72568 - Biochemistry of cellular signalling with laboratory

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

Cyclic AMP and calcium: classic intracellular messengers and innovative methods  for their measurement.

Imaging cAMP in vivo in intact cells: recombinant PKA and FRET; cAMP microdomains; AKAPs.

 "Calcium-signalling toolkit"; 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 and mitochondria: a strict relationship.....

Mitochondria: not only powerhouses  but intracellular signalling crossroad. Supramolecular organization of the respiratory/supercomplexes: methods for isolation, identification and physiological relevance. The mitochondrial genome: general features,  organization in nucleoids,  mutations, mitochondrial diseases. Mitochondrial metabolites as tumorigenic signals: involvement in the activation/inactivation of the transcription factor HIF-1-alpha. Mutations in succinate and isocitrate DH.  mtDNA mutations and cancer: from Warburg effect to the concept of cancer metabolic transformation. Molecular alterations in the glycolytic pathway responsible for the Warburg effect. 

Mitochondrial ultrastructure by tomography and crio-electron microscopy: the cristae junctions and their functional implications.

The mitochondrial network: morphological and functional heterogeneity;  use of fluorescent proteins and dyes for mitochondrial morphology.  

Mitochondrial network shaping proteins: structure and function of DRP1, mitofusins and OPA1; OPA1 isoforms and mtDNA stability; role in regulation of apoptosis.

Interactions of mitochondria with other organelles: interaction with the ER: role on calcium homeosthasis. Calcium microdomains and proteins involved: the case of mitofusin-2. Mitochondrial calcium and energetic metabolism and apoptosis. The quality-control mechanisms in different mitochondrial compartments: PINK-1 and mitochondrial localizatio; interaction with Parkin; optineurin and LC3;  

Mitochondria and lysosomes: role on autophagy.

 

The student will follow theoretical lectures on the techniques utilized in the experimental laboratory. He will become familiar with the basic  methodologies to manipulate  mammalian cells in culture, namely: 1. Trypsinization, counting and seeding of cells; 2. Determination of protein content; 3. Measurement of glucose metabolism rate and lactate production; 4. Analysis of mitochondrial morphology in fixed cells using fluorescence microscopy; 5. Cell seeding in multiwells and determination of cell viability after a metabolic stress, by using a colorimetric assay.

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