Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

AAbbssttrraacctt:: The mammalian organism is entirely dependent on ATP production by oxidative phosphorylation system (OXPHOS) on the inner mitochondrial membrane. OXPHOS is composed of respiratory chain complexes I-IV, ATP synthase and also include two electron transporters cytochrome c and coenzyme Q. Disorders of mitochondrial energy metabolism caused by OXPHOS defects are characterized by extreme heterogeneity of clinical symptoms, variability of tissues affected and the severity of the defect at the level of individual tissues. The mitochondrial disorders are not always clearly expressed at the level of available tissue or most easily available cultured fibroblasts and/or currently available methods are not capable to detect the defects on the fibroblasts level. The aim of this master thesis was to identify by biochemical methods, especially by high sensitive polarography, OXPHOS disorders in cultured fibroblasts. Cell lines from 10 patients with isolated (SURF21, SCO1 ND1, ND5) or combined defects of OXPHOS complexes whose biochemical defect was confirmed in muscle tissue as well as 14 patients with non- mitochondrial diseases (8 patients with Huntington disease, 6 patients with disorder of sulphur amino acids metabolism) were analysed. Furthermore impact of various cultivation conditions on OXPHOS...

Functional analysis of Spr1057 protein Streptococcus pneumoniae The genome of important human pathogen Streptococcus pneumoniae encodes a single gene of an eukaryotic type serine/threonine protein kinase StkP. Analysis of the global transcriptome of a mutant strain with inactivated stkP gene identified spr1057 gene whose expression was significantly repressed in ∆stkP strain. This gene is coding for Spr1057 protein which is a member of haloacid dehalogenase family. The analysis of the substrate specifity of the Spr1057 protein confirmed nucleotidase activity of this protein in vitro. To study the function of this protein in vivo we prepared several mutant S. pneumoniae strains. Growth characterictics of mutant strains were observed in the presence of modified nucleotides, 5-fluoro-2'-deoxyuridine (5-FdU) and 5-bromo-2'-deoxyuridine (5-BrdU). In addition, we monitored the rate of incorporation of 5-BrdU into the chromosomal DNA of the mutant strains in comparison with the wild type S. pneumoniae strain. The growth of the Δspr1057 strain was significantly inhibited in the presence of the modified nucleotides and increased incorporation of 5-BrdU in DNA was showed. Neither growth inhibition nor incorporation of 5-BrdU in DNA was observed for the wild type strain. The expression of an ectopic copy of spr1057...

Acute lymphoblastic leukemia (ALL) is the most common malignant dise- ase in children. Despite great advancements in treatment of this disease, around 15-20 % of patients suffer a relapse. One of the possible reasons for relapse is developed resistance to cytostatic drugs. L-asparaginase is an im- portant chemotherapy component for childhood ALL and resistance to this drug often complicates treatment. To date, causes of developing resistance have not been sufficiently described. This thesis is a part of a greater research project focusing on mechanisms of L-asparaginase's activity and reasons for developing resistance to this chemotherapeutic agent. Differential metabolic requirements of cancerous cells have been described as early as 1924 by O. H. Warburg and they have been subject to scientific inquiry since. This study aimed to describe the relationship between basal metabolic determinants of leukemia cells and their sensitivity to L-asparaginase. For this reason, two metabolic pathways, glycolysis and oxidative phosphorylati- on, were studied in detail using a Seahorse Bioanalyzer. Further, expression of specific genes involved in glycolysis was detected. Content of mitochon- drial reticulum in cells, expression of the asparagine synthetase gene, and cell size were also studied. Experiments were...

Spr0334, new protein of cell division in Streptococcus pneumoniae Streptococcus pneumoniae is an important human pathogen. The geonome of this bacteria encodes a single gene for eukaryotic-like serine / threonine protein kinase called StkP. StkP regulates many physiological processes such as pathogenesis, competence for genetic transformation, resistance to various stresses and resistance to antibiotics. It also affects the transcription of many genes involved in cell wall biosynthesis, pyrimidine metabolism, DNA repair and iron uptake. Recent studies have shown that StkP is located in the cell division septum and significantly regulates cell division and morphology. Its substrates include, among others, cell division protein DivIVA, FtsZ and FtsA. Analysis of phosphoproteome maps of wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the protein Spr0334. Mass spectrometry analysis identified phosphorylation sites of the protein Spr0334: threonine 67 and threonine 78. Furthermore, it was found that the protein Spr0334 is located in the cell division septum, which led to the hypothesis that it could be newly identified cell division protein in S. pneumoniae. The main aim of this thesis was to describe the function of the...

The function of most proteins is regulated by post-translational modifications, of which phosphorylation in particular has been shown to be ubiquitous and of paramount importance to cell signaling. Two enzyme families, protein kinases and phosphatases, regulate phosphorylation, and aberrant activities of family members have been implicated in many diseases such as cancer and neurological disorders. Thus, understanding the function of these enzymes in living cells is important for understanding their biology and for designing new therapies, but a challenging task due to their highly conserved architecture. The major focus of the dissertation is on the development of a new approach to selectively turn-on multiple specific kinases and/or phosphatases using orthogonal ligands as chemical inducers of dimerization (CIDs). Specific kinases or phosphatases were dissected at particular sites into two inactive fragments or split-proteins. The split fragments are attached to interacting protein pairs of CID systems, such that upon addition of the specific ligand they heterodimerize with subsequent reassembly of the split-protein and concomitant activity. We demonstrated the in vitro and in cellulo feasibility of this approach using three orthogonal CIDs, rapamycin, abscisic acid, and gibberellic acid, to turn-on members of the tyrosine kinase group such as Lyn and Src, and of the tyrosine phosphatase group such as PTP1B and SHP1. We have also developed a new synthetic photocleavable di-trimethoprim CID that allows for ligand-gated turn-on of desired kinases in live cells. The new CID can be cleaved or turned-off by UV irradiation which results in a turn-off of kinase activity. Small molecule controlled split-proteins allow for developing logic gates and we demonstrate that the systems we have developed can be used to construct 7 out of the 10 basic, circuit-type Boolean phosphorylation-based logic gates in living cells. These post-translational logic gates may have interesting applications in synthetic biology. Finally, we present an initial approach to use redesigned kinases and redesigned ligands as potential scaffolds for developing new CIDs. Thus, we provide and extend new methodologies that potentially allow for posttranslational control over the activity of user defined split-kinases and split-phosphatases for interrogating and redesigning signaling pathways. The last section of this work focuses on understanding small-molecule selectivity toward protein kinases. We systematically analyzed different reported kinase screens to further understand the reliability of large scale data in the kinome field as the design of selective inhibitors is one the most useful approaches for understanding the function of enzymes or the development of drugs in a natural setting such as a primary cell or an organism.

Streptococcus pneumoniae encodes single serine/threonine protein kinase StkP and its cognate protein phosphatase PhpP. This signalling couple phosphorylates/dephosphorylates many target proteins involved in various cellular processes. So far, only few ot them was characterized in detail. Global phosphoproteomic analysis in the ∆stkP mutant strain background resulted in the identification of protein Spr0175 as phosphorylated on threonine 7. The main aim of this work was to characterize this new substrate. The ∆spr0175 mutant strains were prepared in the wild type genetic background Rx and R6 and then monitored for their growth and cell morphology. Mutant strains exhibited morphological defects revealing potential involvement of Spr0175 in the process of cell division. In the wild type D39 the deletion was unsuccesful, which may entail possible essentiality of Spr0175 in D39 strain. The results obtained also confirmed that the Spr0175 is modified in in vitro and in vivo conditions at threonine 7. In vitro study also confirmed minor phosphorylation at T4 residue. By using co-immunoprecipitation assay we demonstrated that Spr0175 protein can form oligomeric structures. Another aim of this work was cellular localization of Spr0175. By using fluorescent microscopy we showed that GFP-Spr0175 fusion...

Tumor suppressor p53-binding protein 1 (53BP1) is an integral part of a sophisticated network of cellular pathways termed as the DNA damage response (DDR). These pathways are specialized in the maintenance of genome integrity. Recently, it was reported that nuclear import of 53BP1 depends on importin ß. Here, I used fluorescence microscopy and co-immunoprecipitation experiments to identify its nuclear localization signal (NLS). Clusters of basic amino acids 1667-KRK-1669 and 1681-KRGRK- 1685 were required for 53BP1 interaction with importin ß and for its nuclear localization. Short peptide containing these two clusters was sufficient for interaction with importin ß and targeting EGFP to the nucleus. Additionally, the effect of 53BP1 phosphorylation at S1678 on its nuclear import was examined. Mimicking the phosphorylation in the 53BP1-S1678D mutant decreased the binding to importin ß and resulted in a mild defect in 53BP1 nuclear import. However, 53BP1 entered the nucleus continuously during the cell cycle, suggesting that CDK-dependent phosphorylation of S1678 probably does not significantly contribute to the regulation of 53BP1 nuclear transport. Taken together, 53BP1 NLS meets the attributes of a classical bipartite NLS. Although no cell cycle-dependent regulation of its import was observed, the...

p-21-activated kinase 6 (PAK6) is a serine-threonine protein kinase originally identified as an Androgen Receptor (AR) interacting protein. In current study, we determined the subcellular localization of PAK6 through mutational analysis. We have found that the N-terminal CRIB domain is partly responsible for plasma membrane targeting, the region between amino acid residues #292 to #368 is functionally relevant to plasma membrane localization and that amino acid residues #119 through #190 are responsible for nuclear targeting of PAK6, in addition to a stretch of positively charged N-terminal residues (#2-#11) since mutants lacking this sequence mis-localizes to cytoplasm. In junction forming epithelial cells, PAK6 is demonstrated to co-localize with B-catenin at adherens junctions, suggesting that PAK6 is an activation-dependent event and that PAK6 translocates from plasma membrane to the cytoplasm in response activation via the PKA signal pathway. / by Ciny John. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.