Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-18T18:29:45Z No. of bitstreams: 1 Albuquerque, Ricardo Diego Duarte Galhardo de [Dissertação, 2013].pdf: 1940847 bytes, checksum: 38411d5b06ee4d62cbbea2cfa90328df (MD5) / Made available in DSpace on 2017-04-18T18:29:45Z (GMT). No. of bitstreams: 1 Albuquerque, Ricardo Diego Duarte Galhardo de [Dissertação, 2013].pdf: 1940847 bytes, checksum: 38411d5b06ee4d62cbbea2cfa90328df (MD5) / Xylopia ochrantha (Mart.) é uma espécie endêmica do Brasil, ocorrendo principalmente na Floresta de Restinga localizada ao longo da costa brasileira. Os estudos químico e biológico presentes neste trabalho são os primeiros relacionados a esta espécie. No estudo fitoquímico, os óleos essenciais das folhas e frutos foram obtidos através de extração por hidrodestilação e analisados por GC-MS e GC-FID. No total, foram identificadas 36 substâncias, das quais, germacreno D, biciclogermacreno e silvestreno (folhas), α-felandreno, β-felandreno e sabineno (frutos) foram os principais componentes identificados. Os fitoesterois campesterol, estigmaesterol e gama-sitosterol, provenientes do extrato hexânico foliar, além dos flavonoides majoritários quercitrina e luteolina-7-O-rutinosídeo, encontrados no extrato de acetato de etila foliar, foram identificados através de análises em CG-EM e CLAE-DAD-EM. Análises realizadas em CCF demonstraram a presença de terpenos, alcaloides e cumarinas, sendo estas, portanto, classes de substâncias representativas da espécie. A atividade antioxidante do óleo essencial e seis extratos das folhas de Xylopia ochrantha foram avaliadas utilizando o método ORAC, com valores na faixa entre 0,42 ± 0,01 e 1,85 ± 0,03 mmolTE / g. A atividade antimicrobiana do óleo essencial e extratos de Xylopia ochrantha também puderam ser analizados através dos métodos de diluição em placas e microdiluição em caldo. O óleo essencial e as partições diclorometânica e de acetato de etila das folhas de Xylopia ochrantha apresentaram atividade contra diferentes cepas de Staphylococcus aureus, além de serem ativos também contra Enterococcus faecalis, Staphylococcus haemolyticcus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus capitis e Staphylococcus hominis. O extrato diclorometâncio apresentou um MIC de 128 mcg/mL contra ORSA sanguíneo. Nas demais análises, os extratos ativos apresentaram MIC de 512 mcg/mL. O extrato etanólico demonstrou atividade citotóxica contra células de tumor hipofisiário (AtT20), sendo possível observar uma diminuição significativa da proliferação e viabilidade celular dentro de 24 horas / Xylopia ochrantha (Mart.) is a endemic species from Brazil, occurring mainly in the Restinga Forest located along the Brazilian coast. The chemical and biological studies in this work are the first related to this species. In phytochemical study, the extraction of essential oils from leaves and fruits of this species was performed by hydrodistillation and analyzed by GC-MS/GC-FID. In total, 36 compounds were identified, of which germacrene D, bicyclogermacrene, silvestrene (leaves), α-phellandrene, β-phellandrene and sabinene (fruits) were the main components. The phytosterols campesterol, stigmasterol and gamma-sitosterol, from the foliar hexane extract, besides the majority flavonoids quercitrin and luteolin-7-O-rutinoside, found in the foliar ethyl acetate extract, were identified through analysis on GC-MS and HPLC-DAD-MS. Analyzes made in TLC showed the presence of terpenes, alkaloids and coumarins, which are also representative substances of the species. Regarding the study of biological species, the antioxidant activity was held to six extracts and essential oil from the leaves of Xylopia ochrantha using the ORAC method, with values in the range between 0.42 ± 0.01 and 1.85 ± 0.03 mmolTE / g. The antimicrobial activity of essential oil and extracts of Xylopia ochrantha could be analyzed by plate dilution and broth microdilution methods. The essential oil and extracts of dichloromethane and ethyl acetate from leaves of Xylopia ochrantha showed activity against different strains of Staphylococcus aureus, and were also active against Enterococcus faecalis, Staphylococcus haemolyticcus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus capitis and Staphylococcus hominis. Dichloromethane extract showed a MIC of 128 mcg / mL against ORSA blood. In other analysis, active extracts showed MIC of 512 mcg / mL. Ethanol extract showed cytotoxic activity against tumor cells of the pituitary (AtT20), where was possible to observe a significant decrease in cell viability and proliferation within 24 hours

Environ un tiers des cancers est dû à la dérégulation du facteur de transcription c-Myc. Les mécanismes par lesquels ce facteur de transcription est impliqué dans le processus de cancérogenèse commencent à être mieux compris grâce notamment à l'identification de ses gènes cibles. Parmi eux a été identifié, à la fin des années 2000, le gène dnph1 codant pour une protéine surexprimée dans de nombreux cancers. Cependant, à ce jour, le rôle et la fonction biologiques de cette nouvelle cible thérapeutique restent méconnus. Cette protéine a été caractérisée au laboratoire comme étant une 2'-désoxyribonucléoside 5'-monophosphate N-hydrolase, activité jamais décrite jusqu'alors. Si les 2'-désoxyribonucléosides 5'-monophosphates sont substrats de cette enzyme, il a été montré que les ribonucléotides puriques canoniques sont des inhibiteurs compétitifs. Au cours de ces travaux de thèse, nous avons entrepris différentes études de relation structure-activité autour d'analogues ribonucléosidiques 5'-monophosphates dans le but d'identifier des inhibiteurs plus affins. Une première famille d’analogues modifiés en position 6 de la purine par différents groupements de taille et fonction variables a été synthétisée et les premiers inhibiteurs micromolaires de DNPH1 ont ainsi pu être identifiés. La co-cristallisation de certains composés avec l'enzyme a guidé la conception d'inhibiteurs de deuxième génération, modifiés en positions 6 et 2. Un léger effet additif sur les constantes d’inhibition a alors été observé et des inhibiteurs sub-micromolaires ont été identifiés. Des tests in vitro sur des lignées cellulaires cancéreuses surexprimant DNPH1 ont montré une activité cytotoxique micromolaire de certains des composés synthétisés. D'autres modifications portant sur la partie ribose-phosphate et le squelette purique ont été abordées dans le but de renforcer l'affinité et la stabilité biologique des inhibiteurs. L'ensemble de ces travaux a permis d'une part de mieux caractériser cette nouvelle cible et d'autre part d'identifier les premiers inhibiteurs micromolaires cytotoxiques. Ces résultats ouvrent des perspectives pour la conception de molécules plus efficaces. / About one third of the cancers is due to the deregulation of the transcription factor c-Myc. The gene dnph1 was identified a decade ago as a target gene of the c-Myc oncoprotein and encodes for a protein which is frequently over-expressed in several cancers. However, its biological role remains still unknown. It was recently shown that DNPH1 is a novel 2'-deoxyribonucleoside 5'-monophosphate N-hydrolase and that natural purine ribonucleotides act as competitive inhibitors. The aim of this thesis was the synthesis of strong inhibitors in order to study this new potential cancer target DNPH1. Several structure-activity relationships were built around ribonucleoside 5'-monophosphate derivatives. A first series of compounds modified at the 6 position of the purine core has been synthesized and enabled us to identify the first micromolar inhibitiors. Thanks to these inhibitors, X-ray structures of DNPH1 in interaction with inhibitors have been resolved and led us to the development of a new generation of analogues modified at the 6 and 2 positions. A slight additional effect on the inhibitory potency was noticed and some sub-micromolar inhibitors were identified. Among the synthesized compounds, several have shown micromolar cytotoxic effects against human cancer cells over-expressing DNPH1. Other modifications on the ribose-phosphate and the purine moieties have also been considered in order to increase both biological stability and affinity of the inhibitors. This work allowed a better characterization of the enzyme active site as well as the identification of new cytotoxic compounds. These results pave the way for the design of more potent inhibitors.

Un axe de recherche particulièrement développé en chimie organométallique moderneconcerne la recherche d’agents chimiothérapeutiques alternatifs au cisplatine dans lestraitements contre le cancer. Dans ce domaine, plusieurs stratégies ont été développées depuisplusieurs dizaines d’années, parmi lesquelles nous pouvons citer la multinucléarité, consistanten la création d’édifices polymétalliques. L’objectif de la première partie de ce manuscrits’inscrit dans cette thématique, par la conception, l’obtention et l’étude des propriétésbiologiques de complexes hétérobimétalliques "early-late" (Ti-Ru, Ti-Au, Ti-Pt, Ti-Os, Ti-Rh, Ti-Ir) et "late-late" (Pt-Au). Un deuxième concept développé au cours de ce manuscritconsiste en la génération de nouvelles espèces théranostiques, par association au sein d’unmême complexe d’un fragment métallique à activité thérapeutique et d’une sonde fluorescentepermettant de visualiser le trajet et les cibles biologiques de ce premier. Dans ce cadre, deuxséries parallèles de complexes bimétalliques ont été synthétisées à partir des squelettesRu(bipy)32+ et Ru(bipy)2(dipy)2+ dont les propriétés de luminescence ont été largementdécrites et utilisées pour diverses applications. Ces composés bimétalliques ont pu fairel’objet de mesures sur les deux fronts, à savoir leur activité cytotoxique envers des lignéescellulaires cancéreuses et leurs propriétés photophysiques. / A research theme especially developed in modern organometallic chemistry deals withnew chemotherapeutic agents as alternatives to cisplatin for the treatments against cancer. Inthis area, several strategies have been considered since decades, among which one can citemultinuclearity, meaning the creation of polymetallic structures. The objective of the first partof this manuscript deals with this theme, with the design, synthesis and study of the biologicalproperties of "early-late" hetero-bimetallic complexes (Ti-Ru, Ti-Au, Ti-Pt, Ti-Os, Ti-Rh, Ti-Ir) and "late-late" (Pt-Au). A second concept developed in this thesis consists in thegeneration of new theranostic agents by association inside a same unit of a metal fragmentwith therapeutic activity, and of a fluorescent probe allowing the visualization of thebiological targets. In this field, two parallel series of bimetallic compounds have beenobtained based on the skeletons Ru(bipy)32+ and Ru(bipy)2(dipy)2+, whose luminescenceproperties have been widely described and used for several applications. These bimetalliccomplexes have been tested both for their cytotoxic activity against cancer cells lines andtheir photophysical properties.

Le système immunitaire comporte différents mécanismes de tolérance périphérique permettant de contrôler la réponse des cellules T CD8+. Dans certaines conditions encore peu connues, des cellules T potentiellement auto-réactives peuvent contourner les mécanismes de tolérance et se différencier en cellules effectrices, capables d’attaquer différentes organes de l’organisme, dans un processus d’auto-réactivité. En utilisant une souris transgénique exprimant un antigène modèle dans les cellules bêta du pancréas, j’ai étudié deux processus fondamentaux impliqués dans la différenciation des cellules T CD8+ en réponse aux antigènes du soi.1) Rôle de la translocation des lipopolysaccharides (LPS) dans la rupture de la tolérance. Nous avons préalablement démontré dans le laboratoire que des protocoles de lympho-déplétions, tels l’irradiation, étaient capables d’induire une rupture de la tolérance périphérique dans les cellules T CD8+. L’irradiation provoque la translocation des LPS des bactéries commensales vers la circulation sanguine, ce qui induit une activation du système immunitaire inné. Mes données ont montré que la translocation des LPS était corrélée avec l’activation systémique des cellules dendritiques (DC) CD11c+, en particulier les DC CD8+, responsables de la cross-présentation des auto-antigènes pancréatiques dans les tissus lymphoïdes. Alors que le traitement par des antibiotiques avant l’irradiation permet de prévenir la translocation des LPS, l’activation des DC n’est que partiellement affectée, et le développement de l’auto-immunité résultant d’une rupture de la tolérance périphérique des cellules T CD8+ ne peut pas être empêchée par le traitement.2) Visualisation de la coopération entre cellules T CD4+ et CD8+ effectrices dans la destruction des cellules bêta pancréatiques in vivo. En utilisant la microscopie intra-vitale à 2-photons, j’ai pu analyser, pour la première fois, la dynamiques des cellules T CD4+ et CD8+ auto-réactives exprimant un marqueur fluorescent, lors de l’infiltration du pancréas et du développement du diabète auto-immun. J’ai mis en évidence que l’infiltration des cellules T était accompagnée d’un remodelage de la matrice extracellulaire du pancréas, permettant la migration dirigée des lymphocytes. De plus, j’ai montré que l’arrêt MHC classe II-dépendant des cellules T CD4+, dû à des interactions avec des cellules présentatrices d’antigène recrutées au site d’inflammation et impliquant dans certains cas également les cellules T CD8+, contribuait au maintien des fonctions effectrices des cellules T CD8+. / The immune system has evolved multiple mechanisms of peripheral tolerance to control CD8+ T cell responses. Under particular conditions that are not yet well understood, potentially autoreactive T cells may override tolerance and differentiate into effector cells capable of targeting the own components of the organism resulting in self-reactivity. Utilizing transgenic mice expressing a model antigen in the beta cells of the pancreas, I have studied two important processes involved in CD8+ T cells differentiation in response to self-antigens. 1) Role of lipopolysaccharides (LPS) translocation in the breakdown of CD8+ T cell tolerance. It has been previously shown in our laboratory that lymphodepleting protocols, such as total body irradiation, promote breakdown of peripheral CD8+ T cell tolerance. Irradiation induces translocation of commensal bacteria LPS, a potent innate immune system activator, into the bloodstream. My data demonstrated that LPS translocation correlated with systemic activation of CD11c+ dendritic cells (DC), in particular CD8+ DC, responsible for pancreatic self-antigen cross-presentation, in lymphoid tissue. While antibiotic treatment of mice before irradiation prevented LPS translocation, DC activation was only partially affected, and onset of autoimmunity and breakdown of CD8+ T cell tolerance could not be prevented.2) Intra-vital visualization of effector CD8+ and CD4+ T cell cooperation in beta cell destruction in the pancreas. Using two-photon microscopy, I have been able, for the first time, to simultaneously analyze dynamics of fluorescently tagged autoreactive CD8+ and CD4+ T cells as they infiltrated the pancreas and induced autoimmune diabetes. I found that T cell infiltration promoted extracellular matrix remodeling in the pancreas, which in turn served as a scaffold for T cell migration. In addition, I showed that MHC class II dependent arrest of effector CD4+ T cells, due to interactions with antigen presenting cells, occasionally also implicating CD8+ T cells, provided help to effector CD8+ T cells in maintaining their effector functions.

The ability of our immune system to mount a response against non-self-antigens legitimates the semi-allogenic fetus as a target for maternal immune attack. Yet, in a normal pregnancy the fetus stays well protected due to the concerted action of several diverse mechanisms which either suppress the fetal allogenicity or spatio-temporally inhibit maternal immune cells’ growth and functions. One such factor which aids in the establishment, progression and maintenance of pregnancy is the 28 kDa dimeric sialylated glycoprotein Glycodelin-A (GdA). Synthesized by the endometrium and decidua, this protein has myriad functions, the most important being that of immunosuppression. GdA is inhibitory to all hematopoietic cells and also induces programmed cell death in activated T cells and monocytes via the intrinsic mitochondrial pathway. In the Introductory chapter of this thesis, details about GdA and the other isoforms of the glycodelin family of proteins have been presented which highlight the involvement of glycodelins in primate pregnancy, with emphasis on GdA and its pleiotropic functions associated with reproduction in females. Activated T-lymphocytes against paternal antigens are found in the uterine compartment and in the maternal circulation throughout pregnancy. Activated CD8+ T-lymphocytes have been reported to pre-dominate the uterine T-lymphocyte population during pregnancy and unlike the CD4+ T cells, are retained until term. Studies show that activated CD8+ T-lymphocytes are necessary for the establishment and progression of early pregnancy. However, how these lymphocytes harbouring cytotoxic activity are regulated at the later stages of pregnancy is poorly defined. We attempted to uncover a possible mechanism of regulation of CTL (cytotoxic T lymphocyte) activity (if any) during primate pregnancy by GdA. In the absence of established human CD8+ T cell lines, we first standardized the generation of CTLs in-vitro from hPBMCs (human peripheral blood mononuclear cells) by alloactivating them with an ovarian carcinoma cell line OVCAR-3 utilized as a mimic of an allograft. The details of the rationale behind using this method for generating CTLs and the alloactivation methodology have been put together in the Chapter 1 of this thesis. The activation of hPBMCs was confirmed by the surface expression of an early activation marker CD69 and tritiated thymidine incorporation. Differentiation of CD8+ T cells into effector cells was confirmed by the upregulation of perforin and granzyme transcripts by real time RT-PCR analysis. Target-cell specific cytolytic activity of the CTLs was assessed by using a cytotoxicity measurement assay- JAM test, details of which also form a part of chapter 1. Having generated effective CTLs in vitro, we tested the effect of GdA on CTL activity. Our findings, on the effect of GdA on CTLs have also been discussed in the Chapter 1. We observed that the cytolytic activity of CTLs was significantly reduced by GdA treatment albeit at a dose three to four times higher than that required for inhibiting CD8+ T cell proliferation, implying that a mechanism of temporal regulation of CTL activity operated at the feto-maternal interface, thereby contributing to the establishment and progression of pregnancy. Interestingly, in our quest to uncover the mechanism of inhibition of CTL activity by GdA, we found that the inhibition of proliferation was comparable in both CD4+ and CD8+ T-lymphocytes at all dosages of GdA, but unlike CD4 + T cells CD8 + T cells were resistant to GdA-induced apoptosis even at high dosage of GdA. Hence we could rule out that the loss of CTL activity upon GdA treatment was due to CD8+ T cell death. Further, we assessed the functional competence of alloactivated CTLs by quantitating the mRNA transcripts of key cytolytic molecules; perforin and granzyme B, in GdA treated alloactivated hPBMCs and found that there was a significant reduction in the mRNA of these cytolytic molecules. Additionally, we also found that GdA treated CD8+ T cells exhibited impaired release of the cytolytic molecules by the process of degranulation, measured by the surface exposure of LAMPs (Lysosome associated membrane proteins) on the surface of cells by flow cytometry and as seen by the retention of perforin protein in them assessed by intracellular staining and flow cytometry. Intrigued by the observations, we probed for the regulators of perforin and granzymes in CTLs. EOMES (Eomesodermin) and T- Bet are well known transcription factors which control the differentiation of CD8+ T cells into effector and memory cell CD8+ T cell type. Interestingly we found that the expression of EOMES was significantly reduced in activated GdA treated hPBMCs, both at the transcriptional and translational level, however T-Bet did not show any variation in expression upon GdA treatment. All the above findings have been compiled in Chapter 2 along with our studies on the possibility of GdA to induce a tolerogenic phenotype in T cells. We found there was no difference in the mRNA level and surface expression of CD103 and CD28 in alloactivated PBMCs, while FOXP3 mRNA did not show any variation upon GdA treatment, indicating that GdA does not induce a tolerogenic phenotype in T-lymphocytes, further confirming our data that the decreased cytolytic activity of CTLs upon GdA treatment was not due to tolerance but due to impaired function Interestingly, IL-2/IL-2R signaling is known to directly regulate perforin and granzyme expression as well as it plays a role in the expression of T-Bet and EOMES. Therefore, as a read out of IL-2 signaling we checked for the surface expression of the high affinity IL-2R subunit, CD25. As expected, CD25 expression was more pronounced in CD4+ T cells and consistent with published reports in literature that GdA suppresses IL-2 synthesis, we also observed a significant reduction in the CD25bright population in both the T cell subsets (CD4+ and CD8+) upon GdA treatment (addressed in Chapter 3). This finding supports a mechanism of action of GdA, wherein the cytolytic activity of CTLs is compromised by the downregulation of EOMES, triggered by the low IL-2 levels. This translates to aberrant synthesis of key cytolytic molecules perforin and granzyme B, leading to low efficiency CTLs, which are further disabled by defective degranulation machinery induced by GdA. We did not look into the mechanistic aspects of how GdA suppresses degranulation, which can be addressed later as a part of another study. Building up on our observations, and taking cues from existing literature, that IL-2 regulates the expression of pro and anti-apoptotic protein levels within activated cells, we looked at the expression profile of Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) in activated PBMCs upon GdA treatment. There was a significant reduction in the total mRNA and protein level of Bcl-2, while a very significant increase in Bax mRNA and protein was observed. Chapter 3 of the thesis also presents this data and explains a plausible mechanism of the inhibitory effect of GdA on T-lymphocytes. In Chapter 2, we have also addressed the probable reasons for the differences in the responses of CD4+ and CD8+ T-lymphocytes to GdA. Interestingly, surface glycan profile of CD4+ and CD8+ T-lymphocytes upon activation and the surface expression of the most probable receptor for GdA i.e. CD7 was comparable in both the T cell subsets, indicating that possibly the downstream signaling events leading to GdA-induced apoptosis and not the surface binding of GdA may vary in CD4+ and CD8+ T-lymphocytes, due to which we observed a difference in the extent of apoptosis induced in these cell types by GdA although the inhibition of proliferation in both the subsets was comparable. In summary, this study is the first to provide evidence for a possible mechanism of temporal regulation of CTL activity at the feto-maternal interface, where activated CD8+ T cells are abundantly present. We can say with much confidence that binding of GdA to T-lymphocytes causes sub-optimal IL-2 signaling which translates into reduced expression of EOMES and hence downregulation of perforin and granzyme B, leading to impaired CTL activity in CD8+ T-lymphocytes, which is further weakened by the impaired release of the cytolytic molecules from them. Insufficient IL-2 signaling in the presence of GdA can also be a cause of inhibition of proliferation in T-lymphocytes, while the resulting decrease in anti-apoptotic protein Bcl-2 and increase in pro-apoptotic protein Bax seem to contribute to the induction of apoptosis in CD4+ T cell. It will be interesting to explore the mediators involved in the IL-2 signaling pathway that are differentially regulated in CD4+ and CD8+ T cells which confer resistance in CD8+ T cells to GdA-induced apoptosis and also the mechanism by which GdA regulates the degranulation of cytolytic vesicles in CTLs needs to be worked out.

Comparatively, HIV like most viruses is very minute, unadorned organism which cannot reproduce unaided. It remains the most deadly disease which has ever hit the planet since the last three decades. The spread of HIV has been very explosive and mercilessly on human population. It has tainted over 60 million people, with almost half of the human population suffering from AIDS related illnesses and death finally. Recent theoretical and computational breakthroughs in delay differential equations declare that, delay differential equations are proficient in yielding rich and plausible dynamics with reasonable parametric estimates. This paper seeks to unveil the niche of delay differential equation in harmonizing low HIV viral haul and thereby articulating the adopted model, to delve into structured treatment interruptions. Therefore, an ordinary differential equation is schemed to consist of three components such as untainted CD4+ T-cells, tainted CD4+ T-cells (HIV) and CTL. A discrete time delay is ushered to the formulated model in order to account for vital components, such as intracellular delay and HIV latency which were missing in previous works, but have been advocated for future research. It was divested that when the reproductive number was less than unity, the disease free equilibrium of the model was asymptotically stable. Hence the adopted model with or without the delay component articulates less production of virions, as per the decline rate. Therefore CD4+ T-cells in the blood remains constant at 𝛿1/𝛿3, hence declining the virions level in the blood. As per the adopted model, the best STI practice is intimated for compliance. / Mathematical Sciences / Ph.D. (Applied Mathematics)

A general and modular synthetic approach to 4-substituted phenyl, 2-substituted pyridin- 5-yl and 5-substituted pyridin-2-yl 2′-C-methyl-C-ribonucleosides as potential anti-HCV agents was developed. Addition of halo(het)aryllithium reagents to benzylated 2-C-methyl-D- ribonolactone gave the corresponding hemiketals, which were subsequently converted to the β-anomeric benzyl-protected bromo(het)aryl-C-nucleosides via either direct reduction (in the case of phenyl derivative) or acetylation followed by reduction of the resulting hemiketal acetates (in the case of pyridyl derivatives). The key halogenated (het)aryl-C-nucleoside intermediates were further transformed by Pd-catalyzed cross-coupling, hydroxylation and amination reactions affording series of protected C-nucleosides with small hydrophilic and hydrophobic substituents. The final protecting group removal was rather problematic, and different debenzylation methods, such as hydrogenation on Pd/C or treatment with BCl3, had to be optimized for each derivative to minimize the formation of side-products. The final C- nucleosides were also converted into their 5′-O-triphosphates, and biological activity screenings revealed that none of the free C-nucleosides possesses any antiviral activity in the HCV replicon assay, and none of their NTPs...

Le déploiement d’outils de Contrôle de Qualité Analytique (CQA) intégrés à la boucle de soins apparaît comme un fort contributeur à la sécurisation du circuit des médicaments cytotoxiques en milieu de soin. Les techniques analytiques actuellement disponibles ont en commun d’être intrusives, de détruire une fraction des solutions thérapeutiques, d’exposer les personnels et de générer une filière spécifique d’élimination de déchets toxiques. En regard, de ces éléments, l’analyse non intrusive par Spectroscopie Raman (SR) est particulièrement innovante et en capacité d’améliorer significativement le cahier des charges technique du CQA. Le but de ce travail de thèse a été de procéder à une qualification opérationnelle de la SR dans le contexte du CQA hospitalier. Une attention particulière a été portée à la praticabilité de la nouvelle solution technique par SR, à son impact sur la sécurité et sur la sûreté des personnes et des biens ainsi qu’en terme environnemental. / The development of effective tools for the analytical quality control (AQC) of therapeutic objects (TO) appear to be a strong contributor to security of the cytotoxic drugs circuit in health care settings. Our goal is to ensure a high and stable quality in our pharmaceutical preparations for the benefit of patients and caregivers. Presently available analytical techniques have in common to be intrusive, destroying a fraction therapeutic solutions, exposing personal and generate specific toxic waste disposal. Compared to these, the non-intrusive analysis by Raman spectroscopy (RS) appears to us particularly innovative and has the capacity to significantly improve the specification of AQC technical specifications. The purpose of this work was to conduct an operational qualification of RS in the context of AQC. Special attention has been given to the feasibility of the new technical solution for SR, its impact on safety of persons and their working environment.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

Objetivo: Evaluar in vitro el efecto antibacteriano y citotóxico del extracto metanólico del café peruano y colombiano frente a cepas de Streptococcus mutans (ATCC® 25175) y Streptococcus sanguinis (ATCC® 10556). Métodos: El extracto metanólico de café peruano y colombiano fue preparado utilizando una proporción 1:2 (P/V). Para determinar las propiedades antibacterianas de cada extracto se utilizó el método de difusión en pozo y la concentración mínima inhibitoria (CMI) se determinó mediante el método de microdilución. Como control positivo se utilizo la clorhexidina al 2%. La viabilidad celular y la citotoxicidad de los extractos fueron realizados utilizando el ensayo de reducción de MTT en la línea celular MDCK. Resultados: El extracto metanólico del café colombiano (Coffea) tuvo un mayor efecto antibacteriano (18.9 ± 2.8 mm) en comparación al café peruano (15+2.54mm), mientras que para el grupo de Streptoccocus mutans y para el grupo de Streptoccocus sanguinis el café colombiano y peruano mostraron halos de 18.8 ± 2.77 mm y 14.7± 3.8 mm, respectivamente. La concentración mínima inhibitoria (CMI) para los extractos del café colombiano y peruano fue de 3.1 μg/ml y 1.6 μg/ml para Streptoccocu mutans y 6.3 μg/ml para Streptoccocu sanguinis, respectivamente. El ensayo de MTT muestras que ambos extractos no fueron citotóxicos a altas concentraciones. Conclusiones: Los hallazgos que se encontraron en este estudio muestran que los extractos del colombiano y peruano (Coffea) tienen propiedades antibacterianas efectivas contra cepas de Streptococcus mutans y Streptococcus sanguinis. / Objective: To evaluate in vitro the antibacterial and cytotoxic effect of the methanolic extract of peruvian and colombian coffee against strains of Streptococcus mutans (ATCC® 25175) and Streptococcus sanguinis (ATCC® 10556). Methods: An extract was prepared for each type of coffee, carrying out nine tests independently, chlorhexidine 2% was used as a positive control. The well diffusion method was performed to determine the antibacterial properties of each extract. The minimum inhibitory concentration (MIC) was determined by the microdilusion method and the cytotoxicity was analyzed by the MTT reduction test using a MDCK cell line. Results: The methanolic extract of colombian coffee (Coffea) had a greater antibacterial effect (18.9 ± 2.8 mm) compared to peruvian coffee (15 + 2.54mm) for the group of Streptoccocus mutans and for the group of Streptoccocus sanguinis, colombian coffee and peruvian obtained 18.8 ± 2.77 mm and 14.7 ± 3.8 mm, respectively. The minimum inhibitory concentration (MIC) for colombian and peruvian coffee extracts was 3.1 μg / ml and 1.6 μg / ml for S. mutans and 6.3 μg / ml for S. sanguinis, respectively. These extracts were not cytotoxic at high concentrations. Conclusions: The findings found in this study show that colombian and peruvian extracts (Coffea) have effective antibacterial properties against Streptococcus Mutans and Sanguinis strains. Further study with Coffea is recommended as there is very little research on this. / Tesis