- Docente: Michela Rugolo
- Credits: 6
- SSD: BIO/10
- Language: Italian
- Teaching Mode: In-person learning (entirely or partially)
- Campus: Bologna
- Corso: Second cycle degree programme (LM) in Molecular and cellular biology (cod. 8021)
Learning outcomes
At the end of the course students should be able to discuss the main biochemical mechanisms involved in intracellular signalling and in functional interactions among organelles. Students should also become familiar with the basic animal cell culture techniques and with methods to investigate their functions.
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 traffic and 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 laboratory using 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 of mitochondrial morphology and potential using live-cell fluorescence 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
The teacher will provide references (reviews and original papers). Lectures slides will be available to students, after downloading the PDF files from the AMS Campus web site, upon registration to a distribution list and use of a password.
Teaching methods
The lectures will analyze general problems concerning of cellular signalling. The lectures will be supplemented with a set of simple experiments carried out individually in the lab, aimed at giving all students the opportunity to learn the basic methodologies for the study of cellular functions, such as spectrophotometry, fluorimetry, optic and fluorescence microscopy, electrophoresis, etc. The students will also become familiar with basic procedure to manipulate and propagate cell in culture. During each practical session students will be able to provide practical solutions to theoretical problems outlined during lectures.
Assessment methods
Critical assessment of the student' preparation is carried out through a colloquium aimed at evaluating the knowledge of the topics discussed in the lectures.
To evaluate the understanding of the experimental work carried out in the laboratory, a written test will be performed, consisting of 5 questions of the methodologies and topics used in the experimental training.
Teaching tools
ppt presentations; papers
Office hours
See the website of Michela Rugolo