Molecular Cell Biology II

The Molecular Cell Biology II course encompasses several medically important aspects of cell biology such as signal transduction, cell cycle and apoptosis, which are essential to understand the molecular background of a range of common diseases including cancer and neurodegeneration. The subcellular biochemistry module highlights the physiological and pathological role of intracellular organelles. Finally, an insight is provided into cutting-edge methods currently used in cell biology research.

It is obligatory to participate in the lectures, while no lab lessons or seminars will be held. During consultations students can ask their questions with regard to the material of corresponding lectures. The oral final examination is based on the whole material of both semesters of Molecular Cell Biology. Students take one topic from each chapter of the topic list (i.e. four topics in total). Lab topics are not clustered into a separate chapter but evenly distributed among the rest of topics.

Students might participate on a voluntary basis in a midterm exam held in week 10. This is an oral exam based on the lectures of weeks 1-8. Midterm topics correspond to those of chapter 3 of the topic list for the final exam (signalling, intracellular signals, cell cycle, apoptosis, aging). Students obtaining a grade 4 or better will be exempted from taking topics from chapter 3 on their final exam. Details on the midterm will be announced in due course.

I.

1. The chemical structure of nucleotides. The primary and secondary structure of nucleic acids (DNA and RNAs)
2. Condensation of the genetic material in pro- and eukaryotic cells. The role of topoisomerases and chromatin proteins
3. The structure of human chromosomes and its alterations during the cell cycle
4. The structure of the human genome: coding and gene regulatory sequences. Non-coding genomic sequences: introns, pseudogenes, repetitive sequences
5. The role of genetic variations in the pathogenesis of diseases. Methods to study genetic factors
6. Principles of semiconservative DNA replication: replication fork, leading and lagging strand synthesis
7. Comparison of DNA replication in pro- and eukaryotic cells: principles, enzymes, proteins
8. Telomeric repeat sequences: replication of the telomeric regions of eukaryotic chromosomes. Functions and importance of the telomerase
9. The most important types of DNA damage. Repair of base deamination
10. Formation and repair of thymine dimers. Mismatch repair
11. Classification of point mutations. The origin of spontaneous point mutations. DNA polymorphisms. Possible effects of DNA sequence variations on the corresponding mRNA and proteins
12. Structure and function of the prokaryotic RNA polymerase holoenzyme. Transcriptional initiation and termination in bacteria; the prokaryotic transcription unit
13. The different types of RNA and their biological importance. Synthesis and maturation of rRNAs and tRNAs
14. Regulation of transcription in prokaryotes. Strong and weak promoters, constitutive genes, operons, positive and negative regulation
15. Structure of eukaryotic genes. Transcriptional initiation and termination in eukaryotic cells
16. Regulation of transcription in eukaryotes. Specific transcription factors, cis- and trans-acting regulatory sequences, coactivators, corepressors
17. Processing of eukaryotic mRNAs
18. The polymerase chain reaction and real-time PCR: principles and fields of application
19. Genotyping of mutations and polymorphisms by RFLP, allele specific PCR, DNA sequencing and primer extension. Genotyping of the TAS2R38 taste receptor by PCR-RFLP
20. Restriction endonucleases and their biotechnological importance. Digestion of the pGL3 plasmid by restriction endonucleases and analysis of DNA fragments by agarose gel electrophoresis
21. Construction of recombinant DNA by cloning. Reporter and expression vectors
22. Genetic engineering and its biomedical importance (transgenic animals; knock-out, knock-in and knock-down techniques; cloning)

II.

1. Regulation of eukaryotic gene expression at post-transcriptional levels (alternative splicing, RNA editing, regulation of RNA stability, RNA quality control)
2. Regulation of eukaryotic gene expression by RNA interference (miRNA, siRNA)
3. Epigenetic regulation of eukaryotic transcription: the role of DNA methylation and histone modifications
4. The genetic code. Structure and function of tRNAs; aminoacyl-tRNA synthetases; the codon-anticodon hybridisation
5. The structure of prokaryotic and eukaryotic ribosomes. The ribosome cycle. Binding of tRNA to ribosomes
6. Initiation of translation in pro- and eukaryotic cells. Regulation of eukaryotic translation. The role of phosphorylation of eIF2α
7. Elongation and termination of pro- and eukaryotic translation. Pharmacological inhibitors of translation
8. Post-translational modifications of proteins
9. Structure, synthesis and isoforms of collagen (the importance of glycine and proline residues; procollagen, tropocollagen, collagen fibres, hydroxylation, crosslinking)
10. Analysis of proteins I.: reversible and irreversible precipitation, detection of the peptide bond, quantitative determination of proteins by the biuret reaction. Detection of thiol groups using the Ellman’s reaction
11. Analysis of proteins II.: chromatography methods (gel filtration)
12. Analysis of proteins III.: SDS-polyacrylamide gel electrophoresis and western blot
13. Analysis of gene expression in vitro by real-time PCR and DNA microarray
14. Analysis of prokaryotic gene expression in vivo: induction of β-galactosidase in E. coli
15. Proteostasis. Intracellular protein degradation
16. The structure and function of proteasomes and the immunoproteasome. Function of the TAP complex. Proteasome inhibitors
17. The different types of autophagy. The role of lysosomes
18. Lytic and lysogenic cycles of bacteriophages. The role of the phage repressor
19. Classification of animal viruses according to their replication mechanisms. Structure and replication of retroviruses
20. Principles of human gene therapy (in vivo vs. ex vivo; viral vs. non-viral; gene augmentation; targeted genome editing with the CRISPR/Cas9 system)

III.

1. Transcription factors of the nuclear receptor superfamily (steroid, thyroid, retinoid receptors)
2. Classification and signalling of cell surface receptors
3. Classification, signalling mechanisms and regulation of G-proteins. The most important effector proteins of G_s, G_i, G_t, G_q and ras
4. Activation mechanisms of serine/threonine specific protein kinases with examples
5. cAMP signalling. Regulation of gene epression by cAMP
6. Phosphatidylinositol signalling pathways
7. NFκB and TGFβ signalling
8. Structure and signalling of tyrosine kinase receptors. The mechanism of ras activation. Structure and function of the Erkl/Erk2 MAP kinase cascade
9. Insulin receptor signalling
10. The role and functioning of mTOR, a principal integrator of intracellular signals
11. The role of AMPK in metabolic regulation and autophagy
12. Cellular oxygen sensing and signalling mechanisms
13. Chronobiochemistry: the function of the central oscillator and the role of the circadian clock in human pathology
14. Chronobiochemistry: the role and integration of afferent and efferent pathways
15. Regulation of the cell cycle in G₁ and S phases
16. Regulation of the cell cycle in G₂ and M phases
17. Molecular sensors detecting DNA damage and completion of DNA replication during the cell cycle
18. Structure and function of the apoptosome, DISC and PIDDosome complexes
19. Members of the Bcl-2 superfamily and their roles in various apoptotic pathways
20. Characterisation of caspases and their role in the regulation of apoptosis
21. Regulation of p53 levels and activity. The role of p53 and the „survival signal” in determining cell fate
22. Definition and hallmarks of ageing. Molecular mechanisms in ageing (cellular senescence and nutrient-sensing signalling pathways)

IV.

1. Organising principles of eukaryotic cells; compartmentation; main features of subcellular organelles
2. The biogenesis of organelles and its regulation
3. Components of the cytoskeleton
4. Structure and function of motor proteins
5. The protein secretory pathway. The role of the rab cycle in the regulation of vesicular transport
6. Endo- and exocytosis
7. Formation of the proteome of subcellular organelles. Principles and mechanisms of protein sorting and targeting
8. Protein targeting in the secretory pathway
9. Protein targeting into mitochondria and peroxisomes. Uptake of substrates into lysosomes
10. Nuclear import and export of macromolecules
11. The organelle stress concept. Peroxisomal and mitochondrial stress
12. Endoplasmic reticulum stress, unfolded protein response (UPR), endoplasmic reticulum stress-induced apoptosis
13. Protein quality control in the endoplasmic reticulum. The fate of misfolded proteins; ERAD
14. Characteristics of the proteome of subcellular organelles
15. Specific features and maintenance of the internal milieu of subcellular organelles
16. The role of the extracellular matrix in signal transduction, drawing on the example of integrin receptors (molecular background of inside-out signalling; the importance of α and β subunits; peculiarities of focal adhesion kinase signalling)
17. The role of the extracellular matrix in cancer dissemination (epitelial-mesenchymal transition and the molecular background of local invasion, migration and extravasation of metastatic cells)
18. Biological networks: the importance of small-worldness, nodes, network groups and network hierarchy in protein-protein and metabolic, signalling, gene expression and organelle networks
19. Methods in cell biology I.: cell cultures, cell fractionation
20. Methods in cell biology II: in vivo microscopy, flow cytometry, FACS