and cellular gene -> may lead to toxicity because of many copies of the protein present
A (ii)
Also insertion in the promotor region of the cellular gene may also result in genotoxicity due to the disruption of the original promotor and the possibly altered gene activation by the transgene LTR.
B (i)
Insertion of transgene cassette upstream of a cellular expression cassette may result in an overexpression of the cellular gene through transgene promotor / LTR activation
(e.g. if the cellular gene is a proto-oncogene, its increased expression might result in cancer-development)
B (ii)
Downstream activation of the cellular gene through LTR / promotor activation can lead to overexpression of the cellular gene, which often results in genotoxicity.
C
Intragenic insertion may lead to disruption of the mRNA and thus lead to a truncated mRNA. Therefore the gene may be inactivated.
Briefly explain 1 repair mechanism of single stand break and 1 repair mechanism of a double strand break.
Single strand:
-Baseexchange
-Nucleotide excision repair: scans DNA for distorsion in double helix-> cleaves the phosphodiester backbone -> DNA -> DNA helicase removes the sequence -> DNA polymerase and DNA ligase binds again a new sequence
Double strand:
-Homologous repair: newly replicated DNA, use of the sister chromatide as template, takes place once the DNA is repliccated for cell division
-Non homologous end joining: broken ends are brought together by ligases
What happens if the cell doesn’t have a centromere?
The primary role of the centromere is to facilitate the accurate separation of chromosomes during cell division. Without a centromere or with a defective centromeric region, chromosomes may not properly align and segregate during mitosis or meiosis.
What are the key differences between prokaryotes and eukaryotes in protein synthesis ?
Prokaryotes: Lack membrane-bound organelles, including a nucleus. Eukaryotes: Transcription occurs in the nucleus, and the primary transcript (pre-mRNA) undergoes processing, including splicing and capping, before it is transported to the cytoplasm for translation.
What is genotoxicity?
substance or agent to cause damage to genetic information-> genetic mutations, chromosomal aberrations, or other genetic alterations. including the development of cancer.
Which repair mechanisms do you know in eukaryotes?
Single strand:
-Nucleotide excision repair: scans DNA for distorsion in double helix-> cleaves the phosphodiester backbone -> DNA -> DNA helicase removes the sequence -> DNA polymerase and DNA ligase binds again a new sequence
Double strand:
-Homologous repair: newly replicated DNA, use of the sister chromatide as template, takes place once the DNA is repliccated for cell division
-Non homologous end joining: broken ends are brought together by ligases
How does the end mammalian dna sequence looks like?
Telomeres
What happens if the cell doesn’t have a telomere?
protect the ends of chromosomes from degradation and prevent them from sticking together. In the absence chromosomes become unstable. When telomeres become critically short, they can trigger a state of cellular senescence. If telomeres become extremely short, the cell may undergo apoptosis
How are viral vectors used in gene expression and name a genetic disease which can be treated?
AAVs are used to deliver therapeutic genes to target tissues, and they are particularly effective for treating diseases. First there is the genome design, then the transgene is cloned in the adenosin associated virus -> cell culture cells for AVV production -> transfection of cells. pathogenic, single-stranded DNA parvovirus. Recombinant AAV (rAAV) vectors’ excellent safety profile and durable gene expression have made it the preferred vector for gene therapy in the nervous system.
Disease: spinal muscular athrophy
Name the key processes in protein production and which steps influence the concentration of the produced protein?
Transcription-> capping, splicing, and polyadenylation-> Mature mRNA is transported out of the nucleus to the cytoplasm-> Translation -> Post-Translational Modifications -> protein folding
The concentration of the produced protein depends on each step
What is the difference between housekeeping genes and regulated genes?
Housekeeping are expressed all the time, regulated genes are controlled for differentiation
Can you think of one feature that make miRNAs especially useful regulators of gene expression?
ability to target multiple genes, used for gene silencing and inhibition
What is the difference between homodimer and heterodimer?
homodimer is a protein made from two identical proteins, while heterodimer is a protein made from two different proteins.
Many transcription regulators form dimers of identical or slightly different subunits on the DNA. Suggest advantages of dimerization.
Increased DNA Binding Specificity, Enhanced Stability on DNA, Cooperative Binding, Functional Diversity
Consider the argument:
If expression of every gene depends on a set of transcription factors, then the expression of these TF must also depend on the expression of other TF regulating their expression, which are in turn again regulated by a set of TF……..
Why does the cellular genome not code for an infinite number of transcription factors?
Combinatorial control is a common strategy in transcriptional regulation, where multiple transcription factors work together to regulate the expression of a gene. This allows for a diverse range of gene expression outcomes without requiring an individual transcription factor for every possible regulation.
Designing an expression casette:
* What sequence elements are present in the 5‘-UTR and 3‘-UTR elements of
delivered RNAs?
* Can you find out how these elements have been designed for clinical
applications or in laboratory experiments?
internal ribosome entry sites (IRES), and the 5’-cap structure enhance translation initiation and stability. The 3’-UTR includes elements such as polyadenylation signals, termination codons, and stability/localization elements crucial for mRNA stability, termination, and regulation.
For clinical applications or laboratory experiments, several design strategies are employed:
Codon Optimization: Optimizing codons for the host organism improves translation efficiency. Regulatory Elements: Inclusion of enhancers or repressors for modulating gene expression. Cell-Specific Promoters: Choosing promoters active in the target cell type ensures cell-specific expression. Vector Systems: Using viral (e.g., lentiviruses) or non-viral vectors (e.g., plasmids) for efficient delivery. Safety Features: Incorporating safety elements like insulators or inducible systems to enhance safety in clinical applications.
Can you think of differences between bacteria and animal cells that are, or could be, depended on the appearance or presence of the eukaryotic cytoskeleton?
Animal cells, with the support of the eukaryotic cytoskeleton, exhibit diverse shapes and can change their morphology. The cytoskeleton, consisting of actin filaments, microtubules, and intermediate filaments, provides structural support, maintains cell shape, and enables cell motility.
Bacterial cells often have a defined shape, which is maintained by the bacterial cytoskeleton
Bacterial cells lack membrane-bound organelles and a well-defined cytoplasmic structure.
The eukaryotic cytoskeleton, especially microtubules and actin filaments, contributes to the organization of organelles, intracellular transport, and the maintenance of cellular polarity.
The amino acid sequences of actin and tubulin in eukaryotic species are remarkably well conserved, while the numerous proteins that interact with the cytoskeleton are no more conserved than most other proteins.
How can it be that the filament proteins themselves are highly conserved, while the proteins that interact with them are not?
the high conservation of actin and tubulin in eukaryotic species reflects their central and essential roles in cellular structure and function. In contrast, the interacting proteins exhibit more sequence variability due to their diverse functions, evolutionary plasticity, and species-specific adaptations to meet the specific requirements of different organisms and cellular pathways.
How could a centrosome „sense“ when it is located in the „center“ of the cell?
The centrosome contains a pair of centrioles and serves as the main microtubule-organizing center (MTOC). Microtubules emanate from the centrosome and interact with various cellular structures. The dynamic nature of microtubules, influenced by polymerization and depolymerization, contributes to the positioning of the centrosome within the cell.
Kinesin-1 motos are highly processive, meaning they move along the tubulin tracks without dissociating.
In contrast, myosin II motors are not processive and dissociate after „a few steps“.
Think about their biological role, do these characteristics of the motor proteins make sense?
Kinesin-1:
Processivity: Highly processive, meaning it can move along microtubule tracks for long distances without dissociating.
Biological Role: Involved in anterograde transport, efficiently moving cellular cargoes, such as vesicles and organelles, along microtubule tracks to specific destinations within the cell.
Myosin II:
Lack of Processivity: Not highly processive and tends to dissociate after a few steps along actin filaments.
Biological Role: Involved in muscle contraction, cytokinesis, and other actin-based cellular activities. The lack of processivity allows myosin II to maintain control over force generation and localized interactions with actin filaments.
The distinct characteristics of kinesin-1 and myosin II motors are well-suited for their respective biological roles, reflecting the adaptability of motor proteins in facilitating diverse cellular functions with precision.
What are the functions of 3 different types of filaments
Intermediate filaments: * mechanical strength (large deformation without failure)
Microtubules: * determine positions for membrane enclosed organelles
* direct intracellular transport; cilia and flagella
* formation of mitotic spindle (chromosome segregation)
Actin filaments: * shape of a cells surface
* whole-cell locomotion
What is RNA editing?
alteration of RNA sequences after transcription, leading to changes in the information encoded by the DNA. Deamination, insertion/deletion
What are artificial modifications of mRNA used in medicine and transfection?
- Nucleotide methylation: increase stability and protein translation and decreases degradation and immunogenicity
- Codon optimization increases mRNA abundance and translation
- Nucleoside modification: increase stability, decrease degradation and immunogenicity
- Tail elongation: increase stability and protein translation
Cells are able to remodel the chromatin to make transcription happen. How do the cells manage this?
- Direct local alterations in chromatin structurethroug the reader writer complex: increaseaccessibility of DNA, facilitate binding of RNA pol and transcription factors. Nucleosome remodeling, nucleosome removal, histone replacement and certain types of histone modifications favor transcription.
Mechanical stress induces depolymerization of tubulin or of actin?
Actin
How cells sense?
This suggests that actin microfilaments are sensors of mechanical stretch in cells, and can form a feedback loop to control the mechanical tension of tissues.
Mzosinrings can sense small nanoscale structures
Actin associated motor proteins and functions in cells?
Motor proteins: movement of cell
myosin I -> all cells -> pull it to different shape
myosin II -> muscle cells:
- tail-tail interactions
- formation of bipolar
„thick filaments“
contraction of each myofibril (bundle of sarcomeres)
Keratin functions in cells and advantages of it as a biomaterial
Keratins: most diverse intermediate filament family
mechanical strength and integrity of tissues such as skin, hair, and nails. Its functions include providing structural support, maintaining epithelial tissue integrity, influencing cellular shape and rigidity, facilitating cellular adhesion,
can be used for the formation of hydrogel, microcapsules, sponges, nanofibers and films and is used in TE for wound healing, bone and cornea regeneration and drug delivery.
have an intrinsic ability to self-assemble and polymerize into porous, fibrous scaffolds
Explain Riboswitch
short sequences of RNA that change their conformation when bound to small molecules, like metabolites-> regulate gene expression. blocking or permitting progress of RNA polymerase. Eg. A riboswitch that responds to guanine. (A) In bacteria, the riboswitch controls expression of the purine biosynthetic genes. When guanine levels are low, an elongating RNA polymerase transcribes the purine biosynthetic genes, and the enzymes needed for guanine synthesis are therefore expressed. (B) When guanine is abundant, it binds the riboswitch, causing it to undergo a conformational change that forces the RNA polymerase to terminate transcription.
Name chemical Histone modifications and how they are applied and removed?
-acetylation can stimulate transcription initiation and the repressor simply reverses this modification
- methzlation:methylated histones are bound by proteins that maintain the chromatin in a transcriptionally silent form.
Mechanical stress induces depolymerization of …
Actin
Explain Actin Hydrogels and what can be researched with
Actin plays a role in muscle contraction, cellular movement. actin polymerization dynamics in nature vs. active gels, study on the formation of contractile fibers. Actin hydrogels are three-dimensional networks formed by the polymerization of actin monomers. Actin hydrogels allow the investigation of cellular mechanics, adhesion, and responses to mechanical cues.
Artifical phospholipid bilayer vesicle formed from an aqueous suspension of phospholipid molecules?
Liposome
Having both hydrophobic and hydrophilic regions, as in a phospholipid or detergent molecule?
Amphiphilic
The main type of phospholipid in animal cell membranes, with 2 fatty acids and a polar head group attached to a three-carbon glycerol backbone:
Phosphoglyceride
Lipid molecule with a characteristic four-ring steroid structure that is an important component of the plasma membranes of animal cells
Cholesterol
Glycolipids are never found on the cytoplasmic face of membranes in living cells?
True, they are synthesized
in the lumen of the golgi (outside)
Although lipid molecules are free to diffuse in the plane of the bilayer, they cannot flip-flop across the bilayer unless enzyme catalysts called phospholipid translocators are present in the membrane.
True, they can flip flop
but they need enzymatic help
What is meant by “two-dimensional fluid”?
The lipid bilayer can move, can change place with one another
Mention the transmembrane proteins
Amphiphilic: single alpha helix, multiple alpha helices, rolled up beta sheet, they are all covantly attached
Proteins exposed at one only side of the membrane: lipid linked (attached covalently bound lipid), oligosaccharide linked, by noncovalent interations (protein attachment), membrane associated (anchored)
What is the function of p-glycoprotein (P-gp) in normal cells?
ts primary function is to act as a transporter protein, and it is a member of the ATP-binding cassette (ABC) transporter family. The main function of P-glycoprotein in normal cells is to actively pump out a variety of substances from the inside of the cell to the outside, using energy derived from the hydrolysis of ATP. In cancer for an overexpression of ABC transporters the P-gp pumps the drugs out
What does P-gp look like?
Has two transmembrane domains (6alpha helix) and nucleotide binding domains forming catalytic domains, with ATP binding cassete
Which gene codes for human P-gp?
Multidrug resistance gene MDR1
Why are cancer researchers interested in P-gp?
because of its involvement in multidrug resistance within cancer cells. By expelling drugs from cancer cells, it diminishes drug efficacy, leading to resistance against various treatments.
From today‘s point of view, why was success of early clinical trials unlikely?
The early clinical did not consider patient MDR1 expression or potential involvement in
resistance to therapy.
* Gene expression profiling in AML samples showed that only a small percentage of samples
were positive for ABCB1 or ABCG2 expression, indicating that the trials were not targeting
the right patient subsets.
* The trials did not have appropriate tools for the characterization and clinical development
of MDR1 as a therapeutic target.
What are major problems with P-gp inhibitors in clinical trials?
The major problems are toxicity, medication interactions, and questions about clinical trial design.
Several phase III trials have been halted due to the toxicity of P-gp inhibitors, and the efficacy of
previously discovered P-gp inhibitors as potential co-therapeutics for MDR diseases have not been proven in clinical studies.
Which fourth generation inhibitors are currently being investigated?
Natural inhibitors, like dietary supplements, are being investigated as fourth-generation inhibitors. These natural substances and food extracts have been shown to have an influence on P-gp to reverse MDR and to have anticancer properties. Examples are curcumin, quercetin, piperine, capsaicin, and
limonin
Membrane proteins that are extend through the lipid bilayer are called?
Transmembrane and have hydrophobic regions that are exposed to the interior of the bilayer
Other proteins are _________ attached to lipid molecules that are inserted in the membrane
covalent
Membrane proteins are linked to the membrane through noncovalent interactions with other membrane bound proteins
Integral
Animal cells drive most transport processes across the membrane with the electrochemical gradient of
Na+
A transporter mediating passive transport switches between…
random, irreversible and independent of binding state
How do you suppose that proteins with a nuclear export signal get into the nucleus?
It also have a nuclear localization signal, that the import recognized
How is it that a single nuclear pore complex can efficiently transport proteins that possess
different kinds of nuclear localization signals?
importins are capable of recognizing different NLS motifs
Nuclear localization signals are not cleaved off after the transport into the nucleus, whereas the signal sequences for import into other organelles are often removed after import. Why do you suppose it is critical that nuclear localization signals remain attached to their proteins?
Retention of the protein inside the nucleus
If the enzyme dihydrofolate reductase (DHFR), which is normally located in the cytosol, is engineered to carry a mitochondrial targeting sequence at its N-terminus, it is efficiently imported into mitochondria. If the modified DHFR is first incubated with methotrexate, which binds tightly to the active site, the enzyme remains in the cytosol. How do you suppose that the binding of methotrexate interferes with mitochondrial import?
As the N-terminal sequence is binding to the methotrexate the TOM complex can not recognize the sequence so the protein can not be translocated inside of the mitochondria
Compare and contrast protein import into the ER and into the nucleus. List at least 2 major
differences and speculate on why the nuclear mechanism might not work for ER import and viceversa.
Nucleus has NLS meanwhile ER has SRP
Translocation into the ER is co-translational, meaning that the protein is translocated across the ER membrane as it is being synthesized by the ribosome.
Translocation into the nucleus is generally post-translational.
Nuclear pores always open, translocation channels most of the time closed
Why might it be advantageous to add a preassembled block of 14 sugars to a protein in the ER,
rather than building the sugar chains step by step on the surface of the protein by the sequential addition of sugars by individual enzymes?
Is faster, speeds up the glycosylation process, is more controlled
If a lysosome breaks what protects the rest of the cell from the lisosome enzymes?
The ph level, inside of the lysosome is acidic. The enzymes are only active in the pH-level of the lysosome.
Using genetic techniques you create a set of proteins that contain two conflicting signals, which would win for import into the nucleus or into the ER?
This is dependent on various factors (Signal Strength, Protein Folding etc). The Signal peptide for the ER is important for the translocation of the mRNA+Ribosome complex to the ER. After the translation is completed the signal peptide is cleaved off. However, the nuclear import signal is still on the protein. Hence, the mature protein with the nuclear signal sequence would get translocated again in the nucleus and stay there. NLS is not cleaved off in the nucleus!
Most abundant ion inside a mammalian cell
K+
Where are located SPR receptor, SPR protein and proteins translocator
ER membrane, sytosol, ER lumen
Some genetically modified protein with two signal sequences. One for export of the nucleus and one for import into the mitochondria. Where would the protein end up ?
The mature protein has the ER signal peptide cleaved off so only the signal for the mitochondria (MTS) remains. However, for the import to the mitochondria the protein needs to be unfolded. So the protein is unfolded (still with the MTS attached) and then translocated into the mitochondria. Then in the mitochondria the MTS is cleaved off and the protein folds back to its functional conformation. And as we know a folded protein can not pass through the mitochondrial double membrane ( Its the only protein transport where the protein needs to be unfolded).
You wanna make an RNA strand more stable. HOW?
Poly A Tail, G-CAP (Methylation), Alternative Splicing
Describe what the function of these different RNAs is:
Messenger RNA: DNA->mRNA, copying of the genetic information into an RNA to later translate it to proteins. Also has modifications (Poly A Tail and G-Cap Methylation)
Ribosomal RNA: Is crucial for translation and acts as an integral part of the ribosome.
transferRNA: Is needed for the translation of the proteins. Carries the anticodon to the wanted RNA. Creates a polypeptide chain.
small interfering RNA: non-coding RNA. Used to silence genes.
You wanna cause an RNA strand to achieve a higher expression. HOW?
Knock in
Promoter selection (Strong and well characterized)
Enhancer (Introduce enhancer elements, increase transcriptional activity)
What is a symport?
Active coupled transport
It’s a transport mechanism where two molecules are passed through the membrane at the same time.
Why and how do you do differential centrifugation?
To separate cell components with different molecular weights.
Several centrifugation steps with various speeds and durations. After each of them you continue centrifuging the supernatant until you have your desired components.
ER proteins and nuclear proteins are never intermixed with nuclear proteins?
True
Which types of extracellular vesicles do you know?
Microvesicles
Apoptotic bodies
Exosomers
How can EVs be isolated? With the help of …
Kits, ultrafiltration, differential centrifugation, immunoafinitz capture
Engineering of EVs for use as therapeutic delivery vehicles is a promising tool in …
Personaliyed medicine
Which cell type plays a major role in therapeutic strategies using EVs for tissue
repair?
Stem cells
Liposome vs vesicles
Liposomes: Synthetic lipid vesicles.
EVs: Natural vesicles released by cells.
Source:
Liposomes: Lab-synthesized.
EVs: Naturally derived from cells.
Biological Properties:
Liposomes: May be recognized by the immune system.
EVs: Better biocompatibility, with cell-specific markers.
Cargo Delivery:
Liposomes: Encapsulate diverse payloads.
EVs: Naturally carry biomolecules; can be engineered for specific cargo.
Targeted Delivery:
Liposomes: Require surface modifications for targeting.
EVs: Inherently express cell-specific markers for targeted delivery.
Biological Signaling:
Liposomes: Lack intrinsic biological signaling.
EVs: Actively participate in intercellular communication.
Immunomodulation:
Liposomes: May induce immune responses.
EVs: Can have immunomodulatory effects.
application of EVs for bone tissue engineering:
For regeneration of: bone fractures, common age related disorders (e.g. osteoporosis)
- EVs deliver diverse biological cargo (nucleic acids, proteins, bioactive molecules)
- EVs isolated from Osteoblasts promote MSCs to differentiate into osteoblasts
- Immigrating MSCs into the bone defect secrete large amounts of EVs, and it was shown that MSC-EV-based approaches have the potential to promote bone regeneration
- EVs also stimulate angiogenesis (by carrying pro-angiogenic factors)
Challenges:
- EVs to this date commonly harvested from cells cultured in 2D conditions
- > does not applicate in situ conditions (limited surface for cell growth, artificial cell-matrix interactions)
- Limitation on scaffold architecture impact the reproducibility of EVs
- extensive processing if from non-porous materials
Limitations of organoids
Vasculariyation, mimicking complex tissue structures, potential genetic instabilitz, challenges in standariyation, variabilitz between batches, limited immune response representation
How help control organoid formation
Using specific growth factors, incorporating biomaterials or scaffolds techniques, microenvironment engineering, genetic manipulation
CAR stands for:
chimeric antigen receptor. Its modular design consists of a variable region of heavy chain and light chain, a transmembrane domain and 2 intracellular domains: one is zeta chain for activation and the other is costimulatory domain, which is either CD28 or 41BB.
CAR T-cell Therapy Steps:
a. Selection and Collection: A patient’s own T cells are collected through a process called apheresis.
b. Engineering: Collected T-cells are then genetically modified to express CAR.
c. Expansion: The genetically modified T cells are cultured and allowed to multiply in number, which are then frozen and sent to the hospital.
d. Conditioning Therapy: Before CAR T-cell infusion, patients undergo a conditioning treatment: chemotherapy
e. CAR T-cell Infusion: The engineered and expanded CAR T cells are infused back into the patient.
f. Monitoring: Patients are monitored for potential side effects and to evaluate the disease response to CAR T-cell therapy as well as the persistence of the modified T cells.
Most Important Side Effects of CAR T-cell Therapy:
Cytokine Release Syndrome and Toxicity that could be either neurological (Paralysis, Speech …) or Off-target toxicity.
G protein that activates adenylyl cyclase and thereby increases cyclic AMP concentration…
Stimulatory G protein (Gs)
Protein composed of 3 subunits, one of which is activated by the binding of GTP.
Trimeric GTP-binding protein (G protein)
Cell surface receptor that associates with an intracellular G protein upon activation by an extracellular ligand.
G-protein coupled receptor (GPCR)
Second messenger that is released from a phospholipid in the plasma membrane and diffuses to the ER, where it opens Ca2+ release channels.
Inositol 1,4,5-triphosphate (IP3)
Cell surface receptors that, when activated by ligand binding, add phosphates from ATP to tyrosine side chains in their own cytoplasmic domain.
Receptor tyrosine kinase (RTK)
The founding member of a superfamily of monomeric GTPases that help to relay signals from cell-surface receptors to the nucleus.
Ras
A group of monomeric GTPases that regulate both the actin and microtubule cytoskeletons.
Rho family
A kinase that is involved in intracellular signaling pathways activated by cell surface receptors and that phosphorylates inositol phospholipids at the 3 position of the inositol ring.
Phosphoinositide 3-kinase (PI 3-kinase)
A three-component signaling module used in various signaling pathways in eukaryotic cells.
MAP kinase module
A crucial signaling protein in the PI-3-kinase-Akt signaling pathway, so named because it is the target of rapamycin.
Tor or mTor
If the most basic function of the cell cycle is to duplicate accurately the DNA in the chromosomes and
then distribute the copies precisely to the daughter cells, why are there gaps between S phase and M
phase?
The gaps between S phase and M phase in the cell cycle (G1 and G2 phases) serve as critical checkpoints. These gaps allow the cell to ensure that conditions are favorable for progression to the next phase, and they provide opportunities for the cell to assess its readiness for DNA synthesis and mitosis. G1 phase, in particular, acts as a checkpoint where the cell evaluates its size, nutrient availability, and overall health before committing to DNA synthesis in the S phase. G2 phase similarly assesses the accuracy of DNA replication and prepares the cell for mitosis.
Why do you suppose cells have evolved a special G0 state to exit the cell cycle, rather than just stopping in G1 at a G1 checkpoint?
The G0 state is a non-dividing state that cells can enter from G1, and it represents a reversible exit from the cell cycle. Cells enter G0 when conditions are not suitable for division or when they receive signals indicating that cell division is not needed. This state allows cells to conserve energy and resources. The decision to enter G0 instead of stopping at the G1 checkpoint provides the cell with the flexibility to re-enter the cell cycle when conditions become favorable. It is a more dynamic and adaptable strategy for responding to environmental changes.
What are carcinomes?
Carcinomas are types of cancer that typically originate from epithelial cells, such as those lining the organs or tissues. Fibroblasts, inflammatory cells, and blood vessels are not derived from the cancer cell population itself. Instead, they may be recruited or influenced by the cancer cells, creating a complex microenvironment known as the tumor stroma. The components of the tumor stroma play roles in supporting the growth and progression of the carcinoma, but they do not evolve directly from the cancer cell population.
Mortality due to lung cancer was followed in groups of males in the United Kingdom for 50 years.
Figure 1 shows the cumulative risk of dying from lung cancer as a function of age and smoking habits
for four groups of males: those who never smoked, those who stopped at age 30,
those who stopped at age 50, and those who continued to smoke. These data show clearly that
individuals can substantially reduce their cumulative risk of dying from lung cancer by stopping
smoking. What do you suppose is the biological basis for this observation?
when an individual stops smoking, the body’s natural repair mechanisms come into play. The respiratory system has the capacity to regenerate and repair damaged cells, allowing for the gradual restoration of normal cellular function.
The loss of p53 protein makes some cancer cells much less sensitive to irradiation and to many anticancer drugs, which would otherwise destroy tumors by inducing proliferating cells to either stop
dividing or undergo apoptosis?
true. The loss of the p53 protein in some cancer cells can make them less sensitive to irradiation and many anticancer drugs. p53 is a tumor suppressor protein that plays a crucial role in regulating the cell cycle and preventing the formation of tumors. When cells with damaged DNA are detected, p53 can halt the cell cycle, allowing time for DNA repair, or induce apoptosis (programmed cell death) if the damage is irreparable.
Overexpression of the Myc protein is a common feature of many types of cancer cells, contributing to their excessive cell growth and proliferation. By contrast, when Myc is overexpressed in most normal
cells, the result is not excessive proliferation, but cell-cycle arrest or apoptosis. How do you suppose that overexpression of Myc can have such different outcomes in normal cells and in cancer cells?
In normal cells, Myc is tightly regulated, and its expression is controlled to prevent uncontrolled cell growth.
Overexpression of Myc in normal cells can lead to a response known as oncogene-induced senescence or apoptosis, acting as a safeguard mechanism against potential cancer development.
