Dissertation topics suggested by the teacher.
A) CdS LT in Genomics:
Project 1 - Validation of differential proteomics data for the identification of novel respiratory chain complex I interactors and determination of metabolic pathways in response to its ablation.
Respiratory complex I (CI) is the largest multi-protein complex in the OXPHOS system, consisting of 45 subunits, encoded by nuclear or mitochondrial DNA. CI can associate with CIII and CIV, forming 'supercomplexes' (SC), but the assembly pathways of CI or SC are still controversial. In a previous study, we investigated the biogenesis of CI and SCs in cellular models lacking two essential CI structural subunits, namely the mtDNA-encoded ND1, located in the membrane arm, and the NDUFS3-encoded nDNA, located in the matrix arm. These experiments allowed us to identify small amounts of SC even in cells lacking these subunits necessary for CI assembly. In these models, we also performed quantitative differential proteomics experiments using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) in order to identify novel CI interactors with a possible role as assembly factors in the early stages of CI biogenesis. These candidates appear to be involved in certain mitochondrial metabolic pathways. The aim of this project is to validate the proteomics data and to understand the role of these interactors in the assembly process of the CI and SCs, as well as to identify the function of mitochondrial proteins that are overexpressed as a result of the absence of the CI.
Project 2 - Analysis of exomes in patients with mitochondrial diseases for the identification of new genes and pathogenic variants (Co-supervisor Dr. Leonardo Caporali - Laboratory of Neurogenetics - IRCCS Istituto delle Scienze Neurologiche di Bologna)
Project 3 - NGS approaches for the study of mtDNA variants with low heteroplasmy (Co-supervisor Dr. Leonardo Caporali - Laboratory of Neurogenetics - IRCCS Institute of Neurological Sciences of Bologna)
B) LM in Pharmaceutical Biotechnology, Molecular and Industrial Biotechnology and Molecular and Cellular Biology
Project 1 - SURVIVAL MECHANISMS IN TUMOR CELLS DEFLECTED BY OXIDATIVE PHOSPHORILATION (Porcelli - Iommarini)
Cancer cells are characterised by cellular adaptation mechanisms to ensure their survival even under conditions of nutrient and oxygen deprivation. In this context, our research group has shown that cancer cells in which complex I (CI) of the respiratory chain is disassembled are unable to adapt to hypoxia because the metabolic changes induced by the deficiency of CI prevent the stabilisation of the hypoxia-induced factor (HIF1) even under hypoxic conditions. However, after a period of adaptation, these cells are still able to form tumour masses, albeit in a much longer time than their wild type counterparts and although these tumours are morphologically very different. The aim of this project is to study the possible molecular mechanisms that enable the survival of IC-defective tumour cells. In particular, the following mechanisms will be analysed:
(i) the PI3K/Akt-mediated signalling pathway that is known to promote activate survival mechanisms and that we have found to be activated in cells and tumours defective for IC. In these models, this signalling pathway will be studied by analysing the phosphorylation of several target proteins and the use of specific inhibitors of this intracellular signalling pathway in order to demonstrate that its inactivation leads to tumour cell eradication
(ii) autophagy, and in particular mitophagy, a mechanism known to allow cell survival in the event of nutrient deprivation by recycling cellular components and which is finely tuned in relation to nutrient availability and intracellular energy charge
(iii) Endoplasmic reticulum stress and the accumulation of lipid droplets in cells deprived of IC
Project 2 - STUDY OF THE METABOLIC SETTING OF OVARIAN CARCINOMA CELLS IN RELATION TO RESISTANCE TO CHEMOTHERAPY (Porcelli - Iommarini)
Ovarian carcinoma cells have recently been classified according to their metabolic characteristics into high and low-OXPHOS and chemoresistance-related metabolism. Since the main regulators of glycolytic and oxidative metabolism are the transcription factor HIF1a and the transcriptional coactivator PGC-1a, respectively, this project aims to study their involvement in the response to chemotherapeutics in order to identify the molecular determinants of chemoresistance in the context of ovarian carcinoma.
Project 3 - Validation of differential proteomics data for the identification of novel respiratory chain complex I interactors and determination of metabolic pathways in response to its ablation.
Respiratory complex I (CI) is the largest multi-protein complex in the OXPHOS system, consisting of 45 subunits, encoded by nuclear or mitochondrial DNA. CI can associate with CIII and CIV, forming 'supercomplexes' (SC), but the assembly pathways of CI or SC are still controversial. In a previous study, we investigated the biogenesis of CI and SCs in cellular models lacking two essential CI structural subunits, namely the mtDNA-encoded ND1, located in the membrane arm, and the NDUFS3-encoded nDNA, located in the matrix arm. These experiments allowed us to identify small amounts of SC even in cells lacking these subunits necessary for CI assembly. In these models, we also performed quantitative differential proteomics experiments using SILAC (Stable Isotope Labeling with Amino acids in Cell culture) in order to identify novel CI interactors with a possible role as assembly factors in the early stages of CI biogenesis. These candidates appear to be involved in certain mitochondrial metabolic pathways. The aim of this project is to validate the proteomics data and to understand the role of these interactors in the assembly process of the CI and SCs, as well as to identify the function of mitochondrial proteins that are overexpressed as a result of the absence of the CI.
Project 4 - New massive approaches for the genetic definition of patients with rare neurological diseases (Co-supervisor Dr. Leonardo Caporali - Laboratory of Neurogenetics - IRCCS Istituto delle Scienze Neurologiche di Bologna)
Defining the genetic defect in patients with neurological diseases, including optic atrophy, chronic progressive external ophthalmoplegia (CPEO), mitochondrial encephalomyopathies, epileptic encephalopathies, juvenile-onset Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS) and cases of patients with unresolved hereditary neurological diseases. Considering the genetic heterogeneity underlying these diseases, and the experience gained, patients with a genetic suspicion will be subjected to whole exome analysis (WES), prioritising for variants in genes associated with the specific disease and/or on the basis of main and secondary symptoms (HPO terms). In cases negative to exome analysis, an integrated protocol will be applied, where possible, including whole genome analysis (WGS), transcriptome (RNA-seq), proteome and metabolome analysis on target tissue or cell lines derived from the patient. In patients in whom intermediate-sized genetic rearrangements are suspected, which cannot be detected by NGS or CGH-array sequencing techniques, whole-genome analysis by long-read sequencing will be performed. Using the same approach, mitochondrial genome rearrangements in muscle biospecies of patients with mitochondrial myopathy will be characterised in order to identify possible duplications/insertions that cannot be detected by NGS techniques.
C) THESES ABROAD CdS LM in Pharmaceutical Biotechnology, Molecular and Industrial Biotechnology and Molecular and Cellular Biology
Project 1 (Université Claude Bernard Lyon - Lyon - France) - Genetic control of acetylcholine receptor biosynthesis: from C. elegans to human diseases (Co-supervisor Dr. Manuela D'Alessandro) (funding is available for the internship period)
Ionotropic acetylcholine receptors (AChRs) support neurotransmission at the neuromuscular junction and have a neuromodulatory function in the central nervous system. Dysfunction of these receptors is linked to various diseases, including myasthenia, schizophrenia or epilepsy. The amount of receptors present on the plasma membrane is finely regulated and results from a balance between biosynthesis, recycling and degradation. Our research aims to identify and characterise new factors involved in AChR biosynthesis. Our strategy consists of: 1. identify novel factors involved in AChR biosynthesis using the model organism Caenorhabditis elegans, 2. characterise the function of these factors in C. elegans, 3. test for conservation of function in human cell lines, 4. data mine for polymorphisms in the identified genes that could be associated with human diseases; if applicable, introduce the same polymorphisms into the C. elegans genome to test the potential pathogenicity of the human mutation. During the internship, the student will characterise two proteins, TMED7 and TMED2, which were identified from a genetic screening conducted in C. elegans. TMED7 and TMED2 are involved in endoplasmic reticulum-mediated transport to the Golgi. Until now, they have never been linked to AChR biosynthesis. Our preliminary data show that the amount of AChR at the neuromuscular junction is reduced by more than 50% in mutants for TMED7 or TMED2.
More specifically, the student:
1. confirm preliminary data by imaging endogenously labelled receptors with t-RFP in TMED mutants with a confocal spin disc system, AChR Biosynthesis C. elegans
2. will identify and characterise the following models. Mammalian cell lines: conservation Patients: polymorphisms - determine the subcellular localisation of TMED7 and TMED2 by modifying the C. elegans genome using CRISPR/Cas9 technology to insert a fluorescent tag into the loci encoding the proteins, - participate in the knock-out of TMED7 and TMED2 using CRISPR/Cas9 in human cell lines and characterise AChR levels in these cells,
3. search for TMED7 and TMED2 polymorphisms using databases and biobanks for rare diseases, and eventually introduce them into the C. elegans genome to test for pathogenicity. The student will work under the supervision of two permanent researchers
Applicants must have excellent academic performance and be motivated and enthusiastic. Previous experience with C. elegans is not required. We ask applicants to send a CV and a one-page motivation letter (in French or English; the letter should explain the applicant's interest in the project) to manuela.d-alessandro@univ-lyon1.fr.
Project 2 (UCL London - UK) - Generation of in vitro models of primary mitochondrial diseases (Co-supervisor Dr. Micol Falabella)
Primary mitochondrial diseases (PMDs) are genetic disorders caused by mutations in nuclear DNA or mitochondrial DNA (nDNA/mtDNA) that impair mitochondrial energy production and other aspects of cellular metabolism. They are among the most common inherited neurological diseases and are associated with severe disability and shortened lifespan. There is currently no effective treatment and clinical management focuses on treating complications. There is therefore an urgent and unmet need to develop novel treatments with a high likelihood of success in clinical efficacy trials.
This project will focus on generating clinically relevant PMD in vitro models to test small therapeutic molecules. A project in this area of neuroscience research will provide specialist training in mitochondrial biology and bioenergetics, in addition to broad training in cellular and molecular biology approaches.
Project 3 (UCL London - UK) - Characterization of novel genes of primary mitochondrial diseases (Co-supervisor Dr. Micol Falabella)
Mitochondria are fundamental to cellular metabolism and provide the major source of energy in human cells. Mitochondrial dysfunction is a common motif of neurodegenerative and neuromuscular diseases. This MSc project will focus on characterising rare and novel disease-causing gene mutations in people with suspected mitochondrial disease, harnessing data from the 100,000 Genomes Project, to understand the molecular mechanisms that underpin the pathophysiology of mitochondrial dysfunction. To this end, the project will benefit from a wide range of clinically relevant cellular models to establish the impact of mitochondrial gene variants on bioenergetics and the clinical phenotype. A project in this area of neuroscience research will provide specialist training in mitochondrial biology and bioenergetics, in addition to broad training in cellular and molecular biology approaches.
