87604 - BIOCHIMICA DI SISTEMI CELLULARI COMPLESSI E ANALISI DI RETI PROTEICHE CON LABORATORIO

Learning outcomes

At the end of the course, the student will acquire knowledge for the understanding of: 1) biochemical mechanisms that regulate complex cellular systems and their functional interactions; 2) protein interactions in different intracellular signaling pathways. The attending to the practical laboratory will allow the student to acquire skills in the handling of the cell cultures as tool for the biochemical analysis of some cellular functions. In particular, the student will be able to perform in vitro assays on cell cultures useful both for the evaluation of the response to drugs and for the evaluation of their mechanism of action. Finally, the student will be able to analyze and discuss topics of cellular biochemistry and to understand the biochemical methodologies used for such studies.

Course contents

Lectures: Part 1 (4CFU= 32 hours) and Part 3 (3CFU= 24 hours)

1) Structural and molecular characteristics of 7TM receptors; structural interactions between 7TM receptors and heterotrimeric G proteins; intracellular signaling mechanisms mediated by Gs protein; Molecular inactivation of 7TM receptors; role of GRKs and arrestin proteins; arrestin as an example of molecular switches and signaling platform.

2) Structure and function of adenylate cyclase; adenylate cyclase and lipid rafts; in vivo determination of cAMP microdomains; PKA: structure and subcellular location; AKAPs as an example of molecular platforms for cAMP and PKA signalling.

3) The molecular organization and regulation of phospholipases C enzymes; structure and function of IP3 and ryanodine receptor; biosynthesis and function of novel intracellular messengers such as cADPR and NAADP.

4) Ca2+ as an universal and versatile second messenger: spatio-temporal aspects (waves, oscillations and hypothesized mechanisms); mitochondrial calcium homeostasis; structure and function of Ca2+ uniport; calcium microdomains between mitochondria and ER; structural and functional organization of MAMs as subcompartment for Ca2+ homeostasis and lipid biosynthesis.

5) Cell membranes and their supramolecular organization: the mitochondrial cristae.

6) Mitochondrial network: methodologies for the analysis of structure and function. Functional heterogeneity of mitochondrial network. Functional heterogeneity of mitochondrial network. Protein networks involved in mitochondrial dynamics: molecular mechanisms of fusion and fission and GTPases involved. Structural and functional organization of the subcellular compartment "MAMs". MAMs as scaffold for lipid biosynthesis.

7) Network metabolic and metabolic reprogramming during cell proliferation; metabolomics and metabolic fluxes; oncometabolites and HIF1a signaling during the metabolic and hypoxic adaptation of cancer cells.

8) Proteins involved in the maintenance and function of the mitochondrial genome and mitochondrial dynamics; the mitochondrial proteome.

9) Autophagy and mitophagy: protein network involved in the formation and maturation of autophagosome; structure and function of PINK1 and Parkin proteins and their involvment in the regulation of mitochondria quality control.

10) Network of anterograde and retrograde communication between nucleus and mitochondria.

11) Molecular network between nucleus, endoplasmic reticulum and mitochondria in the process of import and sorting of mitochondrial proteins.

12) PI3K/AKT/mTOR signalling in the regulation of cell metabolism and survival. Lysosomes as molecular platform for the crosstalk between AMPK and mTORC1 in the cellular catabolism and anabolism modulation.

Laboratory: Part 2 (2CFU; Prof. Farruggia) and Part 3 (1CFU; Prof. Porcelli)

The credits of Part 2 (2CFU= 30 hours) together with 1CFU (15 hours) of Part 3 will constitute an integrated laboratory in the training activity. The experimental laboratory is placed at the beginning of the course and will introduce the students to an exemplary path of how they can evaluate the effects on colon adenocarcinoma cells of a potential drug that interfere with cellular energy metabolism. During the practical laboratory the following experimental activities will be foreseen:

1) Evaluation of effective doses of the potential drug

2) Analysis of its effects on: a) cell cycle; b) mitochondrial potential; c) nduction of apoptosis

3) Identification of potential molecular targets

At the end of the laboratory a session of analysis of the collected data will be evaluated for each student and as a working group. The guidelines for drafting a scientific report related to the laboratory experience will be discussed with the whole class starting from the collected data.

The practical part will be integrated with a general theoretical part concerning:

1) Techniques for the generation and handling of animal cell cultures

2) Basic principles of the following techniques: a) Visible UV spectroscopy; b) Fluorescence spectrometry; c) Cytofluorimetry; d) Fluorescence microscopy; e) Electrophoretic techniques; f) Western Blot

Readings/Bibliography

Part 1 and Part 2:

Necessary educational supports

The teacher will provide further recommended bibliographical references (reviews and original papers) for a further deepening of the program contents. The lectures will be provided as PDF files and will be available to the students on on IOL platform (https://iol.unibo.it/).

Advised educational supports

The teacher advises to consult the following textbooks for clarifications on the basic concepts of cellular and structural biochemistry: i) “Cells” by Lewin et al; ii) “Protein Structure and Function” by Petsko and Ringe; iii) “Lehninger Principles of Biochemistry” by Nelson and Cox.

Laboratory:

The teacher will be provided the sheets concerning the conduct of individual experiments. Furthermore, all the material used in carrying out the theoretical lessons will be provided as PDF files. No particular textbook is required.

Teaching methods

Part 1 and Part 3: The teaching method used is based on lectures during which the contents of the program will be illustrated using PowerPoint slides. Attendance to such lessons is not mandatory but it is highly recommended as the contents of the program will be presented and explained by the teacher and discussed with the whole class. This teaching method will facilitate the learning of contents and will allow the achievement of the knowledge and skills by the whole class. Students are encouraged to communicate to the teacher any requirements by e-mail as soon as possible. This will allow teacher to evaluate which teaching support tools are most adequate to make the training course accessible to all students of the course.

Laboratory: The practical experience in laboratory will be carried out in single place at the workbench by using several and different laboratory equipments. This will be followed by a working group to discuss the obtained data. Students are encouraged to communicate to the teacher any requirements by e-mail as soon as possible. This will allow teacher to evaluate which teaching support tools are most adequate to make the training course accessible to all students of the course. Attendance at the laboratory is mandatory.

Assessment methods

Part 1 and Part 3: the learning test assessment will consist of an interview that provides at least 3 questions related to the topics covered in the program in order to verify and evaluate the student's knowledge of the contents developed and discussed during the lessons and the experimental laboratory. Moreover, during the interview the student's ability to link and integrate the various topics with particular attention to the scientific terminology used and to correct and accurate exposure will be evaluated. The learning assessment of Part 1 and Part 3 must be verified in the same exam session. In order to obtain a final grade of 30/30 with honors, the student must show that he/she knows and has deepened, using the textbooks and bibliography indicated by the teacher, all the topics covered during the lessons. Further, they must explain and integrate the topics with scientific language properties. To obtain a final grade of 30/30, the student must explain contents covered during the lessons and show the ability to correctly integrate them with scientific language properties. The final grade will be scaled from 30/30 to 18/30 based on the number of questions to which the student answers and her/his ability to integrate the topics with scientific properties language. In particular, to obtain a grade of 18/30 the student must show to have superficial knowledge of all the contents discussed during the lessons and not be able to integrate them with scientific language properties. The vote obtained will be considered valid within the calendar year and will contribute to the final mark, determined as the weighted average of the different Parts of the 87603- Cellular Biochemistry and Physiology (I.C.) teaching.

Laboratory: during the pratical experience, the students will be followed by the teachers in charge of Part 1, 2, and 3 and by tutors, with a constant dialogue aimed at clarifying both the analytical approch and the individual experiments performed, and the metodologies used. Passing the exam requires the presentation of a written report, elaborated according to the procedures presented on the last day of the laboratory, which must be delivered within a week from the end of the workshop. The evaluation expressed in 30/30 will contribute to the final grade, determined as the weighted average of the different Parts of the 87603- Cellular Biochemistry and Physiology (I.C.) teaching.

The final mark for teaching 87603- Cellular Biochemistry and Physiology (I.C.) will be calculated as the weighted average of the marks obtained in the Parts that compose the teaching.

Prof. Porcelli, as responsible of Cellular Biochemistry and Physiology teaching, will communicate to the student the final vote by e-mail and will proceed with the vote verbalization only after receiving an  e-mail in which the student states to accept the vote.

Teaching tools

Part 1 and Part 3:

Students are encouraged to communicate to the teacher any requirements by e-mail as soon as possible. This will allow teacher to evaluate which teaching support tools are most adequate to make the training course accessible to all students of the course. The contents of the lectures will be presented using PowerPoint slides and discussed with the whole class through appropriate teaching materials.

Laboratory:

Students are encouraged to communicate to the teacher any requirements by e-mail as soon as possible. This will allow teacher to evaluate which teaching support tools are most adequate to make the training course accessible to all students of the practical laboratory. The practical experience of single-place laboratory and the use of laboratory equipment will be carried out using appropriate teaching tools in order to make the training accessible to all students of the course.

Office hours

See the website of Anna Maria Porcelli

See the website of Giovanna Farruggia