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ADVANCED CELL BIOLOGY

             
Compulsory/Elective Code Semester Lectures Practicals Credits ECTS
Elective 13B031 6th 4 Hrs/Wk  3 Hrs/Wk 5 6,5
Aims:
 
 

The course deals with concepts related to the way in which fundamental cellular structures, such as membranes, cytoskeleton and organelles, are formed and operate. It examines and analyzes fundamental cellular processes, including protein expression, signal transduction, self-assembly, lateral organization and vesiculation of the membrane, molecular machines, aging, programmed cell death and their regulation. It studies diseases associated with the cell membrane and organelles, giving emphasis to carcinogenesis. At the same time, students will be acquainted to the research methodology (e.g. Confocal Laser Scanning Microscope-CLSM, Electron Energy Loss Spectroscopy-EELS, Pseudo-coloring-3D Electron-Micrograph Processing, Immuno-Electron Microscopy, Cryo-techniques, Atomic Force Microscope-AFM, Cell Cultures, In Situ Hybridization and TUNEL Assay, fluorescence recovery after photobleaching (FRAP)), and they will be able to apply, combine and analyze the results obtained by these techniques.

 
Objectives:
 
 

By the end of the lectures and the laboratory exercises students are expected to: a) distinguish the various types of cellular membranes based on differences in structure, molecular composition and topology, b) understand the inter-dependence of molecular composition and function at the level of cellular structures, c) know the mechanisms involved in the import of proteins into the various cellular organelles, and in the transit and biogenesis of lipids, d) be able to describe complex cellular mechanisms and the way by which their perturbations lead to disease in human, e) be able, at the laboratory level, to choose, combine and apply conventional and modern cellular biology techniques, such as Cell Culture, Scanning Electron Microscopy, Protein Immuno-Assays/Immunoblotting, Immuno-Electron Microscopy, in situ Hybridization and TUNEL Assay, f) interpret the results obtained by applying those techniques.

Knowledge: At the end of the course students should: a) understand the concepts of structure, molecular composition, biogenesis, and organization of  the biological membranes, b) known the molecular properties, biosynthesis and intracellular trafficking of membrane lipids, c) understand the fundamental membrane properties including permeability, asymmetry, fluidity, curvature, signal transduction, lateral compartmentalization and vesiculation with emphasis to the formation and functions of liquid ordered and disordered phases, lipid rafts, caveolae and microvesicles, d) understand why the erythrocytes have been used as a model system in the field of biological membrane science and hereditary disorders that are associated with disturbances in the structural organization of the membrane, e) be able to describe the mechanisms of post-translational protein modification and sorting, f) be familiar with the mechanisms of protein targeting and cellular polarity, including those of exocytosis, proteasome, and generation and trafficking of intracellular vesicles, g) acquire enhanced knowledge on the processes being implicated in protein transport and import into the nucleus through the nuclear pore, with the contribution of karyoferines and the Ran-GTPase cycle, acquire advanced knowledge on the structure, role and function of cytoskeleton, as well as on molecular-motor machines, i) describe and analyze the mechanisms of supra-molecular structures, and the macromolecule, virus and phage assembly courses. Analysis of HIV and infectious prions, k) recognize and describe the regulatory mechanisms of signal transduction pathways (GPCR, TGF-β, Smad, JAK / STAT, NF-κB, Hedgehog and Wnt), i) determine and identify the processes of protein import into peroxisomes and mitochondria, j) acquire advanced knowledge and understanding of the mechanisms being involved in apoptosis (programmed cell death) and cellular aging, k) understand and describe the molecular mechanisms and defects leading to carcinogenesis and mitochondria-, peroxisomes- and lysosomes-related diseases.

Skills: At the end of the course students should: a) interpret the protein-import processes into cellular organelles, like nucleus, peroxisomes and mitochondria, b) explain the defects underlying the development of cancer disease, c) recognize and classify the variability observed in biological membranes, d) develop the ability to examine the inter-dependence of molecular composition and function at the level of cellular structure, e) analyze fundamental cellular mechanisms, such as the protein expression, lateral organization and vesiculation of the membrane, signal transduction, autophagy, molecular motorways, aging and programmed cell death ones, and their regulation, f) be able to select and apply techniques, such as Cell Culture, Protein Immuno-blotting, in situ Hybridization, as well as Scanning and Immuno-Gold Electron Microscopy, g) have the ability to apply and adapt a technical protocol for advanced Cell Biology experiments.

Abilities: At the end of the course students should: a) combine techniques and interpret the results of their application, in order to answer biological questions regarding complex mechanisms and processes, such as protein expression, signal transduction, self-assembling, lateral compartmentalization of the membrane, molecular machines, aging and apoptosis, b) analyze results and make new assumptions with respect to cell organelle-associated diseases, cytoskeleton, carcinogenicity and erythrocyte-membrane disorders, c) be able to perform, analyze and comment on experiments and observations on the mechanisms of post-translational modification, protein sorting, targeting and cellular polarity, d) perform and combine the appropriate research methodology, together with the conventional and modern techniques applied in Cell Biology

 
Lectures:
 
 

RESEARCH METHODOLOGY (4 Hours): Confocal Laser Scanning Microscope (CLSM). Electron Energy Loss Spectroscopy (EELS). Pseudo-coloring - 3D (three-dimensional) electron micrographs. Immuno-Electron Microscopy. Cryo-techniques. Atomic Force Microscope (AFM). Cell cultures. In situ hybridization. TUNEL assay. Analysis of a research article. Literature search and data mining. Preparation, elaboration and presentation of a scientific seminar

BIOLOGICAL MEMBRANES - (6 Hours): Molecular composition, organization and functions of biological membranes. Older and contemporary membrane models. Lipid composition of membranes during the evolution. Order, molecular structure and features of lipids in bilayers. Diffusion, mobility and barriers in the movement of membrane components. Membrane permeability, asymmetry, fluidity and curvature. Lipid phases. Biosynthesis and intracellular trafficking of membrane and membrane lipids.

LATERAL ORGANIZATION AND COMPARTMENTALIZATION OF THE MEMBRANE: LIPID RAFTS AND CAVEOLAE (6 Hours) Formation, molecular composition, structure topology and functions of lipid rafts and caveolae in various biological membranes and cell types. Pathways of intracellular transport and lipid rafts. Caveolins, cavins and other caveolae coat proteins. Signal transduction across membrane through lipid rafts and caveolae. Ceramide rafts and “pathological” rafts. Hybrid lipids, lipid chaperones, linactants and lipid droplets. Rafts and diseases. Methodological approaches.

ERYTHROCYTE: A MODEL SYSTEM IN THE MEMBRANE BIOLOGY (2 hours). Composition, organization and features of red cell membrane. Major proteins and protein complexes.   Arrangement of membrane and skeleton proteins. Submembrane networks of spectrin-actin and junctional complex. Principles of plasma membrane vesiculation: microvesicles and their role in cell homeostasis, aging, intercellular communication and pathophysiology of hereditary membranopathies and other diseases.

POST-TRANSLATIONAL MODIFICATION - SORTING - TARGETING OF PROTEINS AND CELLULAR POLARITY (6 Hours): Targeting and transport of lysosomal proteins. Protein exocytosis. Transport of molecules from extracellular space and plasma membrane inward to the cell. Mechanisms of vesicles formation and their specific fusion to the target membrane. The pathway of protein degradation in proteasome.

NUCLEAR - CYTOPLASMIC TRANSPORT, NUCLEAR IMPORT OF PROTEINS (2 Hours): Nuclear Pore Complex (NPC), Structure, organization and functions of Nuclear Pore Complex. Nucleoporins. Signals and receptors of nuclear transport. Karyopherins. Signaling mechanisms of protein transport into the nucleus. Ran cycle.

CELLULAR FIBRILS - CYTOSKELETON (2 Hours): Dendritic nucleation of actin. Role of tropomodulin. Actin related - interacting proteins. Gelsolin family in mammals. Cadherin - catenin complexes. smGTPases. Dynamics of microtubules. Katanins - Stathmins. The role of Microtubule Organizing Centers (MTOCs). Centrosomes. Profilin. Plant hormones and cytoskeleton. Cytoplasmic filaments-related diseases.

MOLECULAR MOTOR PROTEIN MACHINES (2 Hours): Regulation of interactions between molecular motor proteins - cargo. Skilful molecular motor machines. Structure and function of myosin super-family members. Classification and structure of kinesins. Kinesin shuffling on microtubules. Controlling kinesin activity and function. Dynein structure and function. Regulation of Dynein activity. Dynein shuffling on microtubules. Locomotion of organelles and movement of protein complexes. Transportation of mRNPs.

SELF - ASSEMBLY, SUPRAMOLECULAR  STRUCTURES - VIRUSES - PHAGES - PRIONS (4 Hours): Assembly of macromolecules - supramolecular structures - viruses and phages. Protein self-assembly. Self-assembly of viruses and phages. Lytic and lysogenic cycle of bacteriophages. Aided (Facilitated)-assembly of proteins. Molecular Chaperones of proteins. Self-assembly of collagen. Aided (Facilitated)-assembly of fibrin. Assembly of supramolecular structures. Directed-assembly of bacterial flagellum. The AIDS virus (HIV). Proteinaceous infection particles: Prions.

REGULATORY MECHANISMS OF SIGNAL TRANSDUCTION (4 Hours): Signal transduction and G-Protein-Coupled Receptors (GPCRs). Receptors bearing Serine - Threonine Kinase activities. TGF-β signal transduction. Smad transcription factors. Signal transduction and Cytokine Receptors. JAK (Just {Janus} Another Kinase) tyrosine kinases and STAT (Signal Transducer and Activator of Transcription) transcription factors. NF-kB signal transduction pathway. Principles of Hedgehog and Wnt signaling

PROTEIN IMPORT IN MITOCHONDRIA (4 Hours): Translocation complexes: TOM, TIM23, PAM, TIM22, SAM and export complexes. Targeting systems: amino-terminal pre-sequences for targeting, internal targeting sequences, alternative targeting sequences. Cytoplasmic factors and import of proteins in mitochondria. Contact points. Import of mitochondrial proteins into the mitochondrial compartments. Protein import into the external mitochondrial membrane. Protein import into the internal mitochondrial membrane. Import of proteins harboring an amino-terminal pre-sequence of targeting to mitochondria. Transport of proteins harboring a pre-sequence of targeting to mitochondrial matrix. Variations of mitochondrial protein import pathways.

PROTEIN IMPORT IN PEROXISOMES (4 Hours): Peroxins (Pexs). Assembly of peroxisome membrane. Signals for peroxisome membrane targeting. Roles of Pex19p and Pex3p in peroxisome membrane assembly. Types of peroxisome membrane proteins. Import of proteins into peroxisome matrix. PTS1 and PTS2 targeting signals - Pex5p and Pex7p receptors. Docking on peroxisome membrane. Translocation through peroxisome membrane. Receptor recycling. Formation of pre-entrance complexes. Peroxisome biogenesis. Peroxisome proliferation and division.

CELLULAR ORGANELLES - RELATED DISEASES (4 Hours): Human diseases with mitochondrial origin. Peroxisome and Lysosome-related diseases.

CLONING OF MODEL ORGANISMS - CELLULAR AGING (4 Hours): Aging of cells. The phenotype of cellular senescence. Hayflick limit and telomerase. Organisms cloning. Techniques for cloning model organisms. Future perspectives - Ethical dilemmas.

CELLULAR TRANSFORMATION (NEOPLASIA) - CARCINOGENESIS (4 Hours): Growth characteristics of transformed (neoplastic) cells. Mechanisms promoting cellular transformation. Mutagens. Human Carcinogenesis. Differences between healthy and neoplastic cells. Proteins controlling cell growth and division. Molecular correlation between mortal and immortal cells.

PROGRAMMED CELL DEATH (PCD) - APOPTOSIS (2 Hours): Morphology of apoptosis. The role of Caspases in Programmed Cell Death. Intracellular translocation of proteins. The anti-apoptotic activity of Bcl-2. The participation of Cytochrome-c in Caspase repertoire activation and Apoptosome assembly. The role of neurotrophins. Deregulation of apoptotic mechanisms in mutated and genetically modified model organisms.

 
Practicals:
 
 

1. Immunoblotting (western blotting). 2. Immuno-histochemical localization of antigenic sites through avidin - biotin technology  3. Immuno-electron microscopy. 4. Scanning Electron Microscopy (SEM). 5. In situ hybridization. 6. Cell cultures.

 
Instructors:
 
  Lectures: Ιoannis Trougakos Professor of Animal Cell Biology & Electron Microscopy (Coordinator) - Dimitrios Stravopodis, Associate Professor of Cell Biology & Development - Marianna Antonelou Assistant Professor of Animal Cell Biology - Charalampos N. Alexopoulos, Assistant Professor of Animal Cell Biology
 
  Practicals: Ιoannis Trougakos Professor of Animal Cell Biology & Electron Microscopy - Dimitrios Stravopodis, Associate Professor of Cell Biology & Development - Marianna Antonelou Assistant Professor of Animal Cell Biology - Charalampos N. Alexopoulos, Assistant Professor of Animal Cell Biology - Dr. Ourania Konstandi (Laboratory Teaching Staff) - Dr. Athanasios Velentzas (Laboratory Teaching Staff)
 
Notes:
 
 

There are no prerequisites for the student to choose and attend the course.

The course is offered to Erasmus students: Teaching in Greek language - Exams in English language.

The evaluation process is carried out in Greek language (there is the possibility of written exams in English for Erasmus students) and the final course grade includes: A. Theory: (70% of the final total grade of the course), Short-answer questions, Open-ended questions and Multiple-Choice questions - B. Laboratory exercises: (30% of the final total grade of the course): oral examination and a short-answer question at the end of each respective exercise. The total score is the sum of the two above-mentioned individual evaluations.

 
Contact:
 
  If you require more information, please contact the Course Coordinator, Prof. Ιoannis Trougakos at: Tel +30 210 727 4555 - Email: itrougakos[at]biol.uoa[dot]gr