Cell Biology & Advanced Molecular Biology

Part I — Advanced Cell Biology

1. Cellular organization and compartmentalization
   • Evolution of the eukaryotic cell
   • Prokaryotic vs eukaryotic organization
   • Organelles and functional compartmentalization
   • Protein targeting and sorting
   • Cellular microdomains
   • Membrane-bound vs membraneless organelles
   • Phase separation and biomolecular condensates
2. Biological membranes
   • Fluid mosaic model—modern interpretation
   • Membrane lipid composition
   • Lipid rafts and membrane asymmetry
   • Membrane proteins
   • Membrane curvature and remodeling
   • Membrane fusion and fission
   • Vesicular transport
3. Membrane transport and cellular trafficking

• Simple and facilitated diffusion
• Ion channels
• ABC transporters
• Primary and secondary active transport
• Na⁺/K⁺-ATPase
• Ca²⁺ pumps
• Endocytosis
• Exocytosis
• Clathrin-mediated trafficking
• COPI and COPII vesicles
• Rab GTPases
• SNARE proteins

4. Cytoskeleton and cellular architecture
   • Actin cytoskeleton
   • Microtubules
   • Intermediate filaments
   • Molecular motors
   • Kinesin and dynein
   • Myosin
   • Centrosomes and microtubule-organizing centers
   • Cell polarity
   • Cytoskeletal regulation
   • Mechanotransduction
5. Cell junctions and extracellular matrix
   • Tight junctions
   • Adherens junctions
   • Desmosomes
   • Hemidesmosomes
   • Gap junctions
   • Integrins
   • Collagen
   • Fibronectin
   • Laminins
   • ECM remodeling
   • Matrix metalloproteinases
6. Cell signaling
   • Receptor–ligand interactions
   • GPCR signaling
   • Receptor tyrosine kinases
   • JAK–STAT signaling
   • Ras–MAPK pathway
   • PI3K–AKT–mTOR
   • Wnt/β-catenin
   • TGF-β signaling
   • Hedgehog signaling
   • Notch signaling
   • Calcium signaling
   • cAMP and protein kinase cascades
   • Signal amplification and termination
   • Crosstalk between pathways
7. Cell cycle and its molecular regulation

• G1, S, G2 and M phases
• Cyclins and CDKs
• Restriction point
• DNA-damage checkpoints
• Spindle assembly checkpoint
• APC/C
• SCF complex
• Cyclin degradation
• Mitotic spindle
• Chromosome segregation
• Cytokinesis
• Cell-cycle dysregulation in cancer

8. Apoptosis and programmed cell death
   • Intrinsic pathway
   • Extrinsic pathway
   • Caspases
   • Bcl-2 family
   • Cytochrome c
   • Apoptosome
   • Death receptors
   • Necroptosis
   • Pyroptosis
   • Ferroptosis
   • Autophagy-associated cell death
   • Crosstalk between cell-death pathways
9. Autophagy
   • Macroautophagy
   • Microautophagy
   • Chaperone-mediated autophagy
   • Autophagosome formation
   • ATG proteins
   • ULK complex
   • Beclin-1/VPS34
   • LC3 processing
   • Lysosomal fusion
   • Autophagy in cancer and neurodegeneration
10. Mitochondrial biology
    • Mitochondrial genome
    • Endosymbiotic origin
    • Electron transport chain
    • Oxidative phosphorylation
    • Mitochondrial dynamics
    • Fusion and fission
    • Mitophagy
    • ROS generation
    • Mitochondrial apoptosis
    • Mitochondrial inheritance
11. Endoplasmic reticulum and Golgi apparatus
    • ER protein folding
    • Chaperones
    • ER quality control
    • Unfolded protein response
    • ER-associated degradation
    • Golgi cisternal organization
    • Glycosylation
    • Protein trafficking
    • Lysosomal targeting
    • Mannose-6-phosphate pathway
12. Nucleus and nuclear organization
    • Nuclear envelope
    • Nuclear pore complex
    • Nucleocytoplasmic transport
    • Importins/exportins
    • Ran-GTP cycle
    • Nuclear lamina
    • Nuclear bodies
    • Nucleolus
    • Chromosome territories

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Part II — Advanced Molecular Biology

13. DNA structure and genome organization

• DNA topology
• Supercoiling
• Linking number, twist and writhe
• Topoisomerases
• Nucleosomes
• Histones and histone variants
• Higher-order chromatin
• Euchromatin and heterochromatin
• Chromosome territories
• CTCF and chromatin loops
• Cohesin
• TADs
• 3D genome organization

14. DNA replication

• Replication origins
• Origin licensing
• Pre-replication complex
• Helicases
• Primases
• DNA polymerases
• Leading and lagging strand synthesis
• Okazaki fragments
• Sliding clamps
• PCNA
• Replication fork dynamics
• Telomeres
• Telomerase
• Replication stress
• Fork protection and restart

15. DNA damage and repair

Major pathways:

DNA lesion	Major repair pathway
UV-induced pyrimidine dimers	Nucleotide excision repair
Deaminated bases	Base excision repair
Replication mismatches	Mismatch repair
Double-strand breaks	HR / NHEJ
Interstrand crosslinks	Fanconi pathway
Oxidative DNA damage	BER

Advanced topics:

• DNA damage sensors
• ATM and ATR
• CHK1 and CHK2
• p53
• Homologous recombination
• Non-homologous end joining
• Alternative end joining
• Synthetic lethality
• PARP inhibition
• DNA repair and cancer

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16. Transcriptional regulation

Eukaryotic transcription

• RNA polymerase I
• RNA polymerase II
• RNA polymerase III
• Promoters
• Enhancers
• Silencers
• Insulators
• General transcription factors
• Mediator complex
• Transcription factor binding
• Chromatin remodeling
• Histone modifications
• Transcriptional bursting
• Enhancer–promoter communication

Important concepts

Cis-regulatory elements

• Promoters
• Enhancers
• Silencers
• Insulators

Trans-regulatory factors

• Transcription factors
• Coactivators
• Corepressors
• Chromatin-remodeling complexes

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17. RNA biology

This should be a major Master’s-level section.

RNA classes

• mRNA
• rRNA
• tRNA
• miRNA
• siRNA
• piRNA
• lncRNA
• circRNA
• snoRNA
• snRNA
• enhancer RNA

RNA processing

• 5′ capping
• Polyadenylation
• Splicing
• Alternative splicing
• RNA editing
• RNA modification
• mRNA export
• RNA stability
• RNA localization

RNA modifications

• m⁶A
• m⁵C
• pseudouridine
• RNA editing

RNA surveillance

• Nonsense-mediated decay
• Non-stop decay
• No-go decay
• Exosome-mediated RNA degradation

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18. Translation and translational regulation

Major topics

• Ribosome structure
• Ribosomal biogenesis
• tRNA charging
• Aminoacyl-tRNA synthetases
• Initiation
• Elongation
• Termination
• Ribosome recycling
• Polysomes
• Translational control
• eIF2 regulation
• mTOR signaling
• IRES-mediated translation
• Stress granules
• Ribosome quality control

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19. Protein folding and quality control

• Primary → quaternary structure
• Molecular chaperones
• Hsp70
• Hsp90
• Chaperonins
• Protein folding pathways
• Misfolding
• Aggregation
• Ubiquitination
• Proteasome
• ER quality control
• Unfolded protein response
• Protein degradation
• Proteostasis

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20. Epigenetics

DNA methylation

• CpG islands
• DNA methyltransferases
• DNMT1
• DNMT3A
• DNMT3B
• TET enzymes
• 5-methylcytosine
• 5-hydroxymethylcytosine

Histone modifications

• Acetylation
• Methylation
• Phosphorylation
• Ubiquitination
• SUMOylation

Epigenetic regulation

• Writers
• Erasers
• Readers
• Chromatin remodeling
• Genomic imprinting
• X-chromosome inactivation
• Epigenetic memory
• Epigenetics in cancer

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21. Non-coding RNA and gene regulation

• miRNA biogenesis
• Drosha
• Dicer
• RISC
• Argonaute
• siRNA
• piRNA
• lncRNA
• circRNA
• RNA-mediated gene silencing
• ceRNA concept
• RNA–protein interactions

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22. Recombinant DNA technology

Core techniques

• Restriction digestion
• DNA ligation
• Plasmid cloning
• Transformation
• Selection and screening
• Blue-white screening
• Expression vectors
• Reporter genes
• Fusion proteins

Modern cloning

• Gibson assembly
• Golden Gate assembly
• Gateway cloning
• Seamless cloning

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23. PCR and advanced nucleic-acid techniques

PCR

• Conventional PCR
• RT-PCR
• qPCR
• RT-qPCR
• Multiplex PCR
• Nested PCR
• Digital PCR
• Allele-specific PCR
• Long-range PCR

Quantitative PCR

• Ct/Cq
• Standard curves
• ΔCt
• ΔΔCt
• Reference genes
• Absolute vs relative quantification
• PCR efficiency

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24. DNA sequencing

First generation

• Sanger sequencing

Next-generation sequencing

• Illumina sequencing
• Sequencing by synthesis
• Library preparation
• Adapter ligation
• Indexing
• Read depth
• Coverage
• Paired-end sequencing

Third-generation sequencing

• PacBio
• Oxford Nanopore
• Long-read sequencing
• Single-molecule sequencing

Applications

• Whole-genome sequencing
• Whole-exome sequencing
• Targeted sequencing
• RNA-seq
• ChIP-seq
• ATAC-seq
• Single-cell sequencing

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25. CRISPR and genome engineering

This should be treated as a core Master’s-level topic.

CRISPR-Cas systems

• CRISPR arrays
• crRNA
• tracrRNA
• Cas proteins
• PAM recognition
• Target cleavage

Genome editing

• Cas9
• Cas12
• Cas13
• sgRNA
• HDR
• NHEJ
• Knockout
• Knock-in
• Base editing
• Prime editing

Advanced concepts

• Off-target effects
• Guide-RNA design
• High-fidelity Cas variants
• CRISPR screens
• CRISPR interference
• CRISPR activation
• Functional genomics

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26. Molecular biology of cancer

• Oncogenes
• Tumor suppressor genes
• Proto-oncogenes
• p53
• RB
• MYC
• RAS
• BRAF
• PI3K
• PTEN
• APC
• BRCA1/BRCA2
• Telomerase
• Genomic instability
• Epigenetic alterations
• Tumor microenvironment
• Cancer stem cells
• Metastasis
• EMT
• Precision oncology

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27. Stem-cell biology

• Stem-cell hierarchy
• Self-renewal
• Differentiation
• Potency
• Totipotency
• Pluripotency
• Multipotency
• Embryonic stem cells
• Adult stem cells
• Induced pluripotent stem cells
• Reprogramming
• Yamanaka factors
• Organoids
• Tissue regeneration
• Stem-cell niche
• Epigenetic reprogramming

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28. Single-cell and spatial molecular biology

Single-cell technologies

• scRNA-seq
• scATAC-seq
• Single-cell DNA sequencing
• Single-cell proteomics
• Cell clustering
• Dimensionality reduction
• UMAP
• t-SNE
• Cell-type identification
• Trajectory analysis

Spatial biology

• Spatial transcriptomics
• In situ sequencing
• Multiplex imaging
• Spatially resolved gene expression

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29. Molecular biology experimental techniques

A Master’s student should understand not merely what the technique does, but why each step is performed and how to interpret the result.

Protein techniques

• SDS-PAGE
• Native PAGE
• Western blotting
• ELISA
• Immunoprecipitation
• Co-IP
• Pull-down assays
• Immunofluorescence
• Flow cytometry
• Mass spectrometry

DNA techniques

• Agarose gel electrophoresis
• Southern blotting
• Restriction mapping
• DNA sequencing
• DNA footprinting
• EMSA

RNA techniques

• Northern blotting
• RT-PCR
• qPCR
• RNA-seq
• RNA immunoprecipitation

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30. Advanced microscopy and imaging

• Bright-field microscopy
• Phase contrast
• DIC
• Fluorescence microscopy
• Confocal microscopy
• TIRF
• Super-resolution microscopy
• STED
• PALM
• STORM
• Live-cell imaging
• FRAP
• FRET
• Calcium imaging

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31. Proteomics and systems biology

Proteomics

• Bottom-up proteomics
• Top-down proteomics
• LC-MS/MS
• Label-free quantification
• SILAC
• TMT
• Protein interaction networks

Systems biology

• Gene regulatory networks
• Protein–protein interaction networks
• Metabolic networks
• Network centrality
• Systems-level modeling
• Multi-omics integration

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32. Bioinformatics for molecular biology

A Master's-level course should include practical bioinformatics.

Sequence analysis

• BLAST
• Multiple sequence alignment
• Phylogenetic analysis
• ORF identification
• Motif analysis

Genome analysis

• Genome browsers
• Variant calling
• SNP analysis
• Structural variants
• Annotation

RNA-seq

• Read quality assessment
• Alignment/pseudoalignment
• Transcript quantification
• Differential expression
• Gene ontology
• Pathway enrichment

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33. Molecular evolution

• Mutation
• Genetic drift
• Natural selection
• Molecular clocks
• Neutral theory
• Gene duplication
• Horizontal gene transfer
• Genome evolution
• Comparative genomics
• Molecular phylogenetics

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34. Advanced laboratory methods: experimental design

A strong Master's-level course should end with experimental reasoning, not just memorization.

Students should learn:

1. How to formulate a molecular hypothesis.
2. How to select an appropriate experimental model.
3. How to identify positive and negative controls.
4. How to select biological vs technical replicates.
5. How to distinguish correlation from causation.
6. How to quantify experimental results.
7. How to determine statistical significance.
8. How to identify experimental artifacts.
9. How to reproduce an experiment.
10. How to interpret contradictory results.

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Recommended examination framework

For each chapter, the notes can follow this structure:

1. Learning objectives

2. Core concepts

3. Molecular mechanism

4. Step-by-step pathway

5. Key proteins/genes

6. Experimental techniques

7. Experimental interpretation

8. Clinical/biomedical significance

9. Research applications

10. Conceptual diagrams

11. Master's-level MCQs

12. Short-answer questions

13. Long-answer questions

14. Problem-based questions

15. Viva questions

16. Research-paper discussion

17. Key molecules to remember

18. High-yield examination points

Suggested complete course structure

Module	Major area
I	Cell organization & membranes
II	Organelles & intracellular trafficking
III	Cytoskeleton & cell junctions
IV	Cell signaling
V	Cell cycle & cell death
VI	Mitochondria, ER & Golgi
VII	Genome organization
VIII	DNA replication
IX	DNA repair
X	Transcription
XI	RNA biology
XII	Translation
XIII	Epigenetics
XIV	Recombinant DNA technology
XV	PCR & sequencing
XVI	CRISPR/genome editing
XVII	Cancer molecular biology
XVIII	Stem-cell biology
XIX	Omics & single-cell biology
XX	Proteomics & systems biology
XXI	Bioinformatics
XXII	Experimental molecular biology