CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Seminal plasma is the fluid portion of semen and its presence positively affects the survival and parameters of sperm motility in rabbits. This study aimed (i) to verify the seminal concentrations of sodium, chloride and citric acid as a function of collection month, the color and aspect of the ejaculate, and to find the frequency of ejaculations with the presence of gel fraction, color and aspect of the semen of rabbits on tropical climate, (ii) to meet monthly protein spots and their possible correlation with seminal and biochemical parameters in rabbits. 20 rabbits of New Zealand White, raised in the flat-deck, fed commercial feed were used. The samples were collected twice a week, and then evaluated for volume, color, aspect, vigor, motility and sperm concentration. After the evaluations, the samples were centrifuged to obtain seminal plasma, which was stored in eppendorfs tubes at -18 oC. A monthly seminal plasma pool of each animal was made to evaluate the concentrations of sodium, chloride, citric acid and proteins. Significant monthly variations were found (p <0.05) in the concentrations of sodium, chloride and citric acid in the seminal plasma of rabbits. All mineral constituents analyzed suffered significant influence (p <0.05) by the color of the ejaculate, and the highest concentrations were found in the white-yellowish ejaculates. Correlation studies found a high and significant association between concentrations of sodium and vigor (r = 0.80, p <0.001), and between sperm concentration and citric acid (r = -0.64, p <0.02). Most of the ejaculates of rabbits showed no gel fraction. The results also showed that the white and milky ejaculate are the most common for the species. It was also observed an average concentration of 2.73  0.31 mg / dl total protein in samples of seminal plasma. From the quantification of total protein were prepared two-dimensional electrophoresis gels stained SDS-PAGE with silver nitrate, with a pH gradient between 3 â 10, mesh 15% and a concentration of 100 mg of protein per sample for each month. Gels were analyzed with the Image Master 2D Platinum 6.0.  software. The gel containing the most spots (555 spots) was the gel of the month of May, and the gel with fewer spots (71 spots) was observed in January, but no effect of month on the amount of spots was detected. The majority of spots present in seminal plasma of rabbits have pI below 8, and these spots, about 40% have pI acid, the distribution of the spots as a function of pI was homogeneous throughout the year. The distribution of spots depending on the molecular weight widely varied between the months. Except of a few months, most of the protein had a molecular weight above 100 kDa. The number of protein spot positively and moderately correlated with total protein (r = 0.57, p <0.05) and citric acid (r = 0.59, p <0.05). In silico analysis of 1411 protein spots found compatible proteins with the Swiss-Prote database and TrEMBL (UniProtKB). It is concluded that the composition of the seminal plasma of rabbits showed a broad monthly variation and ejaculated with high concentrations of citric acid are undesirable. In addition, the protein profile of rabbits has great affinity of the proteins to acidic and high molecular weight, no influence of the months of the year on the amount of protein spots identified were found and bioinformatics analysis tool does not provide consistent results with those obtained gel electrophoresis, but it allows an estimate of the probable proteins that can be found. / O plasma seminal representa a porÃÃo fluida do sÃmen e sua presenÃa afeta positivamente a sobrevivÃncia e os parÃmetros de motilidade espermÃtica em coelhos. Este trabalho teve como objetivos (i) verificar as concentraÃÃes seminais de sÃdio, cloretos e Ãcido cÃtrico em funÃÃo do mÃs de coleta, da cor e do aspecto do ejaculado, e encontrar a frequÃncia de ejaculados com presenÃa de fraÃÃo gel, cor e aspecto do sÃmen de coelhos criados em clima tropical; (ii) conhecer os spots proteicos mensais e suas possÃveis correlaÃÃes com parÃmetros seminais e bioquÃmicos de coelhos. Foram utilizados 20 coelhos da raÃa Nova ZelÃndia Branca, criados em sistema flat-deck, alimentados com raÃÃo comercial. Os ejaculados foram coletados duas vezes por semana, e logo avaliados quanto ao volume, cor, aspecto, vigor, motilidade e concentraÃÃo espermÃtica. ApÃs as avaliaÃÃes os ejaculados foram centrifugados para obtenÃÃo do plasma seminal, que foi acondicionado em tubos eppendorfs a -18 oC. Um pool mensal de PS de cada animal foi feito para avaliaÃÃo das concentraÃÃes de sÃdio, cloretos, Ãcido cÃtrico e proteÃnas totais. Foram constatadas variaÃÃes mensais significativas (p<0,05) nas concentraÃÃes de sÃdio, cloretos e Ãcido cÃtrico no plasma seminal de coelhos. Todos os constituintes minerais analisados sofreram influencia significativa (p<0,05) da cor do ejaculado, e as concentraÃÃes mais elevadas foram constatadas nos ejaculados de cor branco-amarelada. O estudo de correlaÃÃes encontrou associaÃÃo alta e significativa entre as concentraÃÃes de sÃdio e vigor (r=0,80; p<0,001), bem como entre a concentraÃÃo espermÃtica e o Ãcido cÃtrico (r=-0,64; p<0,02). A maior parte dos ejaculados de coelhos nÃo apresentaram a fraÃÃo gel. Os resultados tambÃm mostraram que o ejaculado de cor branca e de aspecto leitoso sÃo os mais comuns nessa espÃcie. TambÃm foi verificado uma concentraÃÃo mÃdia de 2,73 Â0,31 &#956;g/dl de proteÃnas totais nas amostras de plasma seminal. A partir da quantificaÃÃo das proteÃnas totais foram confeccionados dois gÃis de eletroforese bidimensional SDS-PAGE corados com nitrato de prata, com gradiente de pH entre 3 â 10, malha de 15% e uma concentraÃÃo de 100 &#956;g de proteÃnas por amostra, para cada mÃs. Os gÃis foram analisados no software Image Master 2D Platinum 6.0. Â. O gel que continha o maior nÃmero de spots (555 spots) foi o do mÃs de maio, e o gel com menor nÃmero spots (71 spots) foi verificado em janeiro, mas nÃo houve efeito do mÃs sobre a quantidade de spots detectados. A maioria dos spots presentes no plasma seminal de coelhos tem pI abaixo de 8 e, destes spots, cerca de 40% tem pI Ãcido, a distribuiÃÃo dos spots em funÃÃo do pI foi homogÃnea ao longo do ano. A distribuiÃÃo dos spots em funÃÃo do peso molecular variou amplamente entre os meses. Com exceÃÃo de alguns os meses, a maioria das proteÃnas apresentaram peso molecular acima de 100 kDa. O nÃmero de spot proteicos correlacionaram-se moderada e positivamente com as proteÃnas totais (r=0,57; p<0,05) e com o Ãcido cÃtrico (r=0,59; p<0,05). A anÃlise in silico dos spots encontrou 1.411 proteÃnas compatÃveis com os bancos de dados Swiss-Prote e TrEMBL (UniProtKB). Concluiu-se que a composiÃÃo do plasma seminal de coelhos apresentou ampla variaÃÃo mensal e que ejaculados com alta concentraÃÃo de Ãcido cÃtrico sÃo indesejÃveis. AlÃm disso, o perfil proteico de coelhos apresenta grande parte das proteÃnas com afinidade a meio Ãcido e com alto peso molecular, nÃo houve influencia de meses do ano sobre a quantidade de spots proteicos detectados, e devido à ausÃncia de dados sobre coelhos, a ferramenta de anÃlise bioinformÃtica nÃo ofereceu resultados coerentes, mas permite uma estimativa das provÃveis proteÃnas que podem ser encontradas.

Estresses abióticos como seca, alta salinidade e baixas temperaturas, afetam o crescimento e a produtividade em culturas de interesse comercial como o feijoeiro comum. Proteínas DREB (Dehydration Responsive Element Binding) são fatores de transcrição que regulam genes específicos envolvidos na tolerância ao estresse abiótico. Para determinar como as plantas toleram condições ambientais adversas, variedades tolerantes, biologia molecular e bioinformática podem ser aplicadas para identificar e caracterizar genes que controlam mecanismos de adaptação a estresses. Baseado nas informações disponíveis nos bancos de dados públicos, a sequência da Orf completa do gene Phvul.009G029600.1| PACid:27146455 contendo 1062 pb foi encontrada e usada para o desenho dos primers e para o sequenciamento. A nova sequência é muito similar ao AtRAP2.4 e foi nomeada como PvDREB6A, segundo a análise filogenética. Ferramentas de predição mostraram que a sequência apresenta 354 aminoácidos e possui uma cópia do domínio AP2, que se dobra em uma estrutura com três ?-folhas e uma ?-hélice apresentando resíduos importantes e motivos específicos de reconhecimento e de ligação ao DNA. Além disso, um peptídeo trânsito foi detectado na porção N-terminal com um sítio de clivagem no resíduo 52. A interação deste fator de transcrição com seu domínio de ligação ao DNA foi validada por Electro Mobility Shift Assay (EMSA). A localização subcelular da proteína foi realizada e expressão da Green Fluorescent Proteín (GFP) foi detectada no núcleo. A transformação genética para a superexpressão do gene PvDREB6A em plantas de Arabidopsis thaliana Columbia-0 e mutantes nocaute para o gene AtRAP2.4 (Salk_020767C) foi realizada. Quatro eventos com cópia única e melhor expressão do gene PvDREB6A denominados Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/ pFEC2.1 #19.7 e Salk_020767C/ pFEC2.1 #23.7, foram selecionados. O evento Salk_020767C/pFEC2.1 #23.7 mostrou melhor expressão do gene PvDREB6A e foi visualizado sob luz UV. A análise funcional revelou que as plantas transgênicas submetidas ao déficit hídrico, à alta salinidade e ao frio, apresentaram maior taxa de sobrevivência. Plantas transgênicas superexpressando o gene PvDREB6A apresentaram menor taxa de desidratação e de vazamento de eletrólitos quando submetidas a estresses abióticos. Uma análise da expressão de genes relacionados à tolerância foi conduzida. A quantificação revelou que a expressão de 18 genes: AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, relacionados a tolerância a seca, sal e frio foram up-regulated devido à superexpressão do gene PvDREB6A de feijoeiro nas plantas transgênicas / Abiotic stresses like drought, high salinity and low temperatures affect growth and productivity in crops of economic interest such as common bean. DREB (Dehydration Responsive Element Binding) proteins are transcription factors that activate specific genes involved in tolerance to abiotic stress. To generate new information on the research for drought and other abiotic stresses, tolerant varieties, molecular biology and bioinformatics can be applied to identify and characterize genes that control plant defense and adaptation mechanisms to water deprivation, to excessive salt and to high/low temperature. Based on public databases, a common bean DREB sequence was found and an in silico study was carried out. A complete Orf sequence Phvul.009G029600.1 |PACid:27146455 containing 1062 bp was found and used for primer design and sequencing. The new sequence was very similar to AtRAP2.4 and named as PvDREB6A, according to phylogenetic analysis. Prediction tools showed that the deduced 354 aa sequence has one copy of the AP2 domain, folding in a three ?-sheets and one ?-helix structure, and presenting important residues and motifs for DNA contacting and binding specificity. In addition, a chloroplast transit peptide was detected at the N-terminal region with cleavage site in the 52 residue. Binding activity of this transcription factor was validated by Electro Mobility Shift Assay (EMSA). Subcellular localization was verified by transient expression of PvDREB6A::GFP in Nicotiana benthamiana and the expression of GFP was detected at the nucleus. Genetic transformation for overexpression of PvDREB6A gene in Arabidopsis thaliana wild type and knockout mutant for AtRAP2.4 gene was conducted. Four single copy events with better expression of the PvDREB6A named Col-0/pFEC2.1 #1, Salk_020767C/pFEC2.1 #13.1, Salk_020767C/pFEC2.1 #19.7 and Salk_020767C/pFEC2.1 #23.7 were selected. The event Salk_020767C/pFEC2.1 #23.7 showed the best expression of PvDREB6A and was visualized under UV light. Functional analysis, revealed that transgenic plants under water deficit, high salt, and cold showed higher survival rate. Transgenic plants overexpressing the PvDREB6A exhibited lower water loss rate and electrolyte leakage rate under abiotic stress. A gene expression analysis with tolerant-related genes was conduted. The quantification revealed that 18 genes, AtDC1.2, AtUSP, AtKIN1, AtERF69, AtGolS3, AtMT2A, AtCAP160, AtNTR1.7, AtGPR7, AtPDC2, AtLTI78, AtCOR15a, AtCOR15b, AtCOR47, AtCOR413, AtLEA6, AtLEA9 e AtLEA14, related to drought, salt and cold tolerance, were up-regulated due to the overexpression of PvDREB6A from common bean in the transgenic plants

Cette étude porte sur l’analyse des séries jurassiques du Shaqra Group (Toarcian to Kimmeridgian) qui affleurent de manière continue en Arabie centrale le long d’un transect de plus de 1000 km de long. Ces séries se sont accumulées sur une vaste plate-forme épicontinentale peu profonde, en contexte tropical. Ces affleurements permettent ainsi d’observer la partie occidentale des séries renfermant des systèmes pétroliers prolifiques exploités en subsurface en Arabie Saoudite. L’analyse sédimentologique de nombreuses coupes et la réalisation de corrélations stratigraphiques de haute résolution sur un transect de 600 km au sud de Riyad, complété par des corrélations avec les données de forage plus à l’est (entre Riyadh et le Rimthan Arch), permettent de distinguer une organisation séquentielle à différents ordres de fréquence et de reconstituer l’évolution de la plate-forme au sein de ces séquences. La plate-forme jurassique évolue d’une plate-forme horizontale caractérisée par des systèmes mixtes à la transition continental-marin du Toarcien au Callovien moyen (formations Marrat et Dhruma) vers un système de type rampe - bassin intrashelf du Callovien au Kimméridgien inférieur (formations Tuwaiq et Hanifa) pour finir par une plate-forme aggradante carbonatée et silico-clastique (Fm. Jubaila) puis carbonatée et évaporitique en contexte aride (Fm. Arab) au Kimméridgien. Les cycles tectono-stratigraphiques de 2ème ordre du Jurassique inférieur et moyen sont limités à la base et au sommet par des discontinuités régionales. Ils occupent un dépôt-centre stationnaire et décrivent un onlap côtier de grande ampleur avec un maximum transgressif au Callovien moyen (Upper Tuwaiq Mb.). Durant le Jurassique supérieur, les dépôts de rampe carbonatée de la Formation Hanifa passent progressivement vers l’ouest à des dépôts plus profonds de bassin intrashelf relativement riches en matière organique (Khurais - Rimthan Arch). La séquence Jubaila – Arab-D montre des variations d’épaisseur qui indiquent une déformation de grande longueur d’onde de la plate-forme arabe à cette période. Les faciès récifaux du membre Arab D sont interprétés comme représentant le maximum d’inondation de ce cycle qui se termine par le développement de systèmes carbonatés – évaporitiques à la fin du Jurassique. / They serve as westernmost reference for adjacent prolific reservoirs and source-rock bearing intrashelf basins. Several hierarchical sequences (second to fourth order) have been recognized in outcrops sections (600 km long south of Riyadh) and correlated with gamma-ray logs of subsurface wells (550 km long from Riyadh to Rimthan Arch). The Jurassic platform evolved from very-flat continental-to-nearshore mixed carbonate-siliciclastic platform (Marrat-Dhruma; Toarcian to Middle Callovian) to differentiated ramp with deep intrashelf basins (Tuwaiq-Hanifa; Callovian to Early Kimmeridgian) to a lowstand followed by flat aggraded platform (Jubaila-Arab-D; Kimmeridgian). Tectonic related siliciclastic influx took place in arid condition during the Kimmeridgian (Jubaila Fm.). The Jurassic platform ends with the mixed carbonate-evaporite systems of the Arab Fm. A first second-order tectono-eustatic cycle (Marrat to Tuwaiq) is bounded at the base and top by regional unconformities. It has a stationary depocenter, and show long-term coastal onlap and marine transgression that reached its maximum extent during the upper Tuwaiq (Middle Callovian). The Hanifa Fm. consists of four 3rd-order sequences aggraded flat-toped platform (outcrops to Khurais) marked at the base by argillaceous limestone and top by pure high-energy carbonates with localized reef buildups. The Jubaila Arab-D is two 3rd-order sequences begin with low-stand deposits followed by long-term transgression. These formed flat successions with lateral thickness variations controlled by differential subsidence increased in the Arabian Basin. The transgression is marked by storm-influenced inner-platform with sandstone quartz, grainstones and restricted lime-mudstone. The Maximum marine transgression is placed in the Arab-D with reef buildups in the westernmost inner-platform. During highstand, the reefs are gently prograding out into Rimthan Arch leaving behind restricted lagoon and sabkhah/salina anhydrite.

L’étude de l’Ordovicien de l’Anti-Atlas oriental s’intègre dans un vaste projet celui de l’étude de la plate-forme nord-gondwanienne qui se caractérise par une sédimentation essentiellement silico-clastique avec une polarité globale qui est Sud-Nord. Cette étude, basée sur les principes de la stratigraphie séquentielle et de la sédimentologie, met en évidence l’existence de deux systèmes de dépôts sur la marge nord-ouest de la plate-forme nord-gondwanienne. A l’Ordovicien moyen et supérieur préglaciaire, à l’emplacement de l’Anti-Atlas oriental, coexistent un système deltaïque (lato sensu) et un système de plate-forme dominée par l’action de la houle et des tempêtes. Ces deux systèmes de dépôts ont une certaine répartition spatiale et temporelle. Le système deltaïque domine le nord-est de l’Anti-Atlas oriental où il prend naissance à l’Est du Tafilalt. Ce système deltaïque est à dominance fluviale, il est actif lors des périodes de régression. Pendant les maximums de régression, il incise le haut du plateau continental en créant d’importantes structures érosives dont le remplissage se fait par des faciès de haute énergie à composante unidirectionnelle. Le système de haute énergie influencé par l’action de la houle et des tempêtes domine le secteur sud de l’Anti-Atlas oriental et se répand au nord au cours des périodes transgressives. La sédimentation durant l’Ordovicien préglaciaire sur la marge nord-ouest du Gondwana a un contrôle tectonique et eustatique avec une polarité des systèmes, dans le Tafilalt-Maïder qui diffère de celle de la plate-forme nord-gondwanienne et est Nord Est-Sud-Ouest. Le profil de dépôt est quasi plat où les clinoformes sont de faible amplitudes et se dessinent à l’intérieur des séquences de 3ème ordre. Le Darriwillien se caractérise par des une subsidence différentielle entre le domaine nord et le domaine sud en raison d’une tectonique active. Alors que, le Sandbien-Katien montre un taux de subsidence faible (x 10m/Ma) en adéquation avec un contexte intracratonique. L’Hirnantien dans le Tafilalt, décrit comme étant discontinu voir absent, est très bien développé il se caractérise par la mise en place d’une sédimentation mixte gréso-carbonaté à bryozoaires de type littoral et ce à l’extrémité oriental de l’Anti-Atlas et d’une épaisse série conglomératique (conglomérat d’Amessoui et d’Imzizoui) et flyschoïde qui peut atteindre les 500m dans le Tafilalt. / The Ordovician Anti-Atlas study is part of a larger project of comprehension the sedimentary system of the north Gondwana platform. This sedimentary area is characterized by essentially terrigenous sediments that come from the south from de West African Craton. This study based on sequence stratigraphy and sedimentology principles highlights two systems juxtaposed during the preglacial Ordovician period (before hirnantian). These two deposits systems have a spatial and temporal distribution. One deltaic occurs on north-eastern Tafilalt (east Anti-Atlas) and widespread during regressive periods. The second is storm dominate system which is developed in the south-western part of eastern Anti-Atlas and transgressive periods. The deltaic system is river dominant, it incise the continental shelf by creating a significant erosive structure whose filling is done by high energy facies unidirectional component. During the Ordovician, the sedimentation was controlled by tectonic and eustatic variation. The polarity of the system in the Tafilalt-Maïder is different from that of the northern Gondwana platform and it is given North East- South West. The Darriwilian characterized by differential subsidence, between the north and the south of the oriental Anti-Atlas, due to active tectonics, while, Sandbian-Katian show a low subsidence rates (x10m/Ma) in the line of intracratonic basin. The Hirnantian, in Tafilalt, is largely well developed and characterized by huge conglomeratic series, flysch deposits and Bryozoan limestone at the northeastern.

Pancreatic cancer is relatively uncommon. Despite its relative scarcity, it is the fourth-ranked cancer killer in the Western world with less than a 5% 5-year survival rate. The high mortality rate is due to the asymptomatic nature of the disease and the advanced stage at which it is usually diagnosed. S100P is a calcium-binding protein that has been shown to be highly expressed in the early stages of pancreatic cancer and has been proposed as a potential therapeutic target via the blocking of its interaction with its receptor RAGE, the receptor for advanced glycation end-products. In this thesis, computational techniques were employed on the NMR ensemble of S100P (PDB Accession code 1OZO) to identify potential inhibitors of the S100P-RAGE interaction in the hope of identifying a series of novel leads that could be developed into clinical candidates for the treatment of pancreatic cancer. In silico studies identified putative binding sites at the S100P dimeric interface capable of accommodating cromolyn, an anti-allergy drug shown to bind to the protein both in vitro and in vivo. Virtual screening of >1 million lead-like compounds using 3D pharmacophore models derived from the predicted binding interactions between S100P and cromolyn, identified 9,408 'hits'. These were hierarchically clustered according to similarities between chemical structures into 299 clusters and 77 singletons. Biological screening of 17 of the 'hits' identified from virtual screening stuidies, 4 of which were synthesised in-house, against pancreatic cancer cell lines identified five compounds that demonstrated an equal or greater capacity to reduce BxPC-3 S100P-expressing pancreatic cells' metastatic potential in vitro relative to cromolyn. Compound 24 in particular, showed significant (p<0.05) inhibition of invasion of these cells at a concentration of 100 μM that was comparable to cromolyn at the same concentration. This compound, structurally distinct from cromolyn, was successfully synthesised, purified and characterised in-house alongside 39 of its analogues. Biological screening of compound 24 and four of its analogues for anti-proliferative activity against BxPC-3 and Panc-1 pancreatic cancer cell lines showed all five compounds significantly (p < 0.0001) inhibiting proliferation in both cell lines at a concentration of 1 μM relative to the non-treated control. Hence, structurally distinct compounds that show promising inhibitory activity on the metastasis and proliferation of pancreatic cancer cells have been identified using a structure-based drug design methodology. These compounds, with further optimisation, could provide good starting points as therapeutic lead candidates for the treatment of pancreatic cancer.

L'objectif de cette thèse est d'étudier les transformations physico-chimiques lors de l'élaboration d'un verre à partir d'un mélange granulaire. Les mélanges vitrifiables industriel formant des verres de vitrage ou de conditionnement sont approchés par le système à trois oxydes SiO2-CaO-Na2O, apportés sous forme de sable, de calcaire et de carbonate de sodium. Cette simplification permet d'étudier le lien entre microstructrure de l'empilement granulaire et transformations pendant le chauffage. La variété des chemins réactionnels entre les grains de matières premières transmet en effet l'hétérogénéité du mélange au verre. Le système ternaire est décomposé en deux sous-systèmes. D'une part, les réactions entre le disilicate de sodium et 4 porteurs de calcium (CaCO3, CaO, CaSiO3, Ca2SiO4) révèlent la formation de couches d'espèces cristallines entre le grain calcique en cours de dissolution et le silicate fondu. D'autre part, la synthèse ex situ de carbonate mixte de calcium et de sodium est réalisée. Ce produit est analysé et caractérisé ; son apparition in situ dans un mélange ternaire est observée, et sa réaction avec la silice est étudiée. Dans un mélange vitrifiable silico-sodocalcique, la tomographie X in situ et post mortem montre l'importance de phénomènes transitoires de mouillage et de dégagement gazeux qui orientent les réactions chimiques. En particulier, un mécanisme local et de courte durée convertit une profondeur limitée des grains de calcaire en carbonate mixte. Enfin, l'homogénéité du verre obtenu à 1300°C est étudiée à l'aide d'une méthode de déconvolution de spectres Raman innovante, pour plusieurs granulométries et espèces porteuses de calcium. / This thesis focuses on the physical and chemical transformations undergone by a granula glass batch during the glass making process. Industrial window or container glass batches are complex mixtures, that we approached by the ternary oxide system SiO2-CaO-Na2O. Industrial raw materials, namely silica sand, limestone and sodium carbonate, are used. This simplification makes it possible to study the coupling between the granular microstructure and the physical and chemical transformations during the heating. Indeed, the multiple possible reaction paths propagate the heterogeneity of the granular batch to the glass. To support our study of the ternary system, two sub-system are investigated. On one hand, reactions between sodium disilicate and 4 calcium-bearing species (CaCO3, CaO, CaSiO3, Ca2SiO4) show the formation of successive layers of crystalline intermediate species between the dissolving grains and the molten silicate. On the other hand, sodium calcium mixed carbonate is synthesized ex situ and characterized. Its formation in a ternary glass batch is observed in situ, and its reactivity with silica is studied. In a ternary soda-lime glass batch, X-ray in situ and ex situ microtomography reveals the significance of wetting and degassing phenomena in orienting the grains towards a reaction path. Conversion of a limited depth of limestone grains into mixed carbonate for a short time is notably witnessed. This mechanism is strongly dependent on the grains surrounding limestone particles. Finally, we use an innovative deconvolution procedure to measure the impact of granulometry and the nature of calcium-bearing raw materials on the final glass homogeneity.

The thesis specializes in the computational description of pharmaceutically important compounds. A substantial number of pharmaceutical drugs are small molecules that are bound to an active site of an enzyme by the "lock (binding site) and key (drug)" model through non-covalent interactions. The association of enzymes with drugs cause an increase or decrease in the activity of enzymes. The main topic is focused on the computational elucidation of the structural basis for the interactions of the purine-like compounds with the enzyme cyclin- dependent kinase 2 that belongs to the protein-kinase enzyme family. These enzymes play an important role in the cell cycle regulation; their increased activity significantly contributes to the loss of control over cell proliferation, which is one of the primary causes of cancer cell formation. The study describes the binding motifs of roscovitine, which shows an inhibitory effect on the function of cyclin-dependent kinases, and its analogues containing bioisosteric central heterocycles in the complex with cyclin-dependent kinase 2. The binding affinity between the cyclin-dependent kinase 2 enzyme and the inhibitors was quantified as calculated binding scores and evaluated in relation to the conformation of the optimized structures. The hybrid model combining the...

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20674号 / 薬科博第85号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 山下 富義, 教授 髙倉 喜信, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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<p>  </p> <p>Epstein-Barr virus (EBV) is a complex oncogenic symbiont. The molecular mechanisms governing EBV carcinogenesis remain elusive and the functional interactions between virus and host cells are incompletely defined. Some of the known mechanisms include viral integration into the host genome, expression and mutation(s) of viral genes and the host response to the virus. Despite decades of research there is a lack of effective treatment options for EBV-positive cancer patients underscoring an urgent need to further investigate the mechanisms underlying tumorigenesis as well as explore and develop personalized treatment strategies for patients with EBV-positive cancers. In Chapter 1, I introduce Epstein-Barr Virus (EBV), the two phases of EBV lifecycle and an overview of certain EBV-associated carcinomas. I will also discuss the underlying mechanisms and few current therapeutic strategies against EBV infection. Next, I will discuss some of the preclinical model systems and high-throughput computation techniques that are commonly used by researchers in the field of EBV.  </p> <p>In chapter 2, we have systematically analyzed RNA-sequencing from >1000 patients with 15 different cancer types, comparing virus and host factors of EBV+ to EBV- tissues to reveal novel insights into EBV-positive tumors. First, we observed that EBV preferentially integrates at highly accessible regions of the cancer genome with significant enrichment in super-enhancer architecture. Second, we determined that the expression of twelve EBV transcripts, including LMP1 and LMP2, correlated inversely with EBV reactivation signature. Over-expression of these genes significantly suppressed viral reactivation, consistent with a ‘Virostatic’ function. Third, we identified hundreds of novel frequent missense and nonsense variations in Virostatic genes in cancer samples, and that the variant genes failed to regulate their viral and cellular targets in cancer. Lastly, we were able to dichotomously classify EBV-positive tumors based on patterns of host interferon signature genes and immune checkpoint markers, such as PD-L1 and IDO1. </p> <p>In chapter 3, we probed the lifecycle of EBV on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from six EBV-immortalized lymphoblastoid cell lines (LCL). While the majority of LCLs comprised cells containing EBV in the latent phase of its life cycle, we identified two additional clusters that had distinct expression of both host and viral genes. Both clusters were high expressors of EBV Latent Membrane Protein-1 (LMP1) but differed in their expression of other EBV lytic genes, including glycoprotein gene GP350. We further probed into the transcriptional landscape of these clusters to identify potential regulators which will be discussed in further detail in the chapter. Importantly, I was able to demonstrate enhancing HIF1-a signaling by using Pevonedistat, a compound that stabilized HIF1-a can preferentially induce the transcriptional program specific to one of the three identified clusters. </p> <p>In Chapter 4, I describe some of my recent work. In this project, we have used an intuitive <em>in-silico </em>drug prediction approach to rapidly screen and identify FDA-approved or clinically available compounds that can be repurposed to induce lytic cycle in different EBV+ tumors. Using this strategy, we identified Ciclopirox, an antifungal drug, as a potent inducer of lytic cycle in EBV+ epithelial cancers. We used EBV+ GC cells to determine the effect of Ciclopirox on EBV reactivation as well as identify the underlying mechanisms. In summary, we discovered that reactivation of EBV lytic cycle by Ciclopirox is mediated by multiple pathways, two of the major ones being the HIF1-a and NF-kB pathways. Although, Ciclopirox treatment enhanced the killing effect of antiviral, further investigation is needed to effectively deliver this drug <em>in vivo.</em> Throughout this chapter, I have discussed findings that needs further investigation and proposed necessary experiments. Finally, in Chapter 5 I have summarized my work and described how our work can provide novel insights that can help delineate some of the complexities of host-pathogen interactions in EBV-associated malignancies. </p>