Le réseau neuronal se base sur l’axone et les dendrites pour former des milliards de connexions, ce qui fait du cerveau l'une des structures les plus complexes existantes. Pour que ce réseau fonctionne bien, il doit être régulé et maintenu. Cela pose de grands défis au cerveau lors du vieillissement, particulièrement dans le cadre d’une maladie neurodégénérative. Les premiers symptômes de plusieurs maladies neurodégénératives corrèlent d’ailleurs plus fréquemment avec le début de la dégénérescence axonale qu’avec la mort cellulaire des neurones. Une meilleure compréhension des mécanismes qui régulent cette dégénérescence axonale pourrait permettre de trouver de nouveaux traitements potentiels agissant dans la phase précoce de la manifestation de ces maladies. Dans cette thèse, nous étudions les mécanismes de dégénérescence et régénérescence axonale impliqués dans la sclérose latérale amyotrophique (SLA) et la maladie de Huntington (MH) en utilisant le nématode Caenorhabditis elegans (C. elegans). Nous nous basons sur les connaissances acquises sur les régulateurs de la régénérescence axonale pour investiguer leur implication et leur potentiel thérapeutique dans la SLA et la MH. Le C. elegans permet d’étudier la dégénérescence axonale dans le cadre des maladies neurodégénératives, puisque la visualisation des axones par fluorescence en facilite l’étude in vivo. Les modèles transgéniques C. elegans de la SLA et de la MH démontrent des bris axonaux pathologiques spontanés lors du vieillissement, permettant ainsi d’évaluer les mécanismes qui influencent cette dégénérescence axonale. Le modèle C. elegans permet aussi de mener des études à plus grande échelle. Nous avons donc pu effectuer un criblage génétique d’environ 40 gènes connus pour être des inhibiteurs de la régénérescence axonale après un dommage axonal mécanique. Selon notre hypothèse, l’inhibition de gènes inhibiteurs de la régénérescence axonale devrait augmenter le potentiel régénérateur des axones et, ainsi, réduire la dégénérescence axonale caractéristique de notre modèle SLA. Effectivement, nous avons pu identifier plusieurs voies de signalisation capables de réduire la dégénérescence axonale pathologique, notamment Dual zipper kinase DLK, la régulation des phosphoinositides et la signalisation de stress des ARNt par le stalled ribosome sensor GCN1. La voie de signalisation de DLK est indispensable dans la régénérescence axonale. Il a été démontré que sa suractivation permet de stimuler la régénérescence. Nous avons prouvé que la suractivation de DLK-1 par l’inhibition de ses inhibiteurs RPM-1 et FSN-1 réduit la dégénérescence axonale ainsi que la paralysie dans le contexte de la SLA. Pour évaluer la meilleure approche thérapeutique, nous avons investigué plus en détail les différents membres de cette voie de signalisation. Ainsi, nous avons trouvé que l’inhibition génétique et, surtout pharmacologique, de PARP1 et PARP2 peut réduire la dégénérescence axonale dans nos modèles de la SLA et de la MH. Les inositol polyphosphate phosphatases (INPP) sont des régulateurs des messagers secondaires d’inositol phosphates et de phosphatidylinositols, qui agissent dans la même voie de signalisation que PTEN, un inhibiteur de régénérescence axonale bien documenté. Dans nos études, nous avons identifié des nouvelles approches et cibles génétiques afin de réduire la dégénérescence axonale reliée à l’ALS et la MH. En résumé, nous avons identifié de multiples gènes qui agissent dans des voies de signalisation qui régulent la dégénérescence axonale, spécifiquement lors du vieillissement, dans le cadre de l’ALS et la MH. Il est primordial de mieux comprendre la signalisation intrinsèque qui régule l’axonopathie dans les maladies neurodégénératives pour établir de nouvelles approches thérapeutiques. Dans cette thèse, nous avons donc identifié plusieurs cibles thérapeutiques potentielles dans la voie de signalisation de DLK, et dans la voie de signalisation des phosphatidylinositols. / The neural network relies on axons and dendrites to form billions of connections, making the brain one of the most complex structures. These connections are both stable and highly dynamic, keeping the network in place while allowing connections to be modulated as needed. This network must be perfectly regulated and maintained for proper functioning, which can be a great challenge for the brain during aging and in the context of neurodegenerative diseases. Indeed, most neurodegenerative diseases show some form of degeneration of their axonal projections in the early stages. Increasingly, it is observed that the first symptoms of many neurodegenerative disorders correlate with the onset of axonal degeneration rather than cell death of neurons. A better understanding of the mechanisms regulating this axonal degeneration could lead to new treatments that could act in this particularly interesting therapeutic window. In this thesis, we aimed to study the genetic mechanisms of axonal degeneration involved in amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). More specifically, we investigated if axonal regeneration-associated genes can be targeted to reduce or even repair the age-dependent axonal damage observed in ALS and HD. To do this, we used Caenorhabditis elegans (C. elegans) models of ALS and HD, as they reproduce age-dependent axonal degeneration well. In addition, the use of fluorescent markers allows the visualization of axons in living animals, which makes it a unique model to study in vivo the dynamics of axonal damage and degeneration. We hypothesized that the stimulation of axonal regeneration pathways should increase the regenerative potential of axons and thus reduce the axonal degeneration characteristic of our ALS and HD models. Another advantage of using C. elegans is the possibility of large-scale genetic screens, allowing us to perform an RNAi-based genetic screen of 40 genes known as inhibitors of axonal regeneration. We identified multiple genes that act as drivers of axonal degeneration. Further analysis allowed us to identify an age-specific signaling network that regulates axonal degeneration through several main pathways, such as the Dual zipper kinase DLK pathway, the regulation of phosphatidylinositol phosphate, and the tRNA-related GCN1 stress response. The DLK signaling pathway is essential for axonal regeneration, and its overactivation has been shown to stimulate regeneration. We demonstrated that overactivation of DLK-1 by inhibiting its inhibitors RPM-1 and FSN-1 reduces axonal degeneration and paralysis in ALS. Furthermore, amongst the DLK pathway, we identified that genetic and pharmacological inhibition of PARP1/2 can consistently reduce axonal degeneration in our models of ALS and HD. Furthermore, we identified that the dysregulation of membrane-bound phosphoinositides is another major regulator of age-dependent axonal degeneration. We identified therapeutic targets similar to PTEN, a well-documented inhibitor of axonal regeneration and modulator of neurodegeneration. In our studies, we identified an alternative therapeutic target to PTEN and a new approach to trat ALS and HD-related axonal degeneration. In summary, we have identified multiple genes acting in an age-dependent network that drives axonal degeneration in ALS and HD. A better understanding of the intrinsic signaling that regulates axonopathy in neurodegenerative diseases is essential to establish new therapeutic approaches. In this thesis, we identified several potential therapeutic targets in the DLK signaling pathway and in the phosphoinositide signaling pathway.

No description available.

<p>Phosphatidylinositol (PI) is a phospholipid that constitutes only a minor component of eukaryotic membranes. However, they are critical in many fundamental cellular processes, such as signal transduction pathways, vesicular trafficking and actin cytoskeletal dynamics. PI is highly enriched in specific acyl chains at both the <em>sn-1</em> and <em>sn-2</em> positions, the major species being 1-stearoyl-2-arachidonoyl. Enzymes required for PI synthesis are believed to play a major role in this enrichment through the selective catalysis of specific substrates. We have studied several aspects of two enzymes involved in PI synthesis, Diacylglycerol kinase ε (DGKε) and CDP-Diacylglycerol synthases (CDS). We have studied the role of the ATP-binding motif of DGKε and showed that this enzyme is not only required for enzymatic activity, but substrate specificity and sub-cellular localization. We have also looked at the region adjacent to the catalytic site, containing a cholesterol recognition motif, and determined that this also affects the enzymes activity and substrate specificity. Finally, we have characterized the enzymatic properties of two CDS isoforms <em>in vitro</em> and demonstrated that these isoforms exhibit different substrate specificities. Taken together, our results serve to further our understanding of both DGKε and CDS1/2 and their roles in PI synthesis and enrichment with specific acyl chains.</p> / Master of Science (MSc)

<p dir="ltr">The Ebola virus (EBOV) is a filamentous RNA virus which causes severe hemorrhagic fever. It is one of the most dangerous known pathogens with a high fatality rate. Multiple outbreaks of EBOV have occurred since the 1970s with the most widespread outbreak starting in December 2013. This outbreak continued through May of 2016 and had a fatality rate of approximately 50%. EBOV outbreaks are recurrent because the virus is still present in animal reservoirs. Despite multiple EBOV outbreaks we still lack a clear understanding of how new viral particles are formed and spread through virus assembly and release. Given the widespread global travel, EBOV now poses a threat to the entire world. EBOV encodes for the matrix protein, VP40, which is one of the most conserved viral proteins. VP40 can form different structures leading to different functions of the protein in different stages of the EBOV life cycle. The VP40 dimer traffics to the inner leaflet of the plasma membrane to facilitate assembly and budding. The VP40 octameric ring has been implicated in transcriptional regulation. This thesis focuses on understanding in further detail the determinates of VP40 plasma membrane assembly and exit from an infected cell.</p><p dir="ltr">The assembly and trafficking of VP40 to the plasma membrane requires a network of protein-protein and lipid-protein interactions (PPIs and LPIs). Studying these interfaces is important for understanding how VP40 structure and function regulates trafficking and assembly and can shed light on therapeutic strategies to target EBOV. The alteration of host cell Ca²⁺ levels is one of the strategies that viruses use to perturb the host cell signaling transduction mechanism in their favor. Evidence has emerged demonstrating that Ca²⁺ is important for the assembly and budding of EBOV in a VP40-dependent manner. The relationship between intracellular Ca²⁺ levels and EBOV matrix protein VP40 function is still unknown. In this work we utilize biophysical techniques to study the role of LPIs and intracellular Ca²⁺ on VP40 dynamics at the plasma membrane and key residues for assembly and budding. This work highlights the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding and a critical interaction between Ca²⁺ and the VP40 dimer that are important for lipid binding at the plasma membrane.</p>

La couche de nacre de la coquille de l'huître perlière Pinctada radiata des côtes tunisiennes est considérée comme un biomatériau ostéogénique prometteur. L’objectif de ce travail intitulé « Ostéogénie, intégration et qualité de la nacre d’un bivalve des côtes tunisiennes : Pinctada radiata (Leach, 1814) » consiste dans un premier temps à valoriser l’espèce P. radiata par sa qualité nutritionnelle par un suivi saisonnier de la composition de sa chair en lipides totaux et en phospholipides particulièrement les PC, PE, PS et PI. Les analyses effectuées ont montré que les lipides de P.radiata sont caractérisés par une richesse en acides gras polyinsaturés (AGPI) de la série n-3 qui dépasse 3 fois celle des AGPI de la série n-6. Ces AGPI de la série n-3 en particulier l’EPA (C20:5n-3) et le DHA (C22:6n-3), sont connus comme étant les AG les plus importants dans l’alimentation humaine puisqu’ils préviennent des maladies cardiovasculaires et des pathologies ostéo-articulaires. Par ailleurs, P. radiata de la région de Maharès présente la meilleure qualité de nacre en Tunisie. Les analyses biochimiques ont montré que cette région, constitue la meilleure localisation de cette espèce qui est loin des zones portuaires et des différentes origines de stress (pêche, exploitation, zone touristique etc…). En plus de cet aspect, la zone de Maharès renferme des pintadines présentant une bonne qualité en termes d’épaisseur de nacre. Nos résultats montrent que la composition, saisonnière, en acide gras des phospholipides et en particulier des glycérophospholipides (PE, PI, PS et PC) de la nacre est riche en acides gras saturés C14 :0, C16 :0 et C18 :0 particulièrement en hiver et dans un moindre degré au printemps. La nacre, substance ostéogénique, a été également caractérisée par un taux élevé de plusieurs AGPI de la série n-3 et n-6, particulièrement (18:3n-3, 18:4n-3, 20:5n-3, 22:5n-3, 22:6n-3 et le 20:4n-6). Pour démontrer les potentialités ostéogéniques des extraits de la nacre, nous avons utilisé un modèle "in vitro" utilisant 4 extraits lipidiques : l’extrait lipidique de la nacre de P.radiata (Ln), l’extrait lipidique de la chair de P.radiata (Lc), l’ESM (Ethanol soluble Matrix) de la nacre de P.radiata (Br) et l’ESM de la nacre de P.margaritifera (Bm). Nous avons comparé, in vitro, le pouvoir ostéogénique des extraits ESM des deux espèces P. radiata et P. margaritifera sur deux types de cellules les préchondrocytes ATDC5 et les préostéoblastes murins MC3T3. Les différents extraits (Ln, Lc, Br et Bm) induisent l’engagement des cellules MC3T3 vers le lignage ostéoblastique par l’activation des promoteurs des gènes spécifiques du tissu osseux, tels que: le collagène de type 1, l’ostéocalcine (OC), l’ostéopontine(OP) et le Runx2. Ces extraits induisent aussi l’engagement des cellules ATDC5 vers la différenciation endochondrale par l’activation des promoteurs des gènes spécifiques du tissu osseux, tels que: le collagène de type 1 alpha-1 (Col1a1), l’Aggrécane et le collagène de type X alpha-1 (ColXA1). De plus, nous remarquons que la fraction organique ou ESMr(Br) en comparaison avec celle de P.margaritifera (Bm) présente également les propriétés stimulantes de la nacre et la stimulation est même beaucoup plus importante. Ces résultats mettent en évidence, dans les modèles expérimentaux mis en oeuvre, l’intérêt des lipides. Ces derniers semblent jouer un rôle important dans cette stimulation. De plus, nous pouvons penser à la possibilité de l’association des molécules de nacre ou de biominéralisation avec les acides gras de la nacre et de la chair dans les défauts osseux à travers les sites actifs de l’os ou du cartilage humain présentant les différentes pathologies ostéarticulaires / The nacre layer of the shell of the pearl oyster Pinctada radiata of tunisian coast is considered a promising osteogenic biomaterial. The objective of this work entitled "Osteogenie, integration and quality of nacre of a tunisian coast bivalve: Pinctada radiata (Leach, 1814)" is a first step to enhance the species P.radiata its nutritional quality by seasonal monitoring of the composition of the flesh of total lipids and phospholipids in particular PC, PE, PS and PI. The analyzes showed that lipids of P.radiata are characterized by rich in polyunsaturated fatty acids (PUFAs) of the n-3 more than 3 times that of PUFAs n-6 series. These PUFAs of the n-3 series particularly EPA (C20: 5n-3) and DHA (C22: 6n-3) are known to be the most important AG in the food as prevent of the cardiovascular disease, and joint/ bone pathologies. Moreover, P. radiata of Mahares region has the best quality of nacre in Tunisia. Biochemical analyzes showed that this region is the best location of this species that is far from the port areas and different sources of stress (fishing, exploitation, tourist area etc ...). In addition to this aspect, the area contains pintadines having good quality in terms of thickness of nacre. Our results show that the seasonal composition of fatty acid of phospholipids in particular glycerophospholipids (PE, PI, PS and PC) nacre is rich in saturated fatty acids C14: 0, C16: 0 and C18: 0 especially in winter and spring in a lesser degree. Nacre, osteogenic substance, was also characterized by a high rate of PUFA of the n-3 and n-6 rate, especially (18: 3n-3, 18: 4n-3, 20: 5n-3, 22 5n-3, 22: 6n-3 and 20: 4n-6). To demonstrate the osteogenic potential of extracts of nacre, we have established an "in vitro" model using 4 lipid extracts: the lipid extract of nacre P.radiata (Ln); the lipid extract of the flesh of P.radiata (Lc), ESM (Ethanol soluble Matrix) of the mother-of P.radiata (Br) and ESM nacre of P. margaritifera (Bm). We compared “in vitro” osteogenic power ESM extracts of both species P. radiata and P. margaritifera on two types of cells the préchondrocytes ATDC5 and the murine preosteoblasts MC3T3. The different extracts (Ln, Lc, Br and Bm) induce engagement MC3T3 osteoblast lineage cells to the activation of the promoters of specific genes of bone tissue, such as collagen type 1, osteocalcin (OC), osteopontin (OP) and Runx2. These extracts also induce the commitment of ATDC5 cells to endochondral differentiation by activating specific genes promoters of bone tissue, such as collagen type 1 alpha 1 (COL1A1), the aggrecan and collagen type alpha 1-X (ColXA1). Moreover, we note that the organic fraction or ESMR (Br) compared with that of P. margaritifera (Bm) also has stimulant properties of nacre and the stimulation is even more important. These results demonstrate, in experimental models used, the interest of lipids. They seem to play an important role in this stimulation. Moreover, we can think about the possibility of the association of molecules or nacre biomineralization with the fatty acids of the nacre and flesh in bone defects through the active sites of bone or cartilage presenting the human osteoarticular different pathologies

Die Funktion und Spezifität von Natürlichen-Killer-T-Zellen (NKT) in angeborener und erworbener Immunität ist nicht vollständig geklärt. Die Mehrheit der NKT-Zellen erkennt alpha-galactosylceramid (alphaGalCer), ein Lipid eines marinen Schwamms mit ungeklärter Relevanz. Verschiedene mykobakterielle Lipide wurden isoliert und auf ihre CD1d-Bindung und NKT-Zell-Aktivierung untersucht. Phospatidylinositol-mannosid (PIM) von Mycobacterium bovis BCG konnte als erstes bakterielles NKT-Zell-Antigen identifiziert werden. PIM aktiviert CD1d-abhängig murine und humane NKT-Zellen zur IFN-gamma aber nicht zur IL-4 Produktion. Mehrere andere Lipid-Fraktionen aktivierten ebenfalls NKT-Zellen. Diese Stimulation war entweder eine direkte, T-Zell-Rezeptor (TZR)-vermittelte und/oder indirekte, Toll-like-receptor 2 (TLR2) vermittelte Aktivierung. Iso-globotrihexosylceramide (iGb3) wurde als das endogene NKT-Zell-Antigen beschrieben. IGb3 ist ubiquitär in Lysosomen vorhanden. Dies wirft die Frage nach der Regulation der Antigen-Verfügkarkeit und der Kontrolle der NKT-Zell-Aktivierung auf. Es konnte gezeigt werden dass die Regulation der Antigen-Verfügbarkeit essentiell für die Regulation der NKT-Zell-Aktivität ist. Unkontrolliertes Auftreten und erhöhte Konzentration von iGb3 führte zu einer substantiellen Reduktion der NKT-Zell-Zahl, vermutlich durch Aktivierungs-induziertem-Zelltod. Mit Hilfe von DNS-Microarray Analysen wurden die Gen-Expressionsprofile von naïven NKT-Zellen und klassischen CD4 T-Zellen, regulatorischen T-Zellen, NK-Zellen und aktivierten NKT-Zellen verglichen. Es konnte sowohl ein NKT-Zell-spezifisches Expressionsmuster etabliert als auch eine gemeinsame Expression von Genen in allen verglichenen Zelltypen identifiziert werden. Naive und aktivierte NKT-Zellen zeigen eine erhöhte Expression von Apoptose-regulierenden Genen welches auf eine starke Selbst-Kontrolle zur präzisen Regulation der eigenen Aktivität hinweist. / The function and specificity of Natural Killer T (NKT) cells in innate and acquired immunity still remains elusive. The vast majority of CD1d restricted NKT cells recognise alpha-galactosylceramid (alphaGalCer), derived from a marine sponge, a lipid of unclear physiological significance. Different mycobycterial glycolipids were isolated and examined for binding to CD1d as well as for their capacity of NKT cell stimulation. Phospatidylinositol-mannoside (PIM) derived from Mycobacterium bovis BCG was identified as the first bacterial lipid antigen presented by CD1d. PIM activated both murine and human NKT cells to secrete IFN-gamma but not IL-4 in a CD1d dependent manner. Additionally, several other lipid fractions with NKT cell activation capacities were identified. This activation was either a direct, T-cell-receptor (TCR) mediated and/or an indirect, toll-like-receptor 2 (TLR2) mediated activation. Iso-globotrihexosylceramide (iGb3) was described as the endogenous NKT cell antigen. iGb3 is a ubiquitously present lysosomal glycolipid which raises the question of regulation of antigen availability and NKT cell activation control. It could be shown that regulation of antigen availability plays a crucial role in regulation of NKT cell activation. Moreover, uncontrolled appearance and increased concentrations of the endogenous antigen iGb3 led to substantial decrease in NKT cell number, presumably by activation induced cell death. Using DNA Microarray analysis, the gene expression profiles of naïve NKT cells and classical CD4 T cells, regulatory T cells, NK cells as well as to activated NKT cells were compared. The profiles revealed a NKT cell specific gene expression pattern as well as expression of genes which NKT cells share with NK cells, conventional CD4+ T cells and Treg cells. Both, naïve and activated NKT cells display elevated expression of apoptosis regulating genes providing NKT cells with high degree of self-control to precisely regulate their own activity.

As septinas fazem parte de uma família de proteínas de ligação ao nucleotídeo guanina. As septinas têm mostrado ter um papel importante na citocinese e outros processos celulares, incluindo a determinação da polaridade celular e reorganização do citoesqueleto. Todos os membros da família de septinas são compostos por três domínios: um N-terminal variável, um domínio central GTPase e uma região C-terminal que inclui sequências de coiled-coil. Septinas possuem uma característica de polimerizarem para formar complexos hetero-oligoméricos altamente organizados, in vivo e in vitro. Estruturas homo-oligoméricas também foram observadas, embora sua função ainda não esteja bem estabelecida. A Septina 2 humana (SEPT2) se acumula no sulco de clivagem de células em divisão, desde a anáfase até a telófase, além de interagir com a actina, e também está envolvida em doenças neurodegenerativas, como mal de Azheimer. Nesse estudo, a ORF que codifica SEPT2, bem como os fragmentos que codificam seus domínios, foram clonados, expressos em E.coli e purificados por cromatografia de afinidade e cromatografia de exclusão molecular. Os produtos foram analisados por espectroscopia de dicroísmo circular, espalhamento de luz a ângulo fixo e espectroscopia de fluorescência extrínseca, usando Tioflavina-T, que é um marcador clássico para fibras amilóides. Em todos os casos, os produtos formaram homodímeros in vitro, e também agregaram em temperaturas fisiológicas. O desenovelamento térmico das proteínas recombinantes revelou a presença de uma população intermediária de desenovelamento, rica em folhas-&beta;, e que ligam Tioflavina-T, sugerindo uma estrutura amiloidogênica para essa proteína, confirmada pelos programas de predição TANGO e WALTZ. Imagens dessas fibras foram obtidas usando Microscopia eletrônica de Transmissão, evidenciando uma agregação organizada das proteínas. Além disso, usando monocamadas de Langmuir, foi possível confirmar a ligação específica de SEPT2 ao fosfolipídeo fosfatidilinositol 4,5-bifosfato (PtdIns(4,5)P2). Essa ligação específica mantém a estrutura secundária de SEPT2, observada pela técnica PM-IRRAS, algo que não ocorre caso o lipídio seja inespecífico, sugerindo uma associação de SEPT2 com a membrana plasmática e podendo ter um papel na regulação das septinas. Por meio da técnica de duplo híbrido em levedura, identificamos proteínas que interagem com a SEPT2, como a MPBI e a DCTN2, auxiliando na elucidação de processos em que a SEPT2 possa participar. O conjunto dos resultados sobre a estabilidade, os processos de agregação de SEPT2 e a identificação de novos parceiros protéicos de interação, obtidos nesse trabalho, contribuíram para o melhor entendimento da função da SEPT2 e de seu envolvimento em desordens neurodegerenativas. / Septins are members of a conserved group of GTP-binding and filament-forming proteins. They are involved in a variety of cellular processes, such as microtubule regulation, vesicle trafficking, the formation of scaffolding platforms and actin dynamics. Human Septin 2 (SEPT2) has an N-terminal polybasic region responsible for lipid binding, a GTPase domain, and a C-terminal domain. SEPT2 is essential for cytokinesis and it is found in many tissues, mainly in the brain. Together with SEPT1 and SEPT4, it is accumulated in deposits known as neurofibrillary tangles in Alzheimers disease, which is evidence that SEPT2 may be involved in this process. In this study, the human SEPT2, and its domains, were cloned, expressed in E.coli and purified by affinity and size-exclusion chromatographies. The proteins form homodimers in vitro, suggesting that the GTPase domain is enough to promote the oligomerization. Thermal unfolding revealed the formation of aggregates under physiological conditions, which have the ability to bind a specific amyloid dye, Thioflavin-T, suggesting them to be an amyloidal fiber. Besides, in silico prediction programs, TANGO and WALTZ, corroborate that SEPT2 contain regions with high probability of aggregation and amyloidogenic formation, respectively. Moreover, we observed 20-50 nm thick filamentous structures by electron microscopy of negatively stained. Using Langmuir monolayers at the cell membrane lipid packing, SEPT2 and SEPT2NG bound to the phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Results from in situ PM-IRRAS experiments indicated that the secondary structure of SEPT2 is preserved upon interacting with PtdIns(4,5)P2, but not when interacting with DPPC - which is not specific for SEPT2 - at the air/water interface suggesting an association with the plasma membrane and a role in septin regulation. Furthermore, we also identified protein partners of SEPT2, from both human leukocyte and brain fetal cDNA libraries, using the yeast two-hybrid system. SEPT2 was shown to interact with: septins 6 and 4; a serine-protease and a MAP inhibitory protein; an ubiquitin-conjugating enzyme; and proteins related to cellular division. Thus, taken together this study contributed for the knowledgment of the stability and the aggregation kinetic of the SEPT2, leading to a better understanding of this protein and their role in neurodegenerative disorders.

Human African trypanosomiasis (sleeping sickness) is a devastating disease which is endemic in parts of sub-Saharan Africa. It is caused by the protozoan parasite T. brucei, which are transmitted by the bite of infected tsetse flies. Although the disease is fatal if left untreated, there is a lack of safe, effective and affordable drugs available; therefore new drugs are urgently needed. The aim of the work presented in this thesis is to develop novel trypanocidal compounds. It is divided into two parts to reflect the two distinct strategies employed to achieve this aim. The first part focuses on the inhibition of glycophosphoinositol (GPI) anchor synthesis by inhibiting the Zn2+-dependent enzyme, GlcNAc-PI de-N-acetylase. Trypanosomes have a variable surface glycoprotein (VSG) coat, which allows them to evade the human immune system. The GPI anchor attaches the VSG to the cell membrane; therefore inhibiting GPI synthesis should expose the parasite to the immune system. Initially, large substrate analogues were synthesized. These showed weak inhibition of the enzyme. Zinc-binding fragments were screened, and small molecule inhibitors based on salicylhydroxamic acid were then synthesized. These compounds showed modest inhibition, but the excellent ligand efficiency of salicylhydroxamic acid indicates this may be a promising starting point for further inhibitors. The second part details the P2 strategy. The P2 transporter is a nucleoside transporter unique to T. brucei, which concentrates adenosine. The transporter also binds and selectively concentrates compounds that contain benzamidine and diaminotriazine P2 motifs, which can enhance the potency and selectivity of these compounds. The sleeping sickness drugs melarsoprol and pentamidine contain P2 motifs. Compounds comprising a P2 targeting motif, a linker and a trypanocidal moiety were synthesized. Initially, a diaminotriazine P2 motif was attached to a trypanocidal tetrahydroquinoline (THQ) protein farnesyl transferase (PFT) inhibitor, with limited success. The P2 strategy was also applied to a non-selective, trypanocidal, quinol moiety. The quinol moiety was attached to diaminotriazine and benzamidine P2 motifs, and an increase in selectivity for T. brucei over MRC5 cells was observed.

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicos / Persistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studies

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain and other peripheral tissues. Phosphatidylinositol- 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. Gasotransmitters, such as carbon monoxide (CO), have been reported to regulate the activity of Kir channels by altering channel-PIP2 interactions. We tested, in a model system, the effects and mechanism of action of another important gasotransmitter, hydrogen sulfide (H2S) thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide (NaHS), as an exogenous H2S source, and expression of cystathionine γ-lyase (CSE), a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. A “tag switch” assay provided biochemical evidence for sulfhydration of Kir3.2 channels. The extent of H2S regulation depended on the strength of channel-PIP2 interactions: H2S regulation was attenuated when strengthening channel-PIP2 interactions and was increased when channel-PIP2 interactions were weakened by depleting PIP2 levels via different manipulations. These H2S effects took place through specific cytoplasmic cysteine residues in Kir3.2 channels, where atomic resolution structures with PIP2 gives us insight as to how they may alter channel-PIP2 interactions. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues into the background of the mutant lacking cytoplasmic cysteine residues, rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights as to how sulfhydration of specific cysteine residues could lead to changes in channel-PIP2 interactions and channel gating.