Dissertations / Theses on the topic 'NRAP'

The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. Nrap is a novel nucleolar protein found to be present in all eukaryotes. Preliminary characterisation of Nrap suggested it was likely to participate in ribosome biogenesis but as with many other nucleolar proteins, the functional role of Nrap is largely unknown. In this study, the role of mammalian Nrap in the nucleolus and in ribosome biogenesis was explored. Initially, a number of tools were generated to investigate Nrap function. This involved raising and purifying a polyclonal antibody against the N-terminal region of Nrap. The anti-Nrap antibody was found to detect two Nrap bands in mouse fibroblast cells, possibly corresponding to the two mouse Nrap isoforms, and . In addition, mammalian expression vectors containing the full Nrap sequence as well as deletion constructs were created. The subcellular localisation of each construct was observed by fluorescent microscopy. It was revealed that recombinant Nrap did not localise to the nucleolus, possibly because it was exported to undergo degradation by the 26S proteasome. Two putative NLSs were found to be responsible for directing Nrap to the nucleus but a region accountable for nucleolar localisation was not identified. The data indicated that multiple domains working together are likely to direct Nrap to the nucleolus. Nrap was also observed to co-localise with nucleolar proteins B23 and p19ARF. Moreover, it was shown by reciprocal immunoprecipitation that these three nucleolar proteins existed in a complex in unsynchronised mouse fibroblast cells. Recent reports demonstrated a complex relationship between B23 and p19ARF although the functional significance remained unclear. Nrap's in vivo association with B23 and p19ARF indicated a specific functional role in the nucleolus. Nrap knockdown using siRNA significantly increased B23 protein levels in a dose-dependent manner and down-regulated p19ARF protein levels at higher siRNA concentration. Preliminary studies also implicated Nrap in cell proliferation through these novel interactions. Both endogenous and recombinant Nrap were found to be highly unstable suggesting that Nrap might regulate B23 and p19ARF through its own tightly regulated stability. Finally, the role of Nrap in rRNA processing was investigated by northern blot analysis. Nrap knockdown was found to affect the levels of 45S, 32S and 28S rRNAs. The changes found may be a consequence of the concurrent perturbation in the levels of B23 and p19ARF caused by Nrap knockdown. As the results were not consistent with previous reports, it was likely that changes to rRNA processing could be contributed to Nrap loss of function. This study demonstrated for the first time a functional role of Nrap in rRNA processing possibly through its association with B23 and p19ARF.

The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. Nrap is a novel nucleolar protein found to be present in all eukaryotes. Preliminary characterisation of Nrap suggested it was likely to participate in ribosome biogenesis but as with many other nucleolar proteins, the functional role of Nrap is largely unknown. In this study, the role of mammalian Nrap in the nucleolus and in ribosome biogenesis was explored. Initially, a number of tools were generated to investigate Nrap function. This involved raising and purifying a polyclonal antibody against the N-terminal region of Nrap. The anti-Nrap antibody was found to detect two Nrap bands in mouse fibroblast cells, possibly corresponding to the two mouse Nrap isoforms, and . In addition, mammalian expression vectors containing the full Nrap sequence as well as deletion constructs were created. The subcellular localisation of each construct was observed by fluorescent microscopy. It was revealed that recombinant Nrap did not localise to the nucleolus, possibly because it was exported to undergo degradation by the 26S proteasome. Two putative NLSs were found to be responsible for directing Nrap to the nucleus but a region accountable for nucleolar localisation was not identified. The data indicated that multiple domains working together are likely to direct Nrap to the nucleolus. Nrap was also observed to co-localise with nucleolar proteins B23 and p19ARF. Moreover, it was shown by reciprocal immunoprecipitation that these three nucleolar proteins existed in a complex in unsynchronised mouse fibroblast cells. Recent reports demonstrated a complex relationship between B23 and p19ARF although the functional significance remained unclear. Nrap's in vivo association with B23 and p19ARF indicated a specific functional role in the nucleolus. Nrap knockdown using siRNA significantly increased B23 protein levels in a dose-dependent manner and down-regulated p19ARF protein levels at higher siRNA concentration. Preliminary studies also implicated Nrap in cell proliferation through these novel interactions. Both endogenous and recombinant Nrap were found to be highly unstable suggesting that Nrap might regulate B23 and p19ARF through its own tightly regulated stability. Finally, the role of Nrap in rRNA processing was investigated by northern blot analysis. Nrap knockdown was found to affect the levels of 45S, 32S and 28S rRNAs. The changes found may be a consequence of the concurrent perturbation in the levels of B23 and p19ARF caused by Nrap knockdown. As the results were not consistent with previous reports, it was likely that changes to rRNA processing could be contributed to Nrap loss of function. This study demonstrated for the first time a functional role of Nrap in rRNA processing possibly through its association with B23 and p19ARF.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text

Al melanoma cutani, els gens que presenten una major incidència mutacional són els gens BRAF i NRAS. Alteracions en aquests gens resulten en l'activació constitutiva de la via de RAS-ERK1/2, contribuint activament així en el desenvolupament i la progressió tumoral del melanoma. Tot i que ambdues mutacions donen lloc a alteracions de la mateixa via de senyalització, està àmpliament descrit que els tumors que es generen d’aquestes constitueixen dues entitats diferents tant a nivell molecular com des del punt de vista de la clínica. Una qüestió rellevant al voltant rau en el fet que mentre els melanomes mutats en BRAF disposen de teràpies específiques dirigides contra aquest oncogèn, els melanomes que presenten mutacions en NRAS no tenen tractaments específics. Com a conseqüència, aquests pacients són tractats amb teràpies antitumorals més genèriques, amb taxes de resposta molt menors i a més amb una elevada toxicitat. En aquest context, desemmascar les diferències moleculars existents entre els tumors amb mutacions en BRAF i en NRAS és essencial per a l'establiment de noves estratègies terapèutiques dirigides a pacients que presenten mutacions en NRAS. Resultats obtinguts amb anterioritat al nostre grup de recerca, juntament amb els d'altres investigacions, han confirmat la presència de diferents patrons metabòlics subjectes a la regulació per BRAFV600E. No obstant això, gairebé no existeix evidència al voltant del paper de les mutacions en NRAS a la regulació metabòlica. L'establiment de característiques metabòliques específiques de melanomes amb mutacions en NRAS podria contribuir al desenvolupament de nous enfocaments terapèutics dirigits contra aquest tipus de tumor. Durant el desenvolupament d'aquest estudi hem investigat les implicacions moleculars derivades de la manca de glucosa en cèl·lules de melanoma mutades en NRASQ61 i BRAFV600E, per tal d'establir si la presència de característiques metabòliques depenent de NRAS podria ser explotada per al desenvolupament de noves teràpies contra aquest tipus de tumor. En aquest estudi, hem demostrat la presència de patrons metabòlics sota el control d'NRASQ61. Les cèl·lules que presenten mutacions en NRASQ61 mostren una resposta diferencial a l'estrès metabòlic en comparació amb les cèl·lules mutades en BRAFV600E, donant com a resultat la hiperactivació de la via de RAS-ERK1/2 i a la sensibilització d'aquestes cèl·lules a l'inhibidor multi-cinasa Sorafenib. PFKFB2, PFKFB3 i PFK-1 són elements clau en la regulació d'aquest procés. Amb això, proposem una nova aproximació terapèutica per al tractament dirigit dels melanomes mutats en NRASQ61, establerta per la combinació de 2-deoxi-D-glucosa (2DG) i Sorafenib. Després dels resultats obtinguts, podem concloure que els tumors que presenten mutacions en NRAS i BRAF són entitats diferents a diferents nivells, no només a nivell clínic i molecular, sinó també a nivell metabòlic, el que implica l'existència de noves finestres terapèutiques per al tractament de tumors que presenten mutacions en NRAS.
Los genes BRAF y NRAS presentan una mayor incidencia mutacional en melanoma cutáneo. Alteraciones en estos genes resultan en la activación constitutiva de la vía de RAS-ERK1/2, lo que contribuye activamente al desarrollo y la progresión tumoral del melanoma. Aunque ambas mutaciones dan lugar a alteraciones de la misma vía de señalización, ha sido ampliamente descrito que los tumores que se generan de las mismas, constituyen dos entidades diferentes tanto a nivel molecular como desde el punto de vista clínico. Una cuestión relevante reside en el hecho de que mientras los melanomas mutados en BRAF disponen de terapias específicas dirigidas contra el oncogén, los melanomas que presentan mutaciones en NRAS carecen de tratamientos específicos. Como consecuencia, estos pacientes son tratados con tratamientos antitumorales más genéricos, que desembocan en tasas de respuesta mucho menores y en una elevada toxicidad. En este contexto, el desenmascaramiento de las diferencias moleculares existentes entre los tumores con mutaciones en BRAF y en NRAS es esencial para el establecimiento de nuevas estrategias terapéuticas dirigidas a pacientes que presentan mutaciones en NRAS. Resultados obtenidos previamente en nuestro grupo de investigación, sumados a los de otras investigaciones, han confirmado la presencia de diferentes patrones metabólicos sujetos a la regulación por BRAFV600E. Sin embargo, apenas existe evidencia sobre el papel de las mutaciones en NRAS en la regulación metabólica. El establecimiento de características metabólicas específicas de melanomas con mutaciones en NRAS podría contribuir al desarrollo de nuevos enfoques terapéuticos dirigidos contra este tipo de tumor. Durante el desarrollo de este estudio hemos investigado las implicaciones moleculares derivadas de la falta de glucosa en células de melanoma mutadas en NRASQ61 y BRAFV600E, con el fin de establecer si la presencia de características metabólicas dependientes de NRAS podría ser explotada para el desarrollo de nuevas terapias contra este tipo de tumor. En este estudio, hemos demostrado la presencia de patrones metabólicos bajo el control de NRASQ61. Las células que presentan mutaciones en NRASQ61 muestran una respuesta diferencial al estrés metabólico, en comparación con las células mutadas en BRAFV600E, que desemboca en la hiperactivación de la vía de RAS-ERK1/2 y en la sensibilización de estas células al inhibidor multi-quinasa Sorafenib. PFKFB2, PFKFB3 y PFK-1 son elementos clave en la regulación de este proceso. Adicionalmente, proponemos una nueva aproximación terapéutica para el tratamiento dirigido de los melanomas mutados en NRASQ61, basada en la combinación de 2-deoxi-D-glucosa (2DG) y Sorafenib. Tras los resultados obtenidos, podemos concluir que los tumores que presentan mutaciones en NRAS y BRAF son entidades diferentes a distintos niveles, no solo a nivel clínico y molecular, sino también a nivel metabólico, lo que implica la existencia de nuevas ventanas terapéuticas para el tratamiento de tumores que presentan mutaciones en NRAS.
BRAF and NRAS are the most commonly found mutated genes in cutaneous melanoma. Alterations in these genes result in the constitutive activation of the RAS-ERK1/2 pathway, contributing to tumor development and progression. Beside both genes are consecutive located in the same signaling cascade, BRAF and NRAS mutated tumors are considered two different entities at clinical and molecular levels, resulting in distinct signaling patterns and different biological behavior. Furthermore, while there is a first line of treatment using targeted therapy against BRAF mutant melanomas, NRAS mutant tumors remain without specific line of treatment, showing low response rates and high toxicity to the currently applied therapies. Thus, the understanding of the molecular differences between BRAF and NRAS mutant tumors is essential to improve therapeutic opportunities for the treatment of patients carrying NRAS mutations. Previous results in our group, together with additional investigations, have highlighted the presence of different metabolic settings subjected to BRAFV600E oncogene regulation. However, little is known about the role of NRAS mutations in metabolic rewiring. Deciphering metabolic settings in NRAS mutant melanomas could provide new avenues for the establishment of specific therapeutic approaches against these, until now, untargetable tumors. In this study, we have investigated the molecular implications of glucose starvation in NRASQ61 and BRAFV600E mutant cells in order to establish whether the presence of NRAS-dependent metabolic settings can be exploited for the development of targeted therapies against NRAS mutant melanomas. Overall, in this study we have demonstrated the presence of NRASQ61 oncogene-dependent metabolic settings. NRASQ61 mutant cells show a differential response to metabolic stress when compared to BRAFV600E mutant cells, which results in the hyperactivation of the RAS-ERK1/2 pathway and the sensitization to the multikinase inhibitor Sorafenib. PFKFB2, PFKFB3 and PFK-1 are key players in the regulation of this process. We also propose a novel approach for the specific targeting of NRASQ61 mutant melanomas based on the combination of 2-deoxy-D-glucose (2DG) and Sorafenib. We conclude that NRAS and BRAF mutant tumors are different entities at different levels, not only at molecular and clinical levels but also at metabolic level and this fact provides a new therapeutic window for the targeting of NRAS mutant tumors.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Bibliography: leaves 53-56.
This paper aims to show the role that NRA can play in environmental and economic accounting, and the procedures that need to be followed when carrying out natural resources accounting. The paper first reviews the SNA and its shortcomings regarding the treatment of the environment and natural capital. This is done by looking at the SNA classification of assets and examining how far environmental attributes are accounted for. Then the paper proposes the use of NRA to correct the deficiencies of the SNA Satellite accounts are suggested for resource based sectors in the Botswana economy, in order to augment national accounts. It is stressed that economic growth is only correctly reflected if the accounting prices used reflect full opportunity costs i.e. correct for externalities.

A subfamília p21 RAS de pequenas GTPases, incluindo KRAS, HRAS e NRAS, participa de muitas redes de sinalização, incluindo proliferação celular, organização do citoesqueleto e apoptose, e é o alvo mais freqüente de mutações ativadoras em câncer. Mutações germinativas em KRAS e HRAS causam graves anormalidades desenvolvimentais levando às síndromes de Noonan, cárdio-facial-cutânea e Costello, porem mutações ativadoras germinativas em NRAS não foram descritas até hoje. A síndrome autoimune linfoproliferativa (ALPS) é o mais comum defeito genético de apoptose linfocitária, cursando com autoimunidade e acúmulo excessivo de linfócitos, particularmente do tipo T + CD4- CD8-. As mutações causadoras de ALPS descritas até hoje afetam a apoptose mediada por Fas, uma das vias extrínsecas de apoptose. Nós demonstramos aqui que os principais achados clínicos de ALPS, bem como uma predisposição para tumores hematológicos, podem ser causados por uma mutação heterozigota ativadora G13D no oncogene NRAS, sem causar prejuízo na apoptose mediada por Fas. O aumento na quantidade intracelular de NRAS ativo, ligado a GTP, induziu a um aumento da sinalização na via RAF/MEK/ERK, o que suprimiu a expressão da proteína pró-apoptótica BIM, e atenuou a apoptose intrínseca mitocondrial. Desta forma, uma mutação germinativa ativadora em NRAS causou um fenótipo clinico diferente do visto em pacientes com mutações em outros membros da família p21 RAS, cursando com um defeito imunológico seletivo, sem distúrbios generalizados do desenvolvimento
The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4-, CD8- ab T cells. Mutations in ALPS typically affect Fas-mediated apoptosis, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair Fas-mediated apoptosis. The increase in active, GTP-bound NRAS augmented RAF/MEK/ERK signaling which markedly decreased the pro-apoptotic protein BIM and attenuated intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects

In mammals, the natural resistance-associated macrophage protein 1 gene, Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay (USA) striped bass, Morone saxatilis, are currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. This dissertation characterizes an Nramp gene in this species (MsNramp ) and provide evidence for induction following Mycobacterium exposure. The striped bass MsNramp gene and 554 amino acid sequence contain all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein specific 'binding-protein-dependent transport system inner membrane component signature,' three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase-C phosphorylation in the amino- and carboxy termini and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely groups MsNramp with other teleost Nramps, and exhibits high sequence similarity with mammalian Nramp2. MsNramp expression was present in all tissues assayed by RT-PCR. Within one day of injection with Mycobacterium marinum, MsNramp expression in vivo was highly induced (17-fold) in peritoneal exudate cells (PE) relative to controls. Levels of MsNramp were increased three- and six-fold on days three and 15, respectively. Injection with Mycobacterium shottsii resulted in two-, five-, and three-fold increases in gene expression in PE over the time course. In vitro, PE expressed significantly higher levels of MsNramp at 4 and 24 hours post-treatment with live and heat-killed M. marinum. MsNramp response to LPS was dose-dependent in these cells, with maximum expression at 4 hr and 20 mug/ml LPS. Treatment of PE with LPS caused an increase in intracellular superoxide anion (O2-) levels, whereas treatment with live M. marinum caused a significant depression. Cultured anterior kidney cells responded to LPS with increased O2 - and MsNramp production, but were uninduced or suppressed relative to controls by mycobacteria. This study represents the first report of induction of an Nramp gene by mycobacteria in vivo or in vitro in a poikilothermic vertebrate, and supports reports of teleost Nramp induction by LPS.

La phytoremediation consiste à utiliser les plantes pour nettoyer des sols contaminés. Jusqu’ici, des plantes naturellement capables de tolérer et d’accumuler les polluants ont été utilisées pour cette approche. Cependant, l’utilisation de plantes transgéniques doit être considérée pour optimiser l’efficacité de la phytoremédiation. Le peuplier est une espèce adaptée pour la phytoremédiation et peut être utilisé pour des approches transgéniques. Néanmoins, son efficacité de phytoextraction est limitée par une forte accumulation de métaux dans les feuilles qui retournent au sol lors de leur chute. L’ingénierie génétique pourrait être utilisée pour résoudre ce problème, en modifiant l’expression de transporteurs de métaux soit pour limiter l’accumulation de métaux dans les feuilles, soit pour stimuler leur accumulation dans le bois.Dans le cadre de cette thèse, trois transporteurs potentiellement impliqués dans la tolérance et l’accumulation de métaux ont été caractérisés : PtIREG1, PtNRAMP3.1 et PtNRAMP3.2. L’expression de PtIREG1 chez la levure et chez Arabidopsis thaliana a montré que ce transporteur contribue à la tolérance au nickel. Des peupliers transgéniques chez lesquels l’expression de PtIREG1 est globalement augmentée ou ciblée dans le bois ont été générés. Des peupliers transgéniques chez lesquels l’expression de PtNRAMP3.1 ou PtNRAMP3.2 est modifiée ont également été générés au cours de cette thèse. Cela a permis de montrer que ces protéines fortement homologues ont des localisations subcellulaires distinctes : la membrane vacuolaire pour PtNRAMP3.2 et un compartiment connecté à l’appareil de Golgi pour PtNRAMP3.1. Des mesures de concentrations en métaux dans les feuilles des peupliers transgéniques ptNRAMP3.1 et PtNRAMP3.2 ont montré des différences avec le type sauvage non transformé, pour le cuivre, le manganèse, le cadmium et le zinc. Les résultats obtenus contribueront à l’élaboration de stratégies biotechnologiques pour réhabiliter les sols pollués
Phytoremediation is the use of plants to clean up polluted soils. Previous approaches have mostly used native plants able to tolerate, degrade and accumulate environmental pollutants such as toxic metals, but transgenic plants may also be considered for phytoremediation in the future. Poplar is well adapted for phytoremediation and suitable for molecular genetic studies. However, high metal accumulation in poplar leaves limits phytoextraction due to toxic metal return to the soil after leaf abscission. In order to circumvent this problem, genetic engineering can be used to limit metal accumulation in leaves or direct metal accumulation in poplar trunks using relevant metal transporter genes under the control of tissue-specific promoters. This thesis focuses on the characterization of 3 candidate metal transporters potentially involved in metal tolerance and accumulation in poplar: PtIREG1, PtNRAMP3.1 and PtNRAMP3.2. Expression of PtIREG1 in yeast and in Arabidopsis thaliana indicated that it contributes to nickel tolerance. Transgenic poplars were generated in which PtIREG1is either ectopically overexpressed or expressed specifically in wood tissues. PtNRAMP3.1 and PtNRAMP3.2 transgenic plants were also generated during this thesis. Despite their high similarity, PtNRAMP3.1 and PtNRAMP3.2 displayed distinct localizations in poplar: PtNRAMP3.2 is targeted to the vacuolar membrane whereas PtNRAMP3.1 localizes in a compartment connected with the Golgi apparatus. Metal concentrations were modified in leaves of transgenic plants grown on metal-contaminated or non-contaminated soil. The results obtained will contribute to develop a biotechnological approach using transgenic plants for the rehabilitation in metal polluted soils

Melanoma is the deadliest form of skin cancer and one of the most difficult cancers to treat. Gene alterations identified in melanoma pointed to distinct molecular subsets of tumors with direct implications in therapeutic strategies. Activating mutations in NRAS, found in 20-30% of melanomas have been associated with aggressive clinical behavior and a poor prognosis. Nevertheless, there is lack of effective targeted therapies for NRAS mutant melanoma.Out of the few MEK inhibitors, pimasertib, a potent inhibitor of both MEK1 and MEK2 has showed promising results in NRAS mutant advanced melanoma. However, as a single agent and similar to other MEK inhibitors, it showed a limited clinical benefit due to its rather cytostatic effect and high toxicity. Our and other preliminary studies clearly indicated a stimulation of MITF (Microphthalmia associated transcription factor), the master transcription factor regulating cell growth and differentiation in the melanocyte, under MEK inhibition challenge. Thus, in a context where the tumor suppressor p53 is largely inactivated in melanoma, the stimulation of MITF may be the cause of the restraint cytotoxic effects of MEK inhibitors. Therefore, we aimed to further investigate the downstream MITF targets that can explain the resistance to the drugs.First, we showed that, MEK inhibition (by Pimasertib) led to a significant inhibition of cell proliferation but with a very limited effect on apoptosis that may be explained by the systematic MITF upregulation in all lines tested. Indeed, Mimicking MITF activation of expression by stimulating cAMP conferred resistance to MEK inhibition and interestingly up-regulated Bcl-2 expression. Further evidence was provided by the fact that, acquired resistance to MEK inhibition is associated with substantial upregulation of the anti-apoptotic signaling MITF/Bcl-2. More importantly, selective Bcl-2 inhibition by ABT-199 or Bcl-2 knock out using CRISPER/Cas9 system restores the sensitivity of NRAS mutant melanoma cells to MEK inhibition and breaks the acquired resistance.Given the known p53 regulating effect on Bcl-2, we evaluated p53 reactivation by PRIMA-1Met (APR-246) under MEK inhibition on the promotion of apoptosis in a panel of Q61NRAS mutant melanoma cells. Strikingly and similarly, this combination not only resulted in a synergistic effect to induce massive apoptosis but also broke resistance to MEK inhibitors both in cells with wild type or mutant p53 alike.In conclusion, we showed that the activation of cAMP/MITF/Bcl-2 pathway is a main anti-apoptotic mechanism associated with resistance to MEK inhibition in NRAS mutant melanoma. We propose drug combinations cotargeting MEK and other proteins regulating apoptosis -p53/Bcl-2- as a promising and clinically relevant therapeutic strategy to not only act in synergy to cause massive apoptosis but also to overcome resistance to MEK inhibitors in NRAS mutant melanoma
Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie)
info:eu-repo/semantics/nonPublished

La mélanomagenèse est un processus complexe sous-tendu par des mécanismes cellulaires et moléculaires variés. L'ensemble de ces mécanismes moléculaires est impliqué dans les réseaux moléculaires permettant une signalisation coordonnée au sein de la cellule. De nombreuses publications montrent que les voies de signalisation MAPK et PI3K/AKT ont un rôle important dans la mélanomagenèse. NRAS et BRAF sont des oncogènes de la voie MAPK mutés respectivement dans 20% et 50% des mélanomes. PTEN est un gène suppresseur de tumeur inhibant la voie PI3K/AKT, dont la perte est souvent associée aux mutations de BRAF. Le traitement récent des mélanomes métastatiques avec les inhibiteurs spécifiques de BRAFV600E donne des résultats exceptionnels, mais ces résultats sont limités aux patients dont le mélanome est porteur de la mutation BRAFV600E, et il existe naturellement des échappements thérapeutiques, parfois lié à l'apparition de mutations NRAS. Nous avons choisi d'étudier le rôle de NRAS et de PTEN, qui sont des protéines majeures des voies MAPK et PI3K. Le but de ce travail est d'évaluer la coopération de NRAS et PTEN au cours de la mélanomagenèse. L'expression de PTEN est fréquemment altérée au cours du mélanome, mais le rôle de PTEN est mal connu. Au cours de ce travail, nous décrivons pour la première fois une mutation de NRAS concomitante à une perte de PTEN dans des prélèvements humains de mélanome et dans des lignées cellulaires humaines. Afin de comprendre l'effet de cette double mutation sur la mélanomagenèse, nous avons étudié des souris transgéniques avec expression d'une forme oncogénique de NRAS et/ou inactivation de PTEN dans le lignage mélanocytaire. L'inactivation isolée de PTEN n'a aucun effet sur la mélanomagénèse. En revanche, en association avec la mutation oncogénique de NRAS, la perte de PTEN accélère le développement des mélanomes, en réduisant le temps de latence et en provoquant l'apparition de métastases plus nombreuses en comparaison aux mélanomes présentant uniquement la mutation oncogénique de NRAS. Nous avons également démontré que la perte de PTEN induit un échappement au phénomène de sénescence. En conclusion, l'inactivation de PTEN coopère avec les mutations de NRAS pour l'initiation et la progression des mélanomes.

Sindbis virus (SINV) is an arthropod-borne Alphavirus in the family Togaviridae. Sindbis virus has a broad host range that includes avian, mammalian, and human hosts; therefore, its receptor(s) is/are highly conserved. When the mosquito imbibes a viremic blood meal, the virus infects the midgut cells, disseminates into the hemolymph, and eventually infects the salivary glands. The midgut is an organ of transmission and the virus must overcome the midgut epithelia infection- and escape-barriers. Sindbis virus infection is determined by the chance collision of the glycoproteins with a compatible receptor. Research has supported the involvement of high-affinity laminin receptor and heparan sulfate in SINV binding to host cells. However, it has been suggested that not all strains of SINV are dependent on heparan sulfate for attachment/entry and that SINV could be utilizing multiple receptors. A study using Drosophila demonstrated that, of the nine genes that encode for proteins that enhance SINV infection, only natural resistance-associated macrophage protein (NRAMP) was conserved. A symporter of divalent metals and hydrogen ions, NRAMP is ubiquitously expressed. Overexpression of NRAMP led to an increase in SINV infection of human cells while deletion of NRAMP in mouse and Drosophila decreased SINV infection. Sindbis virus could be utilizing this protein to overcome the infection barriers of mosquito midgut epithelia. In this study, NRAMP was localized to Aedes aegypti and Anopheles quadrimaculatus tissues via immunofluorescence assay and TR339-TaV-eGFP was detected in the midgut epithelia and visceral muscles. We suspect that NRAMP was detected on midguts and/or Malpighian tubules of Aedes aegypti and Anopheles quadrimaculatus. The similarities between the pattern of NRAMP labeling and TR339-TaV-eGFP infection of the midgut suggest that SINV infection is influenced by NRAMP in the midgut epithelia. Because NRAMP is ubiquitously expressed, this research provides insight into the attachment and entry phase of the arbovirus lifecycle.

The Nramp (natural resistance associated macrophage protein) family of transition metal ion transporters is an important class of secondary active transporters involved in metal homeostasis used by all branches of life. Two Nramp paralogs in mammals are required for the dietary uptake and endosomal recycling of non-heme iron, as well as in the innate immune response to intracellular bacterial infections. Mutations in or mis-regulation of Nramps are associated with iron disorders and immune pathologies causing disease in mammals, and Nramps have been implicated in heavy metal poisoning. The molecular explanations underlying disease phenotypes and metal selectivity by Nramps were poorly understood. Here I present the second X-ray crystal structure of a bacterial Nramp homolog, a mechanism for two disease-causing missense mutations in mammalian Nramps, and detail the role of Nramp’s metal-binding site methionine residue in metal selectivity. Using Deinococcus radiodurans Nramp (DraNramp) as a model system, I determined its crystal structure and successfully resolved the N-terminal half of TM1, which was absent from the previously determined Staphylococcus capitis Nramp structure. By using the DraNramp structure, in vitro proteoliposome reconstitutions, in vivo cell-based cobalt transport assays, and cysteine accessibility experiments, I explain that that a glycine-to-arginine disease-mutation in the N- terminal half of TM1 acts as a steric wedge locking the transporter in the inward state inhibiting its function, while another glycine-to-arginine disease-mutation in TM4 shifts the transporter to the outward state and impairs metal transport in a charge-dependent manner. Using many of the same methods, it is also shown here that Nramp’s metal-binding methionine prevents transport of hard alkaline earth metals including magnesium and calcium, is not required for the transport of softer transition metals, and promotes high-affinity transport of cadmium and manganese. These results provide molecular explanations for the disease phenotypes found in mammals bearing the glycine-to-arginine mutations, and also explain how Nramps specifically discriminate against the highly abundant divalent metals calcium and magnesium, and yet at the same time remain promiscuous in their ability to transport other divalent metals including the toxic metal cadmium.
Biology, Molecular and Cellular

Scalar field theory has been used to develop an accurate beam model for use with the National Radio Astronomy Observatory 91 meter radio telescope and the 6 cm dual feed system. The theoretical beam model was calibrated, to an accuracy of 3% of the beam peak, with a small sample of radio point sources within the declination range 23° ≤ δ ≤ 62°. The new beam model is shown to be effective in deconvolving differential beam maps, to a dynamic range of 30:1, by a maximum entropy deconvolution method.
Science, Faculty of
Physics and Astronomy, Department of
Graduate

This thesis examines the molecular properties of Nramp proteins by centering on the two mammalian orthologs. Nramp1 (Slc11a1) is expressed in phagocytic cells and restricts replication of intracellular pathogens by removing divalent metals from the phagolysosome. Nramp2 (DMT1, Slc11a2) mediates uptake of dietary iron in the duodenum and aids in the acquisition of transferrin-associated iron in many cell types. The first half of this thesis explores structure-function relationships. In Chapter 2, the role of charged amino acids within the membrane-spanning segments of Nramp2 was examined by site-specific mutagenesis. These studies identified several invariant charged residues essential for metal transport and pH regulation of activity. In Chapters 3 and 4, the effects of two NRAMP2 mutations found in human patients suffering from severe congenital hypochromic microcytic anemia and iron overload were characterized in vitro. The first mutation was an E399D substitution in a region known as the "conserved transport motif" of the protein. The second mutation was an R416C substitution at an invariant residue in TM9. The effects of both mutations on expression, activity, and subcellular targeting were characterized. In both cases, a quantitative reduction in Nramp2 expression was found to be the cause of microcytic anemia and iron overload in the patients. The second half of this thesis focuses on the subcellular targeting of Nramp1 and 2. In Chapter 5, cytoplasmic signal(s) in Nramp2 responsible for its subcellular targeting/internalization from the plasma membrane were studied. This work led to the identification of a tyrosine-based motif in the carboxyl terminus of Nramp2 (YLLNT555-559) critical for the transporter's internalization from the cell surface and its recycling back to the plasma membrane. Chapter 6 explored differences in trafficking between two splicing isoforms of Nramp2 and found that one isoform (isoform 1) possessed differences in internalization/recycling which enabeled it to become enriched at the plasma membrane. In Chapter 7, the subcellular trafficking properties of Nramp1, including cytoplasmic sequences responsible for targeting to lysosomes, were investigated by using chimeric Nramp1/Nramp2 proteins. This work led to the identification of a tyrosine-based motif (YGSI15-18) in the amino terminus of Nramp1 that functions as a lysosomal targeting signal.

Background: Rare diseases are chronic and debilitating and while individually they affect less than 1 in 2,000 people, collectively they have a huge impact affect around 1.8 million Australians. Approximately 80% of these have a genetic origin, however only 30% of patients receive a formal molecular diagnosis. RASopathies are a group of rare disease that are caused by a mutations in the genes involved in the RAS-MAPK pathway, the most common of which is Noonan syndrome, which is characterised by heart defects, short stature, chest deformities, and specific craniofacial features. This study focuses on the effects of a novel c.173C>T (p.Thr58Ile) mutation, found in the NRAS gene of a patient diagnosed with Noonan-like syndrome, on the localisation and function of the NRAS protein. In doing so this study aimed to validate the role of mutations in the patient’s disease and provide information toward the creation of an experimental pipeline for the validation of other rare variants. Methods: U87-MG cells were transiently transfected with the NRAS constructs tagged with GFP2, and stained with specific antibody markers to determine localisation of the proteins using confocal microscopy. Functional studies included an Annexin V apoptosis assay, using flow cytometry to detect and quantify levels of apoptosis in transfected and untransfected populations, and the prediction of conserved domains using online bioinformatic tools. Results and Conclusions: Preliminary results from this study suggest that there is a difference between the localisation and function of the mutant and wild type proteins. The mutant protein was seen to co-localise with the Golgi apparatus as expected if the mutant protein was constitutively active. However the mutant protein was also observed to co-localise with the markers for the plasma membrane, nucleus and endoplasmic reticulum indicating that the mutation affects more than just the activation of the protein. An increase in apoptosis observed in NRASMUT transfected population, in comparison to both the wild type and untransfected populations, indicates that the mutation is engaging the pro-apoptotic function of NRAS associated with an increased activation of the RAS–RAF–MAPKK–MAPK pathway. Bioinformatic analyses identified that the mutation is location within a number of motifs involved in the binding of GTP and thus the activation and inactivation of NRAS, directing further studies toward the proliferation, activation and interaction of NRAS with effectors.

Au cours de ma thèse, j’ai entrepris la caractérisation fonctionnelle et physiologique de trois membres de la famille NRAMP (pour Natural Resistance Associated Macrophage Protein) chez Arabidopsis thaliana. Les gènes de la famille NRAMP, présents chez des organismes aussi variés que les bactéries, la levure ou les vertébrés, codent des transporteurs de métaux. Ces protéines sont principalement impliquées dans le prélèvement et la distribution du fer dans les cellules. Chez les plantes, les mécanismes contrôlant le prélèvement du fer sont maintenant bien caractérisés alors que les étapes suivantes de distribution au sein des organes, cellules et organites intracellulaires sont pour l’instant mal caractérisées. Dans un premier temps, j’ai montré que le gène AtNRAMP6 est exprimé dans les tissus conducteurs des feuilles, dans les sépales, le septum des siliques et le funicule de la graine. L’expression hétérologue de AtNRAMP6 dans la levure Saccharomyces cerevisiae provoque une sensibilité accrue au cadmium (Cd). L’étude de plantes mutantes « knock out » ou sur-expresseurs de AtNRAMP6 a montré que ce gène est impliqué, in planta, dans la tolérance au cadmium, en contrôlant vraisemblablement des pools de Cd intra-cellulaires. Ensuite, à travers la complémentation du mutant de levure ?smf2, j’ai montré que le gène AtNRAMP2 code un transporteur intracellulaire de manganèse (Mn). Chez Arabidopsis thaliana, le mutant « knock out » nramp2-1 est dérégulée dans l’homéostasie du manganèse suite à un défaut d’adressage du Mn dans les chloroplastes, affectant son activité photosynthétique. La protéine NRAMP2 n’est pas adressée aux chloroplastes mais à un autre type d’organite intracellulaire, probablement apparentés à des peroxysomes, jouant un rôle crucial dans l’acheminement du Mn aux chloroplastes. Enfin, j’ai montré que le gène AtNRAMP1, exprimé dans le cortex racinaire, code pour une protéine localisée sur le plasmalemme. La protéine AtNRAMP1 est capable complémenter le mutant de levure ?smf1, incapable de prélever le Mn du milieu de culture. Des expériences d’influx réalisées sur des racines de mutants « knock out » nramp1, surexpresseurs et plantes sauvages ont montré que AtNRAMP1 constitue le système de prélèvement de haute affinité du manganèse de la plante
During my thesis I have undertaken the functional and physiological characterization of three members of the NRAMP family (for Natural Resistant-Associated Macrophage Protein) in Arabidopsis thaliana. The members of this gene family, occurring in organisms like bacteria, yeast or vertebrate, code for metal transporters. The NRAMP proteins are mainly involved in the uptake and distribution of iron in the cells. In plants, the mechanisms controlling Fe uptake are quite well characterized whereas the following steps of distribution between organs, cells and organelles are less understood. First, I have shown that the AtNRAMP6 gene is expressed in the vascular system of leaves, in the sepals, the septum of the silique and in the funiculus of the seeds. The heterologous expression of AtNRAMP6 in the yeast Saccharomyces cerevisiaeinduces an increased sensitivity to cadmium. The study of “knock out” and over expressor plants has shown that this gene is involved, in planta, in cadmium tolerance, probably by controlling intra-cellular pools of Cd. Second, through the complementation of the yeast mutant ∆smf2, I have shown that AtNRAMP2 encodes an intra-cellular manganese (Mn) transporter. In Arabidopsis thaliana, the “knock out” mutant nramp2-1 is disregulated in the Mn homeostasis, due to a defect in Mn targeting to the chloroplasts that eventually affects the photosynthetic activity. The AtNRAMP2 protein is not targeted to the chloroplasts but rather to intra-cellular organelles probably related to peroxysomes, and plays a crucial role in the targeting of Mn to chloroplasts. Finally, I have shown that the AtNRAMP1 gene, expressed in the root cortex, encodes a plasma membrane located protein. AtNRAMP1 can complement the ∆smf1 yeast mutant impaired in Mn acquisition from the growth medium. Influx experiments realized on roots from “knock out”, over-expressors and control plants have established that AtNRAMP1 controls the high affinity uptake of Mn in plants

Les mélanocytes sont des cellules productrices de mélanines, à l’origine de la teinte de la peau, des yeux et des cheveux. Elles dérivent d'une population multipotente appelée cellules de la crête neurale, qui génère entre autres également tout le système nerveux périphérique. Une prolifération accrue des précurseurs des mélanocytes durant le développement entraine chez l’homme l’apparition d’un nævus mélanocytaire congénital (NMC). Cette prolifération est due à une mutation somatique au sein d'un de ces précurseurs, dans des gènes de la voie de signalisation des MAP-Kinases, NRAS ou BRAF. Les plus grandes formes, couvrant des parties entières du corps, sont syndromiques. Ils peuvent associer des mélanocytoses, des malformations ou tumeurs cérébrales ou méningées, parfois épileptogènes, ainsi qu'un risque autour de 5% de dégénérer en mélanome, dans un des sites atteints. Durant ma thèse j’ai exploré des modèles murins où les protéines NRAS ou BRAF constitutivement actives sont exprimées très tôt au cours de l’embryogenèse, dans les cellules de la crête neurale. Les embryons mutants BrafV600E connaissent une létalité embryonnaire, probablement due à une superposition de défauts vasculaires et cérébraux. En revanche, les souris NrasG12D sont viables,présentent des mélanocytoses extracutanées dans des sites divers, ainsi qu’une hyperpigmentation cutanée, visible en postnatal. Cette hyperpigmentation est associée à une augmentation de la densité folliculaire, ainsi qu’à un dérèglement du cycle du follicule pileux. Des cultures de cellules de crête neurale murines, BrafV600E ou NrasG12D et contrôles, ont permis d’élucider sur le plan moléculaire les effets de telles mutations
Melanocytes are the vertebrate cells that produce melanin, conferring color on skin, hair and eyes. They arise from a multipotent embryonic cell population called the neural crest, which also gives rise to the peripheral nervous system of the body and many other cell types. Abnormal proliferation of melanocyte precursors before birth can lead to human congenital melanocytic nevus (CMN). CMN are caused by prenatal somatic mutations in the NRAS or BRAF genes of the MAP-Kinase pathway, in one of these precursors. The largest CMN, covering entire segments of the body or head, are syndromic. They are sometimes associated with epileptogenic brain or meningeal malformations, tumors or melanocytosis, and they present a risk of about 5% in all these sites of becoming pediatric malignant melanoma. During my thesis, I explored mouse models expressing constitutively activated NRAS or BRAF proteins in neural crest cell lineages, from very early in embryogenesis. BrafV600E mutant embryos are embryonic lethal at mid-gestation, probably due to coinciding vascular and brain defects. In contrast, NrasG12D mice are viable, present extracutaneous melanocytosis in various sites as well as postnatal hyperpigmentation of the skin. This is associated with increased hair follicle density, and a deregulated hair cycle. Cell culture of mutant or wildtype mouse neural crest cells of both genotypes has permitted the comparison and discovery of molecular differences introduced by these mutations

Chez les plantes, les transporteurs de métaux jouent des rôles essentiels dans l'homéostasie métallique. Pour comprendre les liens entre le transport des métaux essentiels et la détoxication des métaux nocifs chez Arabidopsis, nous avons développé deux stratégies différentes: d'une part une approche ciblée pour comprendre la fonction des transporteurs de métaux AtNRAMP et, d'autre part, une approche de protéomique comparative pour identifier sans a priori de nouvelles protéines de la membrane plasmique régulées lors d'un stress par le cadmium. Les protéines NRAMP sont capables de transporter des métaux essentiels comme le Fe et le Mn et des métaux toxiques comme le cadmium. En associant des approches de génétique moléculaire, de biologie cellulaire et de biochimie, nous avons montré qu'AtNRAMP3 et AtNRAMP4 sont des protéines tonoplastiques. Pendant la germination, elles sont essentielles pour mobiliser les réserves de fer alors que chez la plante adulte, elles permettent le recyclage du Mn à partir de la vacuole. De plus, ces transporteurs jouent un rôle en réponse au stress oxydatif. La membrane plasmique représente la première interface en contact avec le cadmium. Pour identifier de nouvelles protéines membranaires impliquées dans les fonctions de perception, d'acquisition et d'exclusion du cadmium, nous avons développé une stratégie de protéomique quantitative reposant sur un marquage métabolique des cellules d'Arabidopsis thaliana par le 15N. L'analyse comparative entre les protéomes de la membrane plasmique de cellules stressées ou non par le cadmium est en cours. Quelques protéines candidates induites par le cadmium ont déjà été identifiées
In plants, metal transporters play major roles in intracellular metal homeostasis. Many metal transporters are able to transport essential metal as well as toxic metals. To understand the relationship between the transport of essential metals and the detoxification of noxious metals in Arabidopsis, we have used two strategies: a targeted approach with the aim to elucidate the function of NRAMP metal transporters and a global proteomic approach to identify novel plasma membrane proteins regulated under cadmium stress. NRAMP are able to transport a broad range of metals such as Fe, Mn and Cd. Using a combination of molecular genetic, cell biological and analytical approaches, we have shown that AtNRAMP3 and AtNRAMP4 are vacuolar membrane proteins with several redundant functions: during seed germination they are essential to mobilize vacuolar Fe stores while in adult plant they are required to recycle Mn from the vacuole. In addition, the nramp3nramp4 double mutant is also hypersensitive to the oxidative stress generated by cadmium and AtNRAMP3 and AtNRAMP4 accumulates after an oxidative stress. These proteins could be involved in the supply of metal cofactors to reactive oxygen species detoxifying enzymes. The plasma membrane is the first interface where Cd stress is perceived and where cadmium may be taken up or extruded from the cell. In order to characterize novel intrinsic proteins involved in these functions, we have developed a comparative proteomic strategy based on 15N metabolic labeling of Arabidopsis cell. Comparative analysis of the plasma membrane proteome is under way and we have already identified candidate proteins regulated by Cd

La voie de signalisation Ras/Raf/MEK/ERK joue un rôle crucial dans le mélanome, avec des mutations sur le: gènes BRaf et NRas dans 50% et 15% respectivement. De nouveaux traitements sont développés pour les patients porteurs d'une mutation BRaf, mais ils ne peuvent pas être utilisés chez les patients porteurs d'une mutation N Ras. Dans des lignées cellulaires de mélanomes humains, la signalisation en aval de la mutation N Ras passe préférentiellement par CRaf et non par BRaf. Cependant, les rôles spécifiques de BRaf et CRaf au cours du développement tumoral n'avaient jamais été étudiés in vivo. Nous avons développé un modèle murin de mélanome induit par l'expression de NRas°61K dans le lignage mélanocytaire. Dans ce modèle, la perte de BRaf et/ou de CRaf est induite de manière constitutive ou conditionnelle, spécifiquement dans le lignage mélanocytaire. Nous montrons que BRaf est nécessaire à la formation de nevi, mais n'est pas indispensable pour le développement des mélanomes. En revanche, CRaf joue un rôle mineur dans les étapes précoces de la carcinogenèse, alors qu'il est important pour le maintien tumoral. Toutefois, la perte de CRaf peut être compensée par BRaf pour la croissance des tumeurs, démontrant pour la première fois l'existence de fonctions redondantes de ces deux kinases dans le mélanome. Enfin, nous montrons que les cellules de mélanome murin sont capables de développer un mécanisme de résistance en l'absence de BRaf et CRaf, dépendant de la voie MAPK et impliquant ARaf. Les résultats obtenus mettent en évidence l'existence à la fois de fonctions spécifiques et de fonctions compensatoires pour les kinases de la famille Raf au cours de la mélanomagenèse
The RAS/RAF/ERK pathway plays a key role in melanoma, with BRAF and NRAS mutations in 50% and 15% of cases, respectively. The development of chemical inhibitors specifically targeting mutated v600EBRAF represents a major breakthrough in the treatment of metastatic melanoma, although resistance often develops quickly following treatment. Furthermore, such compounds cannot be used to treat half of melanoma patients, especially those mutated on NRAS. In this study, we evaluated the contribution of BRAF and its closely related kinase CRAF downstream of NRAS during tumoral progression in mouse models. We developed NRASQ61K-induced mouse melanoma models in which single or compound ablation of BRAF and CRAF genes can be achieved in the melanocyte lineage upon tyrosinase promoter-driven Cre or CreERT2 expression. These models allowed us investigating the role of both RAF kinases at each step of tumoral progression, from tumor initiation to metastatic melanoma. Temporaly-controlled concomitant ablation of BRAF and CRAF abolished nevi formation and melanoma progression and maintenance, showing that RAF signaling is absolutely required for NRAS-induced melanoma development. However, we will present data showing that BRAF and CRAF play specific roles during the different steps of melanoma progression. In addition, using primary cultures of melanomas, we show that ablation of RAF kinases often lead to the emergence of resistant cells showing reactivation of ERK in the absence of BRAF and CRAF. Our results disclose specific and complementary functions for BRAF and CRAF in NRAS-induced mouse melanoma, and pave the road for further studies on treatment and resistance mechanisms

Les nævi congénitaux mélanocytaires (NCM) sont des tumeurs cutanées bénignes qui résultent de la prolifération in utero des mélanocytes. Les NCM de taille supérieure à 20cm sont associés à un sur-risque de transformation en mélanome. La grande majorité des lésions présentent une mutation somatique unique affectant l'oncogène NRAS (mutations NRASQ61K/R), ce qui aboutit à l’expression une protéine NRAS constitutivement active. Pour les patients atteints de NCM, le seul traitement validé à l'heure actuelle est la résection chirurgicale mais ces exérèses sont souvent incomplètes et n'éliminent pas complètement le risque d'émergence du mélanome. L’objectif de notre travail a été de caractériser les voies dérégulées en aval de NRAS, et de valider in vitro ainsi que dans deux nouveaux modèles d’études précliniques de NCM (culture d’explants ex vivo et xénogreffes sur souris immunodéprimées Rag2-/-) l’effet d’inhibiteurs de ces voies. Quatorze patients porteurs de NCM mutés NRAS ont été inclus dans notre étude. Nous avons pu montrer qu’il existe in vitro une suractivation des voies de signalisation MAPK et PI3K-AKT dans les cellules næviques. In vitro, les inhibiteurs de MEK et AKT (respectivement le Binimetinib et le GSK690693) induisent une diminution de la prolifération et survie cellulaire. Ex vivo et in vivo, les inhibiteurs engendrent une diminution significative du nombre des nævocytes Sox10+ et Melan-A+ et du nombre de cellules prolifératives Ki67. Le but à terme sera de développer une thérapie ciblée intra-lésionnelle pouvant permettre de réduire le volume tumoral, d’éviter au maximum les exérèses itératives mutilantes et réduire le risque de transformation maligne
Congenital melanocytic nevi (CMN) are benign cutaneous tumors resulting from melanocytic proliferation during fetal development. CMN of more than 20cm in diameter are associated with socio-aesthetic issues and an increased risk of malignant transformation into melanoma. Most lesions are characterized by a single mutation affecting the oncogene NRAS, which leads to the expression of a constitutively active protein. For patients harboring CMN, the only validated treatment is surgical resection, though they are often incomplete and do not prevent the risk of melanoma emergence. Our objective was to characterize the molecular pathways deregulated downstream of NRAS, and to validate in vitro as well as in two innovative preclinical models of CMN (ex vivo CMN explants and xenografts on immunocompromised mice Rag2-/-) the effects of inhibitory molecules of those pathways. Fourteen patients harboring NRAS mutated CMN were prospectively included in our study. Our in vitro experiments reveal an overactivation of MAPK and PI3K-AKT signaling pathways in CMN cells. In vitro, MEK and AKT inhibitors (respectively Binimetinib and GSK690693) induce a decrease of proliferation and survival. Ex vivo and in vivo, inhibitors induce a decrease in nevocytic cells Sox10+ and Melan-A+ as well as a decrease in proliferative cells Ki67+, especially when combined. The associated goal of our study is to develop an intra-tumoral therapy, to be used as an adjuvant or neo-adjuvant therapy, and adapted to the genetic profile of these tumors, in order to decrease tumor volume and avoid repeated and mutilating surgeries, and potentially decrease the risk of malignant transformation

Hintergrund Die Strahlentherapie ist neben der radikalen Prostatektomie eine Standardtherapie zur Behandlung von Prostatatumoren und führt zu sehr guten Ergebnissen für die lokale Tumorkontrolle und für das Überleben. Allerdings ist, wie bei der Operation auch, dabei das Risiko eines Rezidivs für fortgeschrittene Tumoren im Gegensatz zu Tumoren in früheren Stadien relativ hoch. Daher besteht eine hohe Dringlichkeit zur Verbesserung der Strahlentherapie vor allem bei fortgeschrittenen Tumoren. Ein Ansatz hierfür ist die Kombination der Bestrahlung mit molekularen Therapien. Ziel dabei ist es, bestimmte Zielproteine zu blockieren, um die Strahlensensibilität der Prostatakarzinomzellen zu erhöhen. Ein potentielles Target könnte hierbei die Blockade des VEGF-C/NRP-2/Akt-Signalwegs (VEGF-C – vascular endothelial growth factor C; NRP-2 – Neuropilin 2; Akt – Proteinkinase B) sein. Im Prostatakarzinom sind die Konzentrationen von VEGF-C und NRP-2 im Vergleich zu normalen Prostatazellen erhöht. Aus Untersuchungen ist bekannt, dass beide Proteine eine progressive Wirkung auf die Tumorgenese haben. In Vorarbeiten zeigen Muders et al. (2009) zudem eine Aktivierung von Akt über die VEGF-C/NRP-2-Achse und eine darüber vermittelte Resistenz gegenüber oxidativem Stress durch H2O2. Akt wirkt in verschiedenen Tumorentitäten außerdem protektiv gegenüber Bestrahlung. Es besteht die Annahme, dass dies auch für Prostatakarzinomzellen gilt. Zielstellung Im Rahmen dieser Arbeit wurde untersucht, ob und über welchen Mechanismus VEGF-C, NRP-2 und Akt die Strahlenresistenz in Prostatakarzinomzelllinien beeinflussen. Methoden Es wurden in vitro- und in vivo-Experimente in den humanen Prostatakarzinomzelllinen PC-3, DU145, LNCaP sowie in PC-3-Xenografts durchgeführt. Der Einfluss von VEGF-C und NRP-2 auf die Strahlenresistenz wurde in vitro nach Herunterregulierung der entsprechenden Gene mittels siRNA beziehungsweise nach Supplementierung mit humanem rekombinanten VEGF-C in Koloniebildungsassays untersucht. Zur Ermittlung des Einflusses von VEGF-C und von NRP-2 auf mögliche Zellüberlebensmechanismen wurden der autophagische Flux nach Blockade der Autophagie mit Bafilomycin A1 mittels Western Blot, die DNA-Doppelstrangbruch-Reparatur mittels Quantifizierung der γH2AX Foci sowie die Zellzyklusverteilung mittels Durchflusszytometrie untersucht. Die Signalweiterleitung von VEGF-C über Akt sowie, als weitere Möglichkeit, die Signalweiterleitung über ERK1/2 wurden nach siRNA-Transfektion mit und ohne Bestrahlung mittels Western Blot geprüft. Weitere Versuche zu Akt erfolgten in vitro und in vivo mit dem PI3K/Akt-Inhibitor Nelfinavir in PC-3-Zellen. Der in vitro Effekt von Nelfinavir auf die Strahlenresistenz wurde dabei mithilfe eines Koloniebildungsassays nach Behandlung der Zellen mit 10 µM Nelfinavir getestet. In vivo wurde die Wirkung von Nelfinavir ohne sowie in Kombination mit Bestrahlung in PC-3-Xenografts in Nacktmäusen untersucht. Für die Bestimmung der Tumorwachstumszeit wurden die Mäuse mit 80 mg Nelfinavir/kg Körpergewicht 30 mal innerhalb von 6 Wochen behandelt. In einem weiteren Versuch wurde die lokale Tumorkontrolle bei gleichzeitiger fraktionierter Bestrahlung mit Gesamtdosen von 30 bis 120 Gy und einer Nachbeobachtungszeit von 180 Tagen bestimmt. Ergebnisse Die Untersuchungen zur Strahlenresistenz über den VEGF-C/NRP-2/Akt-Signalweg haben ergeben, dass in den drei Prostatakarzinomzelllinien PC-3, DU145 und LNCaP VEGF-C signifikant Strahlenresistenz vermittelt. Für NRP-2 hingegen wurde festgestellt, dass es in Abhängigkeit von der Zelllinie entweder zur Strahlenresistenz (DU145) oder zur Strahlensensibilisierung (PC-3) führt. Weiterhin wurde nachgewiesen, dass durch VEGF-C in PC-3 und DU145 weder über Akt noch über ERK1/2 Strahlenresistenz vermittelt wird. Die Versuche zu Strahlenresistenz vermittelnden Mechanismen ergaben, dass VEGF-C in unbestrahlten PC-3-Zellen die Autophagie fördert, NRP-2 jedoch nicht. Unter Bestrahlung war ein Effekt von VEGF-C und NRP-2 auf die Autophagie nicht reproduzierbar nachweisbar. Ein weiterer Versuch hat gezeigt, dass in PC-3 Autophagie keinen Einfluss auf das klonogene Überleben nach Bestrahlung hat. Außerdem wurde festgestellt, dass VEGF-C in PC-3 die DNA-Doppelstrangbruch-Reparatur nicht beeinflusst. Darüber hinaus wurde nachgewiesen, dass eine Verminderung des VEGF-C-Gehalts in PC-3 zum G2/M-Arrest führt. In DU145 konnte jedoch kein Effekt beobachtet werden. In den Untersuchungen zum Einfluss von Akt auf die Strahlenresistenz unabhängig von VEGF-C und NRP-2 wirkte Nelfinavir inhibierend auf die Akt-Phosphorylierung am Ser473 und beeinflusste das klonogene Überleben von PC-3-Zellen minimal. In PC-3-Xenografts führte Nelfinavir zu keiner Tumorwachstumsverzögerung und wirkte in vitro und in vivo nicht strahlensensibilisierend. Schlussfolgerung In den Versuchen konnte gezeigt werden, dass VEGF-C in Prostatakarzinomzellen Strahlenresistenz vermittelt. Diese Erkenntnis könnte als ein Forschungsansatz zur Entwicklung einer kombinierten Therapie aus VEGF-C-Blockade und Bestrahlung dienen. Ein potentieller Mechanismus, über den VEGF-C die Strahlenresistenz vermittelt, ist, in Abhängigkeit von der Zelllinie, die Aufhebung des G2/M-Arrests. NRP-2 wirkt in der Vermittlung von Strahlenresistenz beziehungsweise sensibilität je nach Zelllinie unterschiedlich. Hierzu sollten weitere Untersuchungen bezüglich möglicher Interaktionen innerhalb anderer Signalwege mit strahlensensibilisierendem Einfluss erfolgen. Innerhalb des untersuchten Signalwegs konnte weiterhin festgestellt werden, dass VEGF-C Strahlenresistenz nicht über Akt vermittelt. Die vorliegende Arbeit enthält die erste Studie sowohl zur Untersuchung des Einflusses von Nelfinavir in Kombination mit Bestrahlung auf das Überleben von Prostatakarzinomzellen in vitro als auch auf die Tumorwachstumszeit und die lokale Tumorkontrolle in vivo. Hierin konnte keine strahlensensibilisierende Wirkung von Nelfinavir nachgewiesen werden. Da Nelfinavir in Zellen anderer Tumorentitäten strahlensensibilisierend wirkt und außerdem bekannt ist, dass es in eine Reihe von Signalwegen eingreift, die das Zellüberleben fördern oder hemmen, sollte weiter geklärt werden, ob Tumorzellen mit einem bestimmten genetischen Profil besser auf die Behandlung mit Nelfinavir ansprechen
Background In addition to radical prostatectomy, radiotherapy is a standard therapy for the treatment of prostate tumours and leads to good results for local tumour control and survival. However, as with the resection, the risk of recurrence for advanced tumours is relatively high compared to tumours in earlier stages. Therefore, there is a high urgency to improve radiotherapy especially for advanced stages. One approach is the combination of irradiation with molecular therapies. The aim is to block certain target proteins to increase the radiosensitivity of the prostate carcinoma cells. A potential target could be the blockade of the VEGF-C/NRP-2/Akt signalling pathway (VEGF-C – vascular endothelial growth factor C; NRP-2 – neuropilin 2; Akt – protein kinase B). In prostate cancer the concentrations of VEGF-C and NRP-2 are increased compared to normal prostate cells. Studies have shown that both proteins have a progressive effect on tumourigenesis. In preliminary work Muders et al. (2009) also showed the activation of Akt via the VEGF-C/NRP-2 axis and a resistance to H2O2 induced oxidative stress. Akt also has a protective effect against irradiation in various tumour entities. It is assumed that this also applies to prostate carcinoma cells. Aim of the study Within the framework of this thesis, it was investigated whether and via which mechanism VEGF-C, NRP-2, and Akt affect the radioresistance in prostate carcinoma cell lines. Methods In vitro and in vivo experiments were performed in the human prostate carcinoma cell lines PC-3, DU145, LNCaP, as well as in PC-3 xenografts. The influence of VEGF-C and NRP-2 on the radioresistance was examined in vitro after knock down of the corresponding genes using siRNA or after supplementation with human recombinant VEGF-C in colony formation assays. In order to determine the influence of VEGF-C and NRP-2 on possible cell survival mechanisms, the autophagic flux was examined after the blockade of autophagy with bafilomycin A1 using western blot, the DNA double strand break repair by quantification of the γH2AX foci, and the cell cycle distribution by flow cytometry. The signal transduction of VEGF-C via Akt as well as, as a further possibility, the signal transduction via ERK1/2 were tested after siRNA transfection with and without irradiation using western blot. Further experiments on Akt were performed in vitro and in vivo with the PI3K/Akt inhibitor nelfinavir in PC-3 cells. The in vitro effect of nelfinavir on radioresistance was tested using a colony formation assay after treatment of the cells with 10 μM nelfinavir. In vivo, the effect of nelfinavir without and in combination with irradiation in PC-3 xenografts was investigated in nude mice. For the determination of the tumour growth time, the mice were treated with 80 mg nelfinavir/kg body weight 30 times within 6 weeks. In a further experiment, the local tumour control was determined with simultaneous fractionated irradiation with total doses of 30 to 120 Gy and a follow-up time of 180 days. Results The investigations on radioresistance via the VEGF-C/NRP-2/Akt signalling pathway showed that in the three prostate carcinoma cell lines PC-3, DU145, and LNCaP VEGF-C significantly mediates radioresistance. For NRP-2 however, it was found that, depending on the cell line, it either leads to radioresistance (DU145) or radiosensitization (PC-3). Further, it was shown that in PC-3 and DU145 VEGF-C does not mediate radioresistance via Akt or ERK1/2. The experiments on radioresistance mediating mechanisms revealed that VEGF-C promotes autophagy in untreated PC-3 cells, but NRP-2 does not. Under irradiation, an effect of VEGF-C and NRP-2 on autophagy could not be detected reproducibly. A further experiment has shown that in PC-3 autophagy has no influence on the clonogenic survival after irradiation. In addition, it was found that VEGF-C does not affect the DNA double strand break repair in PC-3. Furthermore, it was shown that a reduction in the VEGF-C content leads to a G2/M arrest in PC-3. However, no effect could be observed in DU145. In studies regarding the influence of Akt on radioresistance independent of VEGF-C and NRP-2, nelfinavir inhibited Akt phosphorylation at Ser473 and minimally affected the clonogenic survival of PC-3 cells. In PC-3 xenografts, nelfinavir did not lead to any tumour growth delay and did not have a radiosensitizing effect in vitro or in vivo. Conclusion In the experiments, it was shown that VEGF-C mediates radioresistance in prostate cancer cells. This finding could serve as a research approach for the development of a combined therapy of a VEGF-C blockade and irradiation. A potential mechanism by which VEGF-C mediates radioresistance is the reverse of the G2/M arrest, depending on the cell line. NRP-2 acts differently in the mediation of radioresistance or radiosensitivity, depending on the cell line. On this, further investigations should be carried out with regard to possible interactions within other signalling pathways with a radiosensitizing influence. Within the investigated signalling pathway, it was further shown that VEGF-C does not mediate radioresistance via Akt. The present work contains the first study examining the effect of nelfinavir in combination with irradiation on prostate cancer cell survival in vitro as well as on growth time and local tumour control in vivo. Herein no radiosensitizing effects of nelfinavir could be detected. Since nelfinavir radiosensitizes cells of other tumour entities and is also known to interfere with a series of signalling pathways that promote or inhibit cell survival, it should be clarified whether tumour cells with a particular genetic profile are more responsive to treatment with nelfinavir

Varias técnicas de física nuclear tem sido aplicadas no estudo de artefatos arqueológicos e de arte contribuindo para seu restauro e preservação. A aplicação destas técnicas são indicadas por não serem destrutivas, preservando o material a ser analisado. Neste trabalho, propomos um procedimento para a investigação indireta da dureza de espadas antigas, por meio de técnicas não destrutivas. Com este proposito as técnicas PIXE, NRA, XRD e RBS se adequam ao nosso estudo de espadas antigas, especificamente uma espada Indiana (Damascena) e outra Japonesa (Wakizashi). Com a técnica PIXE esperávamos identificar os elementos presentes nas laminas das espadas e em suas empunhaduras. Na espada Indiana os elementos identificados foram: Cr, Mn, Fe, Ni, Cu, Zn e As. Para a espada Japonesa somente o elemento Fe foi identificado, mas com o auxílio da técnica RBS identificamos também, um filme fino de carbono na superfície da lamina, medindo 0; 75 _m de espessura. Nas empunhaduras foram identificados os elementos Cr, Fe, Cu e Au para a espada Indiana; Fe, Cu, As e Ag na peca Habaki e S, Cl, K, CA, Fe, As e Au na peca Fuchi, estas duas pecas fazem parte da empunhadura da espada Japonesa. A técnica XRD foi usada para verificarmos as estruturas cristalinas que se formam na superfície das laminas durante o processo de forja (variação de 6 temperatura e deformações plástica). Estas informações possibilitam inferir sobre a temperatura de forja e consequências da deformação plásticas. A fase cristalina da superfície das laminas foi identificada como ferro na forma cristalina cubica de corpo centrado. Nesta estrutura, ha a formação de pequenos cristais orientados (cristalitos), que apresentaram tamanho médio da ordem de 200 _A. Foi verificado que ha também uma fase amorfa do ferro na espada Japonesa, sugerindo que o processo de forja alcançou temperaturas menores quando comparada com a espada Indiana. A espada Damascena _e muito famosa pela dureza e ductibilidade apresentada por sua lamina. Um elemento que pode contribuir para estas caraterísticas e o nitrogênio, que pode ser identificado usando a técnica NRA, mais especificamente a reação 15N(p; _)12C. O nitrogênio poderia ser introduzido na lamina durante o processo de endurecimento da região de corte. Neste processo a lamina era resfriada em urina animal, composta principalmente por acido úrico (C5H4N4O3) e ureia (NH2)2CO. Não foi possível identificarmos a presença de nitrogênio dentro do nosso limite de detecção (acima de 0; 263(4)% em massa de nitrogênio, valor referenciado para a amostra padrão CRM-298).
A set of physical techniques have been applied to characterize archaeological and art artifacts and contribute to its preservation and restoration. The application of these techniques are indicated because they are non invasive methods, preserving the material to be analyzed. In this work, we propose a procedure to investigate indirectly the hardness of ancient swords, by nondestructive techniques. With this aim, we decided to apply the techniques PIXE, NRA, XRD and RBS in the study of ancient swords, specially Indian (Damascus blade) and a Japanese (Wakizashi) swords. With PIXE we identified the major compounds in the blades and in their grips. In the Indian blade were identified the following elements: Cr, Mn, Fe, Ni, Cu, Zn e As. In the Japanese only iron was identified, although, with RBS we could identified a thin _lm of carbon on its surface. The grips were also analyzed and the results indicated to Indian were: Cr, Fe, Cu and Au; and to Japanese: Fe, Cu, As e Ag in the peace Habaki, and S, Cl, K, CA, Fe, As e Au in the peace Fuchi, those pieces are part of the grip. The XRD technique was applied to verify the crystalline structure which were formed during the forging process (hammering and quenching). These information can help to understand more about the quenching and hammering process. The crystalline phase in the surface of the blade was identified as iron. The surface is composed by crystallites oriented with grain size in order to 200_A, oriented as the result of hammering process. Also there is an amorphous phase in the Japanese blade, suggesting that in the forje process the temperature achieved was lower when compared with the Indian sword. The Damascus blade is famous due its hardness and ductility. An element that can improve these characteristics is the nitrogen. Its determination is possible using NRA technique, more specially the reaction 15N(p; _)12C. The nitrogen could been insert in the blade during the edge hardness process (in this process the blade was quenched into animal urine that its main compound are uric acid (C5H4N4O3) and urea (NH2)2CO, or in a brine). It was not possible to identify the presence of nitrogen within our limit of quantification.

This study identified current trends in website design and development for college and university recreation programs accredited by the NRPA/AALR Council on Accreditation. Emphasis was on design, content, governance, and development. The study was an attempt to provide insight for program administrators by identifying current practices related to website design and development. This was the first study to identify current trends in website design and development at nationally accredited recreation programs. This information can be used to evaluate existing websites and plan future website development. Data were collected using an electronic survey instrument created with E-listen software. Three invitations were emailed to program administrators at college and university recreation programs accredited by the NRPA/AALR Council on Accreditation (n=98). Basic descriptive statistics including measures of central tendency and frequency distributions were calculated to address the research questions. The results of this study showed that faculty were most often responsible for their program’s website construction, content and design input, and maintenance. Students did not play a large role in these responsibilities. The websites were most commonly maintained monthly. The most common components available on program websites were informative and served as marketing and recruiting tools. The majority of websites did not contain components for student entertainment or communication. Over half of the websites included links to professional organizations’ homepages. Just under half of the websites did not include a link to the National Recreation and Park Association.

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
O tipo mais comum de câncer de tireoide é o Carcinoma Papilífero (PTC), que representa 80% de todos os casos de neoplasias que acometem essa glândula. O PTC é um tumor maligno, de evolução lenta, que ocorre em qualquer idade, com maior frequência entre 30 a 40 anos. A via metabólica MAPK é a via mais associada ao PTC. Dentre as muitas proteínas que atuam nessa via e que se encontram alteradas nos casos de PTC, destacam-se aquelas codificadas por genes pertencentes às famílias RAS e BRAF. Considerando que na Amazônia brasileira, os estudos genéticos e clínicos sobre o câncer de tireoide são raros, o presente trabalho teve como objetivo investigar possíveis alterações nos genes HRAS, NRAS, KRAS e BRAF em pacientes portadores de PTC oriundos de um hospital público da cidade de Belém (PA), e fazer a associação entre a mutação encontrada e os achados bioquímicos e clínicos. Para isso, foram utilizadas como ferramentas a Reação em Cadeia da Polimerase (PCR) e a técnica de sequenciamento automático direto. A análise estatística foi realizada pelo pacote de software SPSS versão 21.0. Os dados contínuos foram expressos em média e desvio padrão e os dados categóricos foram expressos em porcentagem. O teste t de Student foi utilizado para avaliar as variáveis contínuas e os testes exato de Fisher e Qui-quadrado foram usados para analisar as variáveis categóricas. Foi considerado p<0,05 como significativo em todas as análises. Como resultados, a análise por sequenciamento do gene BRAF revelou a presença da mutação BRAFV600E em 21 de 53 pacientes (16 mulheres e 5 homens, 39,6%), assim como uma mutação nova no códon 38 no gene K-RAS (p.D38E). Em relação aos dados clínicos, houve uma associação significativa entre a mutação BRAFV600E e rouquidão, assim como entre a mutação BRAFV600E e metástase linfonodal. Adicionalmente, a observação de uma nova mutação no gene K-RAS indica que o número de alterações gênicas envolvendo a via metabólica MAPK encontra-se ainda incompleto. Os dados obtidos podem ser utilizados para uma melhor avaliação pré-cirúrgica de tumores tireoidianos, com o objetivo de aumentar a sensibilidade para a detecção do câncer e evitar cirurgias desnecessárias de lesões erroneamente identificadas como malignas.
The most common cancer of the thyroid is the papillary carcinoma (PTC), which represents 80% of the cases of cancer affecting this gland. PTC is a malignant tumor, with slow evolution, found in any age, but with a higher occurrence in patients between 30-40 years old. The metabolic pathway MAPK is the most associated with PTC. Among the several proteins which have a role in this pathway, we highlight the ones encoded by the genes belonging to families RAS and BRAF. Considering that there are few clinical and genetic studies focusing on thyroid cancer from Brazilian Amazonian region, the aim of this study was to investigate the occurrence of alterations in genes HRAS, NRAS, KRAS and BRAF in patients with PTC treated in a public hospitals, from Belém (PA), seeking to make an association between the mutations found and the biochemical and clinical findings. To achieve this goal, polymerase chain reaction (PCR) and direct automatic sequencing were used. Statistical analyses were performed using the software SPSS version 21.0. Continuous data were expressed as means and standard deviation and categorical data were described in terms of percentages. Student t Test was used to evaluate the continuous variables, while Fisher exact test and Chi-square were used to analyze categorical variables. We considered p<0.05 as significant value in all the analyses. Our results showed that, among the analyzed genes, only BRAF showed a mutation, BRAFV600E, in 21 out of the 53 patients (16 female and 5 males, 39.6%). Additionally, a new mutation in codon 38 of gene K-RAS was found (p.D38E). Considering clinical data, we found a significant association between the BRAFV600E mutation and hoarseness, as well as between this mutation and lymph node metastasis. In addition, the observation of a new mutation in the K-RAS gene indicates that the number of gene changes involving the MAPK pathway is still incomplete. The data obtained can be used for a better pre-surgical evaluation of thyroid tumors, in order to increase the sensitivity for the detection of cancer and avoid unnecessary surgeries of lesions erroneously identified as malignant.

Les protéines Ras jouent un rôle majeur dans le développement cellulaire. Faisant partie de la catégorie de petites GTPases, elles sont dotées d'un mécanisme fonctionnant tel un interrupteur moléculaire qui, dans leur cas, contrôle la transmission de signaux de croissance cellulaire. Liées au GTP, ces protéines adoptent une conformation leur permettant d'interagir avec des effecteurs en aval et, ainsi, activer la réplication et différenciation cellulaires. La réaction d'hydrolyse du GTP qui se déroule en leur centre, est accompagnée d'un changement conformationnel qui met fin à ces interactions, conduisant ainsi à l'état inactif de Ras, lié au GDP. Des mutations spécifiques de résidus bien déterminés entraînent une baisse du taux d'hydrolyse, laissant ainsi Ras liée au GTP. Or, de fortes concentrations de cette forme active de Ras ont été associées à une prolifération cellulaire anormale, caractéristique de la dissémination de tissus cancéreux. Il apparaît alors que l'élucidation des mécanismes employés par Ras pour accélérer le clivage du GTP constitue une étape majeure dans le développement de thérapies ciblées contre le cancer. Elles consisteraient à rétablir, au sein des mutants oncogéniques, un taux d'hydrolyse proche à celui mesuré au sein du type sauvage. Dans le but de mieux comprendre au niveau atomique les propriétés catalytiques de Ras, nous avons mené des simulations de dynamique moléculaire (MD) en décrivant le domaine G à différents niveaux de théorie (Mécanique Moléculaire (MM), Semi-empirique et Théorie de la Fonctionnelle de la Densité (DFT)). Ces calculs ont été réalisés pour les formes sauvage et mutées au niveau du résidu 61 de NRas. Ils ont été couplées à des caractérisations biomécaniques des complexes protéine-ligand étudiés, en utilisant la méthode des modes statiques. Cette méthode permet d'identifier des points chauds, réactifs, de la biomolécule et qui, suivant le critère de contrainte choisi, ont une influence mécanique sur la fonction GTPase de la protéine. Par conséquent, ils pourraient servir en temps que sites appropriés pour héberger des molécules médicamenteuses contenant des groupes chimiques spécifiques qui faciliteraient l'hydrolyse du GTP. Tout d'abord, les résultats obtenus montrent que le positionnement des molécules d'eau dans le cite actif est crucial pour catalyser efficacement la réaction. En effet, la répartition précise du solvant, observée dans le type sauvage, est perdue au sein des mutants de NRas considérés ici. Cette distribution différente des molécules d'eau ainsi que les modifications structurales du site actif engendrées par les substitutions du résidu Gln 61, ont un impact direct sur la densité électronique du GTP. Cette dernière présente un profil de type GDP au sein de la protéine de type sauvage uniquement, comme déterminé expérimentalement dans des études précédentes. Il apparaît donc que les mutations oncogéniques de Gln 61 perturbent cet effet catalytique majeur de NRas. Parmi trois propositions faites au cours de cette thèse sur des modifications à apporter à la forme mutée Q61R de NRas, une est présentée pendant la soutenance tandis que toutes les trois sont décrites dans le manuscript. Les groupes chimiques insérés au niveau du site identifié permettent de rétablir une distribution de l'eau comme celle observée dans le type sauvage. Pour terminer, lors de la soutenance uniquement, un chemin réactionnel alternatif de l'hydrolyse enzymatique du GTP est proposé
Ras subfamily of small GTPase proteins holds a key position in cell proliferation pathways. Indeed, the transmission of cell growth signals is controlled by proteins belonging to it. In their GTP-bound conformation, these proteins interact and activate downstream effectors of cell replication and differentiation. The hydrolysis reaction that takes place in their center, terminates these interactions, thereby leading to the GDP-bound inactive state. Point mutations of key residues lead to a hydrolysis rate drop that keeps Ras in a GTP-bound active state. Now, high concentrations of active Ras have been associated to abnormal cell proliferation, emblematic of cancerous tissues dissemination. With this into consideration, the elucidation of Ras mechanisms for accelerating GTP cleavage appears as a major step in the development of cancer targeted therapies that would consist in restoring the hydrolysing capabilities within oncogenic Ras to a wild-type rate. In an attempt to gain insight into Ras catalysing properties at the atomic level, unconstrained Molecular Dynamics (MD) simulations describing the G domain at different levels of theory (Molecular Mechanics (MM), Semi-empirical and Density Functional Theory (DFT)) were carried out for NRas member in its wild-type and Gln 61 mutated forms. These simulations were coupled to biomechanic characterisations of the complexes under inspection employing the static modes approach. The latter method, allows the identification of hot spots {\it i.e.} responsive residues of the biomolecule, that have a mechanical influence on the GTPase function of the protein. Hence, they could serve as suitable sites to host drug-like molecules containing specific chemical groups that would facilitate GTP hydrolysis. The obtained results show that water molecules positioning is crucial for efficiently catalysing the reaction that takes place in NRas center. Indeed, the precise positioning observed within the wild-type is lost within the mutants studied here. Furthermore, the active site structural modifications undergone upon Gln 61 substitutions, together with solvent distribution in it, impact directly GTP electronic density. The latter is accommodated to a GDP-like state within the wild-type protein only, as experimentally determined in previous investigations. Thus, oncogenic Gln 61 mutations impair this major catalysing effect. Among three engineered NRas proteins of the Q61R mutated form, proposed during this thesis, one is presented during the defence while the three are described in the manuscript. The chemical groups inserted at the identified site enable the recovery of water distribution as within the wild-type. To end, during the defence only, an alternative reaction pathway of the enzymatic reaction is proposed

Thanks for economic improvements since the ‘open-door policy’ in 1978; China’s international status has been improving economically and even politically. For instance, she has become a major contributor of BRIC which is a group of four most rapidly developing countries in the world, i.e. Brazil, Russia, India and China, and the largest buyer of U.S. treasury bonds in the world. Nevertheless, a number of intellectuals like Liu and Shirk pointed out that there are still a number of difficulties – both exogenous and endogenous – underneath.1 This research would like to concentrate on one of the aspects which Chinese investigators frequently highlighted: the hydropower development. This thesis will concentrate on the most controversial dam construction projects in China – Three Gorges Project in central China and the Nu River Project in Yunnan (hereafter abbreviate as ‘TGP’ and ‘NRP’ respectively) to examine the roles, influences and linkages of several critical actors in midst of decision-making processes – i.e. central leadership, provincial governments and the civil society in diverse perspectives. By utilizing the model of ‘Fragmented Authoritarianism’ (hereafter abbreviate as ‘FA’) suggested by Kenneth Lieberthal, I argue that the central leadership is still the most critical stakeholder who determines the success or failure of mega hydropower development projects albeit the increasing decentralization and rapid emergence of environmental non-governmental organizations (NGOs) in the recent decades after economic liberalization of China. Besides, the thesis would sketch out the tactics of participating and negotiations in the policy making processes of local leaders and social organizations and explain the apparent deliberation for hydropower development of the central authorities and the abilities of social organizations to halt dam constructions in the latest NRP in spite of being the most deterministic stakeholder in the polity. In the end, I would summarize general findings and enrich the FA model to facilitate the predictability of the model after social and economic changes associated with economic reforms.

Pour les réacteurs de génération IV, les carbures mixtes (U,Pu)C, avec leur grande densité en atomes fissiles et leurs excellentes propriétés thermiques, sont potentiellement des combustibles à la fois économiques (coeurs plus compacts et plus efficaces) et sûrs (marge à la fusion élevée). Un bon simulant de l’(U,Pu)C pour des études R&D sur son comportement est l’UC, puisqu’ils possèdent des structures très similaires. La synthèse par carbothermie a été utilisée car elle est la plus étudiée et celle actuellement envisagée industriellement. Cependant, elle implique la manipulation de poudres : sous air, le carbure peut réagir très violemment à température ambiante, et sous atmosphère contrôlée il est susceptible d’absorber les impuretés. Une installation inertée sous Ar, BàGCARA, a donc été utilisée. Les améliorations du procédé de fabrication ont notamment portés sur l’atmosphère de frittage afin d’évaluer l’impact sur la pureté des échantillons (vis-à-vis des quantités d’oxygène). La méthode originale d’analyse par faisceau d’ions a permis de déterminer la composition de surface (profils d’oxygène en profondeur dans les premiers 1 μm et stoechiométrie moyenne). Elle a pour la première fois été mise en oeuvre pour l’analyse de l’oxygène dans les matériaux carbonés. Les analyses DRX ont montré le passage par un intermédiaire réactionnel lors de la carbothermie et une meilleure cristallisation des échantillons fabriqués dans BàGCARA. Ils possèdent aussi une meilleure microstructure, densité et aspect visuel que ceux fabriqués par le procédé de référence. Un frittage sous vide mène à un UC plus dense avec moins de secondes phases que les frittages sous Ar, Ar/H2 ou sous contrôle de PC. Cependant, il n’a pas été possible d’analyser les carbures sans passage sous air ce qui peut impacter leur paramètre de maille et mener à leur détérioration. Lorsque l’UC est initialement exempt d’oxygène, il s’oxyde plus vite et plus intensément, de manière hétérogène. Les contraintes mécaniques induites entre les grains mènent à la fracturation du matériau et à une corrosion fissurante, puis à la décohésion du matériau. Une étude des mécanismes d’oxydation serait intéressante afin de valider et de comprendre l’évolution du matériau lorsqu’il est en contact avec l’oxygène. Une étude des mécanismes mis en jeu pourrait être envisagée par couplage des techniques d’EBSD et d’analyse par faisceau d’ions afin de vérifier s’il existe un lien entre une oxydation préférentielle des grains et leur orientation cristallographique
Mixed carbides (U, Pu)C, are good fuel candidate for IVth generation reactors because of their high fissile atoms density and excellent thermal properties for economical (more compact and efficient cores) and safety reasons (high melting margin). UC can be imagine as a surrogate material ror R&D studies on (U,Pu)C fuel behavior, because of their similar structures. The carbothermic reaction was used because it is the most studied and now consider for industrial process. However, it involves powders manipulation : in air, carbide can strongly react at room temperature and under controlled atmosphere it can absorb impurities. An inerted installation under Ar, BàGCARA, was therefore used. Process improvements were carried out, including the sintering atmosphere in order to evaluate the impact on the sample purity (about oxygen content). The original method by ion bearn analysis was used to determine the surface composition (oxygen in-depth profiles in the first microns and stoichiometry). This oxygen analysis was set for the first time in carbonaceous materials. XRD analysis showed the formation of an intermediate compound during the carbothermic reaction and a better crystallization of the samples fabricated in BàGCARA. They also have a better microstructure, density, and visual appearance if compared to former samples. Vacuum sintering leads to a denser UC with fewer second phases if compared to Ar, Ar/H2 or controlled PC atmospheres. However, it was not possible to analyze carbides without air contact which may impact their lattice parameter and lead to their deterioration. When the carbide is initially free of oxygen, it oxidizes faster, more intensely and heterogeneously. The mechanical stress induced between the grains lead to fracturing the material, to corrosion cracking and then a debonding of the material. A study of oxidation mechanisms would be interesting to validate and understand the evolution of the material in contact with oxygen. A study of the mechanisms involved could be considered by coupling EBSD technique and ion beam analysis to check whether there is a link between a preferential oxidation of the grains and their crystallographic orientation

L’interaction plasma-flamme résulte d’une interaction complexe entre processus physico-chimiques et hydrodynamiques. Toutefois, dans la littérature, l’influence des effets hydrodynamiques est souvent négligée au profit des effets thermiques et chimiques. L’objectif de nos travaux est de mettre en évidence expérimentalement le rôle des effets hydrodynamiques des décharges NRP dans l’interaction plasma-flamme. La PIV est d’abord mise en oeuvre pour caractériser l’écoulement d’air et étudier l’interaction plasma-écoulement inerte. Cette démarche a permis de mettre en évidence les effets hydrodynamiques des décharges NRP, caractérisés par une onde de choc (1-30 μs) et un noyau chaud (30-500 μs). Ensuite, une chambre de combustion à volume constant est utilisée pour réaliser des expériences d’allumage par décharges NRP de mélange méthane-air en régimes laminaire et turbulent. Nous avons effectué simultanément la PLIF OH et la chimiluminescence OH*. Une analyse basée sur la vitesse apparente du front réactif est conduite afin de comprendre le mécanisme d’allumage et le plissement du front de flamme à mesure que le nombre de décharges augmente
Different phenomena are involved in plasma-assisted ignition/combustion and result in a complex interaction of physico-chemical and hydrodynamic processes. However, in the literature, the influence of the hydrodynamic effects is often neglected and most of studies support chemical and thermal effects as the main mechanisms of interaction. The aim of this experimental study is to highlight the role of the hydrodynamic effects of NRP discharges for a better comprehension of the main mechanisms involved in plasma-flame interaction. PIV is performed to characterize the airflow and study plasma-inert flow interaction. This approach enabled highlight hydrodynamic effects of NRP discharges, namely a shock wave (1-30 μs) and a hot kernel (30-500μs). A constant volume combustion chamber is then used in reactive case to conduct single shot experiments of methane-air mixture ignition by NRP discharges in laminar and turbulent configurations. Simultaneous PLIF and chemiluminescence respectively on OH and OH* radicals are performed. An analysis based on apparent flame velocity of the reactive front is conducted in order to understand the ignition process as well as the observed flame front wrinkling as the number of discharges pulses is increased

Le médulloblastome (MB) est la plus fréquente des tumeurs cérébrales malignes pédiatriques qui représentent la première cause de mortalité par cancer chez l’enfant. Malgré les avancées des nouveaux traitements, les risques de récidive, séquelles et décès après traitement restent importants. Le récepteur de neuropiline-1 (NRP-1) a été récemment impliqué dans la progression tumorale des MBs et semble jouer un rôle important dans le phénotype des cellules souches cancéreuses (CSCs). Le ciblage de cette molécule pourrait ainsi présenter un intérêt thérapeutique dans le traitement des MBs. Nous avons sélectionné des cellules souches de MB capables de former des médullosphères (MS) à partir de 3 lignées cellulaires (DAOY, D283-Med et D341-Med). Ces modèles ont été caractérisés par l’expression de neuropilines (NRP-1 and NRP-2) et de marqueurs phénotypiques (CD133，CD15 et NF-M). Les résultats ont montré une augmentation significative de l’expression de NRP-1 par les cellules cultivées en médullosphères confortant notre stratégie de ciblage. L’impact du traitement de ces cellules par un composé innovant ciblant spécifiquement NRP-1, le MR438, a été ensuite évalué in vitro seul et association avec la radiothérapie notamment sur l’étude de la capacité d’auto-renouvellement des CSCs de MBs. Nous avons mis en évidence une diminution de la capacité d’autorenouvellement des cellules souches de MBs après exposition au MR438 avec une radiosensibilité augmentée pour les 3 modèles cellulaires. In vivo, le composé MR438 a été evalué sur des modèles de xénogreffes hétérotopiques chez la souris nude et montre un effet radiopotentialisant significatif pour les tumeurs issues de la lignée Daoy avec une tendance à la diminution de la progression tumorale pour les 2 autres lignées. De façon intéressante, le composé MR438 induit une diminution significative du nombre de cellules souches pour l’ensemble de nos modèles. Par conséquent, le composé semblerait induire les cellules souches vers un phénotype différencié au moins pour la lignée DAOY, même si les mécanismes n’ont pas pu être clairement élucidé. En conclusion, l’inhibition de NRP-1 via MR438 semble stimuler la différenciation des cellules souches cancéreuses pouvant à terme réduire la progression du MB et apporter un bénéfice en association avec la radiothérapie. L’evaluation du composé sur des modèles orthotopiques de MB permettrait d’obtenir des informations quant à son efficacité sur des modèles plus proche de la physiopathologie tenant compte de sa distribution au niveau cérébral
Medulloblastoma (MB) is the most common malignant pediatric brain tumors which is the leading cause of cancer death in children. Despite the progress of new treatments, the risk of recurrence, morbidity, and death after treatment remain important. The neuropilin-1 receptor (NRP-1) has recently been implicated in tumor progression of MBs, which seems to play an important role in the phenotype of cancer stem cells (CSCs). Targeting this molecule could thus present an interesting therapeutic value in the treatment of MB. We have selected cancer stem like cells of MBs in the form of medullospheres (MSs) from 3 cell lines (DAOY, D283-Med and Med-D341). These models were characterized by expression of neuropilins (NRP-1 and NRP-2) and phenotypic markers (CD133, CD15 and NF-M). Results showed a significant increase of the expression of NRP-1 by our CSCs models cultured in MSs that confirms our targeting strategy. The impact of the treatment of these cells with an innovative compound specifically targeting NRP-1, MR438, was then evaluated in vitro alone and in association with radiotherapy, especially on the study of the capacity for self-renewal. A decrease of self-renewal capacity for MB stem cells after exposition of MR438 with an increase of radiosensitivity for the 3 cell models in vitro was demonstrated. In vivo, MR438 was evaluated on heterotopic xenograft models in nude mice and showed a significant augmentation of radiosensitivity for DAOY tumors with a tendency to decrease tumor progression for the other 2 cell lines. Interestingly, the compound MR438 induced a significant decrease in the number of stem cells for all of our models. The compound appeared to induce CSCs to a differentiated phenotype at least for the DAOY cells, although mechanisms could not be clearly elucidated. In conclusion, inhibition of NRP-1 via MR438 seems to stimulate the differentiation of CSCs that may eventually reduce the progression of MB and bring a benefit in association with radiotherapy. Evaluation of this compound on orthotopic models of MB would provide information on its effectiveness on models closer to the physiopathology taking into account its distribution at the cerebral level

La prise en charge du mélanome métastatique a été bouleversée par les thérapies ciblées comme les inhibiteurs de RAF (RAFi) et les inhibiteurs de MEK (MEKi). Les RAFi permettent dans le mélanome BRAF V600 muté d’améliorer la survie mais leur effet est limité en monothérapie par l’hyperactivation paradoxale de la voie des MAPK dans les cellules non BRAF V600 mutée qui est responsable de l’apparition de mélanomes primitifs induits. Dans ce travail nous montrons la différence entre les modifications transcriptomiques induites par les RAFi dans des lignées cellulaires de mélanome BRAF mutées par rapport à celles induites dans des lignées non BRAF mutées ce qui a permis d’identifier des gènes potentiellement impliqués dans le développement de mélanomes induits. L’efficacité des RAFi est également limitée dans le temps par la survenue de résistances acquises. Nous montrons qu’une charge allélique mutée (CAM) de BRAF V600 élevée est potentiellement associée à une bonne réponse au traitement par RAFi et que la plasticité cellulaire induite par ZEB1 est associée à la résistance aux RAFi. Il n’existe pour le moment pas de thérapie ciblée approuvée dans le mélanome métastatique NRAS muté. Les MEKi sont limités par des résistances qui ne leur ont pas permis d’améliorer significativement la survie des patients. Nous montrons qu’une amplification de NRAS et la mutation MEK P124L sont responsables de résistance aux MEKi dans le mélanome NRAS muté. Nous avons testé le tramétinib (MEKi) en association avec d’autres thérapies ciblées dans des PDX de mélanome NRAS muté afin de mettre en évidence des combinaisons qui améliorent l’efficacité anti tumorale des MEKi dans cette indication
Targeted therapies like RAF inhibitors (RAFi) and MEK inhibitors (MEKi) have completely changed the therapeutic landscape in metastatic melanome. RAFi offer increased survival in patients with BRAF V600 mutated metastatic melanoma but they are limited in their use as single agents by an hyperactivation of the MAPK signaling pathway in non BRAF mutated cells that is responsible for the developpement of treatment induced primary melanomas. In this work we show the differences in the transcriptomic modifications induced by RAF inhibitos in BRAF mutated melanoma cell lines and NRAS mutated cell lines and we identified potential genes implicated in the development of treatment induced melanomas. The anti-tumor efficacity of RAFi is also limited by the acquired resistances that appear after several months of treatment. We show that a high BRAF V600E mutant allelic burden is potentially associated with a good response to RAFi and that ZEB1 induced cell plasticity is responsible for resistance to RAFi. No targeted therapy is approuved for NRAS mutated melanoma. Innate and acquired resistances limit the developpment of MEKi in this setting. We show that NRAS amplification and a MEK P124L mutation are responsible for resistance in NRAS melanoma. Finally, we tested five targeted therapies in combination with trametinib in NRAS mutated PDXs to identify combinations that improve the anti-tumoral effect of MEKi in this setting

Thesis (Ph.D.)--Boston University
Metastatic melanoma is the major cause of skin cancer death, and the annual incidence of melanoma continues to increase. Despite the impressively high rates of response to BRAF inhibitors in patients with melanomas harboring BRAF mutations, most of these patients eventually relapse after developing resistance to the drug, due in part to secondary mutations in NRAS. Although NRAS mutation is the second most common genetic mutation in melanoma patients (after BRAF mutation), there is currently no treatment option that targets NRAS-mutated melanomas. Previous reports have demonstrated the sensitivity of cancer cell lines carrying RAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCδ), suggesting the possible association between RAS mutational status and sensitivity to PKCδ inhibition. I therefore hypothesized that PKCδ inhibitors might also be cytotoxic in melanomas with primary or acquired NRAS mutations. In this project, the effect of PKCδ inhibition, and the efficacy of a new PKCcS inhibitor, BJE6-106 (B106), in melanoma were investigated. Inhibition of PKCδ inhibited the growth of multiple human melanoma cell lines carrying NRAS mutations, and induced apoptosis mediated by terminal caspase activation. Analysis of the molecular mechanisms demonstrated activation of the JNK pathway after PKCδ inhibition, leading to the activation (phosphorylation) of H2AX, a histone H2A variant. Activation of H2AX was attenuated when JNK1/2 levels were repressed, indicating that H2AX activation is mediated by the JNK pathway in response to PKCδ inhibition. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. To explore the potential of B106 further, melanoma cell lines harboring BRAF mutations that had evolved resistance to a BRAF inhibitor, PLX4032 (vemurafenib), were developed. B106 effectively induced cytotoxicity in these cells, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with PLX4032. Taken together, this work suggests that inhibition of PKCδ causes caspase-dependent apoptosis in melanomas with NRAS mutations and in PLX4032-resistant BRAF mutant melanomas. This apoptosis is mediated via activation of the JNK-H2AX pathway, which involves a novel role for phospho-H2AX in the execution of apoptosis.

De maneira a melhorar o desempenho de um Transistor de Efeito de Campo Metal-Óxido-Semicondutor (MOSFET), o germânio (Ge) é um forte candidato para substituir o silício (Si) como semicondutor, devido a sua alta mobilidade dos portadores de carga. Contudo, o filme de dióxido de germânio (GeO2) sobre Ge é solúvel em água e suas propriedades elétricas inferiores. Nesse sentido, a proposta desta dissertação de Mestrado é oxinitretar termicamente filmes de GeO2 em atmosfera de óxido nítrico (15NO), de maneira a melhorar as propriedades elétricas e físico-químicas dessas estruturas. Inicialmente, as amostras foram limpas quimicamente usando uma mistura de peróxido de hidrogênio (H2O2) e ácido clorídrico + água (HCl + H2O, 4:1). Os filmes de GeO2 foram crescidos termicamente sobre Ge usando atmosfera de oxigênio enriquecido 97% no isótopo de massa 18 (18O), com parâmetros na qual geraram um filme com espessura de ~5 nm. As oxinitretações foram realizadas em um forno térmico rápido com atmosfera de 15NO, nas temperaturas variando de 400-600°C, nos tempos de 1 a 5 minutos. O objetivo da oxinitretação foi criar um filme de oxinitreto de germânio (GeOxNy) com propriedades físico-químicas satisfatórias para a indústria de microeletrônica. Também foram realizados recozimentos térmicos em atmosfera inerte com objetivo de testar a estabilidade térmicas dos filmes de GeOxNy. Análise com Reação Nuclear (NRA) e Espectrometria de Retroespalhamento Rutherford em geometria de canalização (RBS-c) foram utilizadas para quantificar a quantidade total de oxigênio 18O e 16O, respectivamente. NRP também foi utilizada de modo a determinar o perfil de concentração em função da profundidade para as espécies de 18O e 15N. De modo a investigar a composição química das amostras, Espectroscopia de Fotoelétrons induzidos por raio-X (XPS) foi utilizada. Pelas análises por RBS e NRA do 18O, podemos observar que ocorre troca entre os isótopos de 18O e 16O para todas das temperaturas de oxinitretação. Este resultado corrobora com estudos recentes da literatura. Para as amostras oxinitretadas em 5 minutos a 500°C e todas as amostras oxinitretadas a 550°C e 600°C, ocorre troca isotópica completa. Observamos ainda por NRP que o 15N é incorporado mais superficialmente para as temperaturas de oxinitretação até 550°C. Resultados de XPS indicam formação maior de GeOxNy próximos da superfície das amostras e para temperaturas e/ou tempos maiores. Testes de estabilidade térmica indicam que a incorporação de nitrogênio mais próximo das superfície da amostra inibe a dessorção das espécies de GeO. As amostras que não foram oxinitretadas acabam dessorvendo quase por completo o filme de GeO2 quando realizados os recozimentos térmicos. Este efeito do nitrogênio incorporado próximo da superfície tem grande potencial para uso em camadas interfaciais entre semicondutor e dielétricos de porta.
In order to improve the performance of Metal-Oxide-Semiconductor Field Effect Transistor (MOSFET), germanium is a good candidate to replace silicon as semiconductor due to its higher charge carrier mobility. However, the germanium dioxide (GeO2) film over Ge is water soluble and produces poor electrical characteristics. In this way, this Master dissertation proposes thermal oxinitridation of the GeO2 films in nitric oxide (15NO) atmosphere in order to improve its electrical and physico-chemical characteristics. Samples were first cleaned using a mixture of hydrogen peroxide (H2O2) and hydrogen chloride + water (HCl + H2O, 4:1). GeO2 films were thermally grown on Ge using oxygen enriched in 97% in the isotope of mass 18, which generated ~5 nm thick film. Oxinitridation was performed in a rapid thermal furnace under 15NO atmosphere, at the 400-600°C temperature range, and 1-5 minutes time range. The goal was to form a germanium oxinitride film (GeOxNy) with physico-chemical properties that are satisfactory for microelectronics industry. We also performed thermal annealing in inert atmosphere to test the thermal stability of GeOxNy films. Nuclear Reaction Analysis (NRA) and Rutherford Backscattering Spectrometry in channeled geometry (RBS-c) were used to quantify the total amount of oxygen 18O and 16O, respectively. NRP was also performed to determine the 18O and 15N depth distribution. In order to investigate the chemical composition of the samples, X-ray Photoelectron Spectroscopy (XPS) was performed. RBS and NRA analysis showed isotopic exchange between 18O and 16O for all temperatures investigated. This result corroborates previous literature studies. Samples oxynitrided in 5 minutes at 500°C and all the samples oxinitrided at 550-600°C showed complete isotopic exchange. We also observed by NRP that nitrogen incorporation occurs more superficially until 550°C. XPS results indicate more formation of GeOxNy near the surface of the samples and for higher temperatures and/or time of oxinitredation. Thermal stability results indicated that the nitrogen incorporation near the sample surface inhibit the GeO desorption. On the other hand, samples that were not oxynitrided have almost all the GeO2 desorbed when thermal annealing is performed.

Dans la conception des futurs réacteurs de fusion, l’impact des interactions plasma – paroi pèse grandement sur le choix des matériaux à utiliser en première interface. L’utilisation du tritium en tant que combustible impose de plus des limites de sécurité quant à la quantité totale contenue dans le réacteur. L’analyse d’échantillons de parois de Tokamaks a montré une pénétration et une rétention du deutérium (utilisé à la place du tritium) au sein des matériaux carbonés; cette rétention est problématique car contrairement à la rétention dans les couches co-déposées, on ne peut espérer l’éliminer facilement. De part l’accès difficile aux échantillons réels, l’étude de ce phénomène se limite souvent à des analyses post-mortem.Afin d’accéder à la dynamique du phénomène et de s’affranchir de potentielles redistributions des éléments lors du stockage, un dispositif couplant micro analyse nucléaire (µNRA) et implantation basse énergie simultanée, visant à reproduire l’interaction entre le deutérium et les matériaux de la première paroi, a été mis en place. L’analyse µNRA permet de caractériser les profils de répartition en trois dimensions du deutérium en temps réel, à des échelles micrométriques. Des tests ont permis de confirmer le caractère non-perturbateur du faisceau d’analyse.On observe sur l’ensemble des données obtenues que la surface de l’échantillon (0-1 µm) présente une teneur en deutérium élevée et quasi constante ; la répartition du deutérium y est uniforme. A contrario, le deutérium piégé en profondeur (1-11 µm) se concentre dans des sites préférentiels liés à la microstructure du matériau. L’inventaire deutérium en profondeur semble augmenter avec la fluence incidente, malgré une grande dispersion des données attribuée à la variation de structure des zones étudiées. La saturation surfacique comme la migration en profondeur sont instantanées ; le stockage sous vide entraine une légère désorption du deutérium.Les observations faites par µNRA ont été croisées avec celles obtenues via d’autres techniques expérimentales. La µtomographie X a permis d’identifier clairement les porosités comme sites de localisation préférentielle du deutérium en profondeur. La micro-spectrométrie Raman a révélé la formation d’une couche amorphe fine (~30 nm) et saturée en deutérium à la surface du CFC suite à l’exposition au faisceau de deutérium. Enfin, la caractérisation expérimentale de la migration du deutérium dans les CFC obtenue est confrontée aux modèles existants, et un modèle simplifié original est proposé. Considérant que le dépôt en profondeur se produit par le biais de l’implantation et de la diffusion coulombienne du deutérium à la surface des porosités, il permet de reproduire qualitativement les profils de migration observés
Plasma-wall interactions play an important part while choosing materials for the first wall in future fusion reactors. Moreover, the use of tritium as a fuel will impose safety limits regarding the total amount present in the tokamak. Previous analyses of first-wall samples exposed to fusion plasma highlighted an in-bulk migration of deuterium (used as an analog to tritium) in carbon materials. Despite its limited value, this retention is problematic: contrary to co-deposited layers, it seems very unlikely to recover easily the deuterium retained in such a way. Because of the difficult access to in situ samples, most published studies on the subject were carried out using post-mortem sample analysis.In order to access to the dynamic of the phenomenon and come apart potential element redistribution during storage, we set up a bench intended for simultaneous low energy ion implantation, reproducing the deuterium interaction with first-wall materials, and high energy microbeam analysis. Nuclear reaction analysis performed at the micrometric scale (µNRA) allows characterizing deuterium repartition profiles in situ. This analysis technique was checked to be non-perturbative.We observed from the experimental data set that the material surface (depth 0-1 µm) displays a high and nearly constant deuterium content, with a uniform distribution. On the contrary, in-bulk deuterium (1-11 µm) localizes in preferential trapping sites related to the material microstructure. In-bulk deuterium inventory seems to increase with the incident fluence, in spite of the wide data scattering attributed to the structure variation of studied regions. Deuterium saturation at the surface as well as in-depth migration is instantaneous; in-vacuum storage leads only to a small deuterium global desorption.Observations made via µNRA were combined with results from other characterization techniques. X-ray µtomography allowed identifying porosities as the preferential trapping sites for in-depth deuterium retention. Raman µspectrometry disclosed the formation of an amorphous layer at the surface, very thin (~30 nm) and deuterium saturated, following deuterium irradiation.At last, we confronted the experimental characterization obtained with existing models for deuterium behaviour in carbon materials and proposed a simple and original one. Considering that in-depth retention is due to deuterium implantation and Coulombian diffusion at the open porosity surfaces, it allows reproducing qualitatively the observed experimental profiles

In humans, Salmonella infections cause two major clinical diseases: salmonellosis and typhoid fever. Silent carriage of the bacteria is frequent and contributes to disease dissemination. Using a genomic approach, we have reported the identification of ten loci (Ses1-Ses10) affecting Salmonella persistence in mice. A major locus, Ses1, was validated using a congenic approach. Nramp1 remains a strong candidate gene for Ses1 although we did not detect a significant interaction between Ses1 and Nramp1-/-. We also present the creation of new double congenic strains (Ses1/Ses4 and Ses1/Ses5) that will be used to validate the inheritance model of Salmonella clearance in females. Furthermore, the influence of Nramp1 on the transcriptome of Salmonella was investigated and diverse virulence mechanisms were shown to be involved. Notably, differential phoP expression, and the resulting differential expression of PhoP-regulated genes, was observed in the presence of Nramp1. Our results confirm the importance of host-pathogen interactions in determining the outcome of infection.
Les infections à salmonelles regroupent différentes maladies dont la salmonellose et la fièvre typhoïde. Le portage asymptomatique des salmonelles est fréquent et contribue à la dissémination de la maladie. En utilisant une approche de criblage génomique par locus, nous avons identifié dix loci (Ses1-Ses10) affectant la persistance de Salmonella chez la souris. Un locus majeur, Ses1, a été validé en utilisant des souris congéniques. Nramp1 demeure un gène candidat de choix pour Ses1 quoiqu’un test d’interaction Ses1/Nramp1-/- se soit avéré non significatif. Le modèle proposé de portage de Salmonella incluant des interactions entre les loci Ses1/Ses4 et Ses1/Ses5 sera exploré par la création de nouvelles lignées congéniques combinatoires qui ont été créées durant la préparation de cette thèse. L'influence de Nramp1 sur le transcriptome de Salmonella a été étudiée au niveau des mécanismes bactériens de virulence. En particulier, nous avons observé une expression différentielle de phoP, et par conséquent l’expression différentielle de gène dont l’expression est contrôlée par PhoP, en présence de Nramp1. Nos résultats confirment l'importance des interactions hôte-pathogène dans l’issue de l'infection à salmonelles.

O Laboratório de Implantação Iônica (LII) do IF-UFRGS possui uma linha de microfeixe instalada há poucos anos e que se encontra em fase de otimização e desenvolvimento. Esta é a primeira e única linha de microfeixe de íons instalada no Brasil e, portanto, é de grande interesse torná-la eficiente e disponibilizá-la para a comunidade científica. Algumas técnicas já estão bem desenvolvidas na linha de microfeixe: micro-PIXE, STIM e PBW (proton beam writing). A grande variedade de técnicas baseadas em feixes de íons de grande dimensão estimula estudos para a operacionalização dessas mesmas para o caso de feixes micrométricos. O estabelecimento e a operacionalização da técnica micro-NRA (reação nuclear) é importante para a consolidação de uma técnica capaz de determinar e mapear elementos leves que não são detectados com as técnicas atualmente em operação. Tais elementos, como por exemplo, carbono, nitrogênio e oxigênio, são os constituintes majoritários de grande parte dos materiais em estudo no LII. Neste estudo, apresentamos os parâmetros experimentais utilizados, bem como os resultados analíticos obtidos com a técnica de micro-NRA. Uma primeira avaliação sugere que a técnica tem grandes potencialidades para caracterização e mapeamento do carbono, enquanto que os resultados para o oxigênio e nitrogênio indicam a necessidade de uma técnica complementar. Adicionalmente, os resultados obtidos com micro-NRA foram comparados com os resultados provenientes de medidas com micro-PIXE utilizando um detector SDD com janela ultrafina, recentemente instalado na linha de microfeixe. Uma avaliação dos mapas elementares obtidos pelas duas técnicas indica que as medidas realizadas com micro-PIXE oferecem resultados com melhor estatística e mapas elementares de qualidade superior.
A couple of years ago, the Ion Implantation Laboratory (LII) at IF- UFRGS installed a microprobe station and ever since it has been under development. This is the first and unique ion microprobe station installed in Brazil and therefore it is of great interest to make it efficient and available to the scientific community. Some techniques are well developed in the microbeam station: micro-PIXE, STIM and proton beam writing. A wide variety of techniques based on broad ion beams stimulates further work for the implementation of such techniques for the case where the beam spot size is of the order of few micrometers. The study of the micro-NRA technique (Nuclear Reaction Analysis) is important since it can be used for the detection of light elements that are not detected with the techniques already in operation at the microprobe station. Such elements, such as carbon, nitrogen and oxygen are the major constituents of most materials under study in LII. In this study, we present the experimental parameters as well as the analytical results obtained with the micro-NRA technique. A preliminary assessment suggests that the technique has a great potential for the characterization and mapping of carbon, while the results for oxygen and nitrogen indicate the need for a complementary technique. Additionally, the results obtained with micro-NRA were compared with the results from measurements with micro-PIXE obtained with a SDD detector with ultra-thin window. An evaluation of the elemental maps obtained by the two techniques indicates that the measurements made with micro-PIXE provide results with better statistics and elemental maps with higher quality.

No âmbito da revisão do regime de arrendamento urbano (NRAU), foi desenvolvido no Laboratório Nacional de Engenharia Civil (LNEC) o Método de Avaliação do Estado de Conservação de imóveis (MAEC). Este método permite determinar, de forma expedita, o estado de conservação de edifícios para efeitos de atualização de rendas, mediante a realização de inspeções visuais ao local, efetuadas por técnicos qualificados. O MAEC está em vigor desde novembro de 2006, tendo sido realizadas mais de 30 000 vistorias durante os primeiros quatro anos de implementação. Em junho de 2010, cerca de 2400 técnicos avaliadores estavam inscritos para realizar vistorias com o MAEC. Considerou se assim oportuno realizar um estudo que analisa a experiência obtida com a aplicação do MAEC. Para o efeito, foram analisados os resultados das vistorias realizadas com o MAEC entre janeiro de 2007 e maio de 2010 e a opinião que os principais intervenientes têm sobre o MAEC (i.e., técnicos avaliadores e técnicos das Comissões Arbitrais Municipais). Como complemento, foi realizado um estudo comparativo do MAEC com 16 outros métodos que avaliam o estado de conservação de imóveis (7 portugueses e 9 estrangeiros). Com base nos resultados destas análises, foram avançadas propostas de aperfeiçoamento do MAEC de dois tipos: (i) propostas para simplificar a aplicação da atual metodologia de avaliação e aumentar o nível de informação recolhida e de rigor dos resultados; e (ii) propostas para que a metodologia de avaliação do MAEC permita obter resultados complementares para além daqueles para os quais foi originalmente concebido mas que a experiência de aplicação tem vindo a provar necessários. Como conclusão geral, verifica se que o MAEC cumpre os objetivos principais para os quais foi concebido. Porém, é possível introduzir alguns aperfeiçoamentos que contribuirão quer para a simplificação quer para o aumento do rigor da aplicação do método.

Ces travaux s’articulent autour de dérivés saccharidiques de type exo-glycals ou C-glycosides pour lesquels de nouvelles méthodologies synthétiques ainsi que des applications dans le domaine de la biologie ont été développées. Dans un premier temps, l’addition de nucléophiles soufrés et carbonés sur le carbone anomérique de différents exo-glycals activés a été réalisée, permettant un accès efficace à de nouveaux S-glycosides tertiaires ainsi qu’à des γ-glycoamino acides anomériques. Ces derniers ont été utilisés pour l’élaboration de peptides linéaires mixtes α/γ dont les propriétés de structuration ont ensuite été étudiées par RMN, IR, CD et modélisation moléculaire. De nouvelles plates-formes glycopeptidiques multifonctionnelles ont été préparées par cyclisation de ces peptides. Dans un second temps, le développement de peptidomimétiques ciblant le récepteur neuropiline-1, impliqué dans l’angiogenèse tumorale, a été entrepris. En s’appuyant sur des études de modélisation moléculaire, certains composés ont montré une bonne affinité pour le récepteur NRP-1 et l’un des composés a montré des propriétés prometteuses pour l’inhibition de la formation de tubules
This work is focused on the development of new synthetic pathways for exo-glycals functionalization and synthesis of bioactive compounds. The first part of this manuscript describes the efficient preparation of new tertiary S-glycosides and γ-glycoamino acids via Michael addition of thiols derivatives and carbanions on anomeric carbon of exo-glycals. The obtained γ-glycoamino acids were then incorporated in α/γ mixed peptides and their structural properties were studied by NMR, IR, CD and molecular modelling studies. Furthermore, cyclic multivalent platforms were built by intramolecular cyclization of these entities. The second part of the manuscript concerns the elaboration of peptidomimetics targeting neuropilin-1 receptor, implicated in tumor angiogenesis. Based on molecular modeling studies, some compounds showed interesting binding affinity for NRP-1 receptor and one of them displayed promising properties toward inhibition of tubule formation