65737 - Molecular Cell Biochemistry

Course contents

Rivisiting the mitochondria. The mitochondrial genome and mitochondrial diseases. Role of mtDNA mutations in tumorigenesis: involvement of the transcription factor HIF-1-alpha. Warburg effect and current concepts on cancer metabolic transformation. New acquisitions on mitochondrial ultrastructure and functional implications. The mitochondrial network: morphological and functional heterogeneity assessed by use of fluorescent proteins and dyes.Mitochondrial network dynamics:structure and function of DRP1,mitofusins and OPA1;role in apoptosis and autophagy. PINK and Parkin.

G-protein coupled receptors: signalling cascades dependent on G protein activation. Signalling strategies of GPCR independent of G protein. Receptor desensitization: the G protein-coupled receptor kinases family. Beta-arrestins: multiprotein scaffolds and molecular signalling switches; intracellular trafficand activation of Erks from class B receptors. Beta-arrestin and other receptors: tyrosine kinase receptor transactivation, crosstalk with insulin and IGF1-R receptors.

Concept of second messengers. Cyclic AMP,structure and isoforms of adenylate cyclase and phosphodiesterases. Measurements of cAMP in vivo with recombinant fluorescent proteins. PKA, EPAC and AKAPs.

Calcium as universal intracellular messenger: intracellular homeostasis; "calcium-signalling toolkit"; channels and transporters; molecular organization of intracellular IP3 and ryanodine receptors.Other messangers as cADPR e NAADP: biosynthesis and function. Spatial and temporal aspects of calcium signalling: waves, oscillations and related mechanisms. Capacitative calcium entry: Stim and Orai proteins. Calcium in mitochondria: uniporetr and exchangers:calcium measurements in the matrix with recombinant mt-targeted aequorin. Microdomains between mitochondria and ER: proteins involved in membrane tethering. Mitochondrial calcium and cellular metabolism.

The students will carry out an experimental laboratoryusing basic methodologies to identify and characterize the mitochondrial dysfunctions, using cultured human cells,namely: 1. Trypsinization, counting and seeding of cells; 2. Determination of protein content;3. Measurement of anaerobic glucose metabolism rate; 4. Analysis ofmitochondrial morphology and potentialusing live-cellfluorescence microscopy; 5. Evaluation of reactive oxygen species production in intact cells; 6.Determination of cell viability after a metabolic stress; 7.Identification of a mtDNA mutation in human cells: genomic DNA extraction, mtDNA amplification, digestion and separation by agarose gel electrophoresis.

Readings/Bibliography

Reviews and papers from scientific journals

Teaching methods

Lectures

Assessment methods

The critical assessment of the student's preparation is carried out through a colloquium aimed at evaluate the knowledge of the topics discussed in the lectures.

To evaluate the understanding of the experimental work carried out in the laboratory, a written testwill be performed, consisting of 5 questions of methodologies and topicsanalyzed in the experimental training.

Teaching tools

Ppt presentations through a beamer driven by a PC; scientific papers.

Office hours

See the website of Michela Rugolo