Recent advancements in pharmaceutical technology based on principles of nanotechnology, polymer chemistry, and biomedical engineering have resulted in the creation of novel drug delivery systems with the potential to revolutionize current strategies in cancer chemotherapy. In oncology, realization of significant improvements in therapeutic efficacy requires minimization of drug exposure to healthy tissues and concentration of the drug within the tumor. As such, encapsulation of chemotherapeutic agents inside nanoparticles capable of enhancing tumor-targeted drug delivery is a particularly promising innovation. Yet, initial investigations into the intratumoral fate of nanomedicines have suggested that they may be heterogeneously distributed and achieve limited access to cancer cells located distant from the tumor vasculature. As such, uncovering the determinants of nanoparticle transport at the intratumoral level is critical to the development of optimized delivery vehicles capable of fully exploiting the therapeutic potential of nanomedicines. In this work, the chemotherapeutic agent, docetaxel (DTX), was incorporated into nano-sized, biocompatible PEG-b-PCL block copolymer micelles (BCMs). Encapsulation of DTX in micelles via chemical conjugation or physical entrapment resulted in a dramatic increase in drug solubility and customizable drug release rate. The use of multicellular tumor spheroids (MCTS) was established as a viable platform for assessing the efficacy and tumor tissue penetration of nanomedicines in vitro. A series of complementary assays was validated for analysis of DTX-loaded micelle (BCM+DTX) toxicity in monolayer and spheroid cultures relative to Taxotere®. Cells cultured as spheroids were less responsive to treatment relative to monolayer cultures due to mechanisms of drug resistance associated with structural and microenvironmental properties of the 3-D tissue. Computational, image-based methodologies were used to assess the spatial and temporal penetration of BCMs in spheroids and corresponding human tumor xenografts. Using this approach, the tumor penetration of micelles was found to be nanoparticle-size-, tumor tissue type- and time- dependent. Furthermore, spheroids were found to be a valuable platform for the prediction of trends in nanoparticle transport in vivo. Overall, the results reported herein serve to demonstrate important determinants of nanoparticle intratumoral transport and to establish computational in vitro and in vivo methodologies for the rational design and optimization of nanomedicines.

This dissertation brings to the fore the “Characterization of the SULT1A1 polymorphism in a South Africa Tswana population group.” The primary experimental group studied came from South African homogeneous Tswana individuals who participated voluntarily in an ongoing large-scale epidemiological Prospective Urban and Rural Epidemiological (PURE) study the North-West University (Potchefstroom Campus) participates in, as one of the 16 low- middleand high-income countries across the world. The primary aspect investigated was the comprehensive profile of the single nucleotide polymorphism (SNP) and copy number variation (CNP) of the SULT1A1 gene. Using the PCRbased RFLP method, SULT1A1 genotypes, and allele frequency distributions in an experimental group of 1 867 individuals were determined. According to the literature this is by far the largest and most homogeneous group from which such information has been acquired to date. The SULT1A1*1, SULT1A1*1/*2 and SULT1A1*2 genotypes were found to be present at a percentage of 43.76, 47.12 and 9.11 respectively. In comparison to similar studies in other population groups, results from this study indicate that there are ethnic differences in the SULT1A1 genotypes incidence. Asian group differs from Caucasian and Tswana groups because of its exceptionally high prevalence of individuals with the SULT1A1*1 genotype and a very low incidence of the SULT1A1*2 genotype. The SULT1A1*1 genotype profiles of Caucasian and Tswana groups were comparable, but notable differences were observed for the SULT1A1*2 genotype. Using a quantitative multiplex PCR method for the CNV study, the numbers of copies of the SULT1A1 gene in the Tswana population were determined, and the results showed 1 to ~5 copies: only 0.65% of the subjects had a single copy, whereas 59.69% of the subjects had 3 or more copies. This result shows a significant discrepancy between the Caucasian-American samples, which showed that only 26% from that group had more than three copies. However, there is a significant relationship with the African-American population, which presented 63% with 3 or more copies. This finding confirms results from a much smaller African-American study, and suggests a possible genetic link between the African Tswana and the heritage of the African-Americans. These findings were submitted for publication to the South African Journal of Science, as that journal specializes in publication of new knowledge that has a regional focus on Africa. Simultaneous phenotypic consequences of the SNP and CNP of the SULT1A1 gene, as well as the thermo-stable and thermo-labile forms of the sulfotransferases were determined. For this, the formation of [35S]-4-nitrophenyl sulphate from 4-nitrophenol and [35S]-3’-phosphoadenosine- 5’-phosphosulfate ([35S]-PAPS) in platelet homogenates were measured, with the data normalized to a common platelet count. This investigation required fresh blood for enzyme activity. These samples came from 98 Caucasian subjects who voluntarily participated in this part of the study. The experimental data presented a unique challenge to develop a statistical model to accommodate the complexity of the distribution of the data in the phenotype and genotype components, which could be achieved by the development of a mixed model. The model indicated that product formation increased through increasing copy number, but did not differ for SULT1A1*1 and SULT1A1*1/*2. However, the rate of increase in product for the thermo-stable forms of the SULTs was greater than that of thermo-labile forms. In contrast, copy number effect for SULT1A1*2 differed considerably from that of the other two genotypes. Since genotype is also a significant factor, it was concluded from Tukey post-hoc tests that the population group means for product formation differ significantly (for all levels). These results are presently being prepared for publication in an accredited international journal. Finally, perturbations in 23 biochemical parameters measured in the PURE study were analyzed as a function of the SULT1A1 SNP and CNP were evaluated. No group separation in this regard could be found. It could be shown however, that sulfonation of the iodothyronines, which are endogenous substrates for the SULTs, was influenced by the SULT1A1 genotype. The relative concentrations in plasma of the sulphonated iodothyronines may be expressed as T2S > T3S >> T4S, which coincides with the substrate preference of the SULT1A1 enzymes. This observation may, however, only be qualitatively interpreted as (1) the targeted metabolomics mass spectrometric method used for the quantitative analysis of these substances needs further development, and (2) the influence of deiodonation was not taken into account in these studies. In conclusion, three perspectives are given at the end of the thesis which might be considered for further investigations. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

This dissertation brings to the fore the “Characterization of the SULT1A1 polymorphism in a South Africa Tswana population group.” The primary experimental group studied came from South African homogeneous Tswana individuals who participated voluntarily in an ongoing large-scale epidemiological Prospective Urban and Rural Epidemiological (PURE) study the North-West University (Potchefstroom Campus) participates in, as one of the 16 low- middleand high-income countries across the world. The primary aspect investigated was the comprehensive profile of the single nucleotide polymorphism (SNP) and copy number variation (CNP) of the SULT1A1 gene. Using the PCRbased RFLP method, SULT1A1 genotypes, and allele frequency distributions in an experimental group of 1 867 individuals were determined. According to the literature this is by far the largest and most homogeneous group from which such information has been acquired to date. The SULT1A1*1, SULT1A1*1/*2 and SULT1A1*2 genotypes were found to be present at a percentage of 43.76, 47.12 and 9.11 respectively. In comparison to similar studies in other population groups, results from this study indicate that there are ethnic differences in the SULT1A1 genotypes incidence. Asian group differs from Caucasian and Tswana groups because of its exceptionally high prevalence of individuals with the SULT1A1*1 genotype and a very low incidence of the SULT1A1*2 genotype. The SULT1A1*1 genotype profiles of Caucasian and Tswana groups were comparable, but notable differences were observed for the SULT1A1*2 genotype. Using a quantitative multiplex PCR method for the CNV study, the numbers of copies of the SULT1A1 gene in the Tswana population were determined, and the results showed 1 to ~5 copies: only 0.65% of the subjects had a single copy, whereas 59.69% of the subjects had 3 or more copies. This result shows a significant discrepancy between the Caucasian-American samples, which showed that only 26% from that group had more than three copies. However, there is a significant relationship with the African-American population, which presented 63% with 3 or more copies. This finding confirms results from a much smaller African-American study, and suggests a possible genetic link between the African Tswana and the heritage of the African-Americans. These findings were submitted for publication to the South African Journal of Science, as that journal specializes in publication of new knowledge that has a regional focus on Africa. Simultaneous phenotypic consequences of the SNP and CNP of the SULT1A1 gene, as well as the thermo-stable and thermo-labile forms of the sulfotransferases were determined. For this, the formation of [35S]-4-nitrophenyl sulphate from 4-nitrophenol and [35S]-3’-phosphoadenosine- 5’-phosphosulfate ([35S]-PAPS) in platelet homogenates were measured, with the data normalized to a common platelet count. This investigation required fresh blood for enzyme activity. These samples came from 98 Caucasian subjects who voluntarily participated in this part of the study. The experimental data presented a unique challenge to develop a statistical model to accommodate the complexity of the distribution of the data in the phenotype and genotype components, which could be achieved by the development of a mixed model. The model indicated that product formation increased through increasing copy number, but did not differ for SULT1A1*1 and SULT1A1*1/*2. However, the rate of increase in product for the thermo-stable forms of the SULTs was greater than that of thermo-labile forms. In contrast, copy number effect for SULT1A1*2 differed considerably from that of the other two genotypes. Since genotype is also a significant factor, it was concluded from Tukey post-hoc tests that the population group means for product formation differ significantly (for all levels). These results are presently being prepared for publication in an accredited international journal. Finally, perturbations in 23 biochemical parameters measured in the PURE study were analyzed as a function of the SULT1A1 SNP and CNP were evaluated. No group separation in this regard could be found. It could be shown however, that sulfonation of the iodothyronines, which are endogenous substrates for the SULTs, was influenced by the SULT1A1 genotype. The relative concentrations in plasma of the sulphonated iodothyronines may be expressed as T2S > T3S >> T4S, which coincides with the substrate preference of the SULT1A1 enzymes. This observation may, however, only be qualitatively interpreted as (1) the targeted metabolomics mass spectrometric method used for the quantitative analysis of these substances needs further development, and (2) the influence of deiodonation was not taken into account in these studies. In conclusion, three perspectives are given at the end of the thesis which might be considered for further investigations. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

EGFR is a transmembrane tyrosine kinase receptor from the HER family which, upon ligand stimulation, activates different signaling pathways involved in tumorogenesis. EGFR can be targeted by monoclonal antibodies, as cetuximab and panitumumab, which bind to EGFR preventing ligand stimulation of the receptor. Cetuximab and panitumumab are approved for colorectal cancer treatment. However, its clinical success is uniformily limited by the development of acquired drug resistance. We describe a new mechanism of acquired resistance to cetuximab in colorectal cancer that was due to a missense mutation in the EGFR ectodomain (S492R mutation). Upon chronic exposure to cetuximab, colorectal cancer cell lines acquired S492R mutation and became resistant to the treatment. We observed that cetuximab was not able to bind mutant EGFR. Notably, this amino acid change did not affect the ability of panitumumab to bind to EGFR, and panitumumab effectively suppressed growth of mutant cells. EGFRS492R mutation was detected in 2 out of 10 tumor specimens from patients following progression on cetuximab. One of these patients was subsequently treated with single agent panitumumab yielding a partial response. The S492R mutation defines a novel biomarker of resistance to cetuximab but not to panitumumab in colorectal cancer / EGFR és un receptor transmembrana tirosina cinasa de la família HER el qual, després de l’estimulació mitjançant lligands, activa vies de senyalització involucrades en processos tumorogènics. L’EGFR es pot inhibir amb anticossos monoclonals, com cetuximab i panitumumab, que s’uneixen al receptor prevenint-ne l’activació per part dels lligands. Cetuximab i panitumumab estan aprovats per al tractament del càncer colorectal, però el seu ús es veu limitat per el desenvolupament de resistència adquirida al tractament. Nosaltres describim un mecanisme de resistència adquirida a cetuximab en càncer colorectal degut a l’adquisió d’una mutació en el domini extracel•lular de l’EGFR, la mutació S492R. Durant l’exposició crònica a cetuximab, linies cel•lulars de càncer colorectal van adquirir la mutació S492R tornat-se resistents al tractament. Cetuximab no era capaç d’unir-se a l’EGFR mutat. Aquests canvi d’aminoàcid no afectava a l’habilitat que té panitumumab a unir-se al EGFR, pertant, panitumumab suprimia el creixement de les cèl•lules tumorals mutades. Vam detectar la mutació EGFRS492R en 2 de 10 mostres tumorals de pacients que havien recaigut al tractament amb cetuximab. Un d’aquest pacients va ser posteriorment tractat amb panitumumab obtenint-ne una resposta tumoral parcial. La mutació S492R defineix un nou mecanisme de resistència a cetuximab però no a panitumumab en el tractament del càncer colorectal.

O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

Les thérapies ciblées telles que les inhibiteurs de la tyrosine kinase ont permis d'améliorer le traitement du cancer du sein en ciblant HER2. Cependant, il a été observé que le lapatinib était moins efficace chez les patients obèses ou en surpoids que chez les patients de poids normal.Nous avons d'abord reproduit l'effet de résistance des cellules de cancer du sein au lapatinib en présence de tissu adipeux tel qu’il a été observé pour d’autres thérapies. Les cellules tumorales qui surexpriment HER2 étaient partiellement résistantes au lapatinib mais également à d'autres inhibiteurs de la tyrosine kinase lorsqu'elles étaient en contact avec le milieu conditionné d’adipocytes. En implantant du tissu adipeux humain et des tumeurs humaines chez la souris, nous avons pu étudier la résistance du cancer du sein au lapatinib in vivo.Pour comprendre le mécanisme de cette résistance, nous avons exposé les adipocytes à plusieurs modulateurs du métabolisme. La cytotoxicité cellulaire induite par le lapatinib était plus faible pour les cellules tumorales exposées à un milieu conditionné d'adipocytes préalablement incubés avec les alpha-bloquants qu'à un milieu conditionné à partir d'adipocytes seuls. De la même manière, cette toxicité était inférieure pour les agonistes des récepteurs alpha adrénergiques, pour les bêtabloquants et pour les inhibiteurs de lipolyse. Au contraire, la cytotoxicité a été augmentée pour les cellules tumorales en contact avec le milieu conditionné d’adipocytes exposés aux agonistes du récepteur bêta-adrénergiques. Au niveau des cellules cancéreuses, l'arrêt du cycle cellulaire induit par le lapatinib était réduit pour les cellules tumorales exposées au milieu conditionné d’adipocytes en ce qui concerne le pourcentage de cellules dans la phase G0/G1. Ceci s’est vérifié en étudiant l'expression des gènes codant pour plusieurs protéines impliquées dans la progression du cycle cellulaire / Targeted therapies as tyrosine kinase inhibitors permitted an improvement of breast cancer therapies by targeting HER2. However, resistance has been observed in obèse patients for lapatinib treatment.We reproduced the effect of resistance of breast cancer cells to laaptinib in presence of adipose tissue as observed for other therapies. Tumor cells overexpressing HER2 was partly resistant to lapatinib but also for other tyrosine kinase inhibitors when in contact with adipocyte-conditioned medium. By impnating human adipose tissue nad human tumors in mice, we were able to study rhe resistance of breast tumor cells in vivo.In order to elucidate the mechanism of such resistance, we exposed the adipocytes to several metabolism modulators. The lapatinib-induced cell cytotoxicity was lower for the tumor cells exposed to the conditioned medium from adipocytes earlier exposed to alpha blockers than to the conditioned medium from adipocytes alone. In the same manner, the toxicity was lower for the agonists of alpha-adrenergic receptors , for beta-blockers and for the lipolysis inhibitors. At the opposite, the cytotoxicity was enhanced for tumor cells in contact with the conditioned medium of adipocytes exposed to the agonists of beta adrenergic receptors.At the tumor cell level, the laaptinib-induced cell cycle arrest was reduced for the tumor cells exposed to the conditioned medium regarding the G0/G1 phase. That was verified by the study of the expression of genes involved in the cell cycle progression

O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

abstract: Earth-system models describe the interacting components of the climate system and technological systems that affect society, such as communication infrastructures. Data assimilation addresses the challenge of state specification by incorporating system observations into the model estimates. In this research, a particular data assimilation technique called the Local Ensemble Transform Kalman Filter (LETKF) is applied to the ionosphere, which is a domain of practical interest due to its effects on infrastructures that depend on satellite communication and remote sensing. This dissertation consists of three main studies that propose strategies to improve space- weather specification during ionospheric extreme events, but are generally applicable to Earth-system models: Topic I applies the LETKF to estimate ion density with an idealized model of the ionosphere, given noisy synthetic observations of varying sparsity. Results show that the LETKF yields accurate estimates of the ion density field and unobserved components of neutral winds even when the observation density is spatially sparse (2% of grid points) and there is large levels (40%) of Gaussian observation noise. Topic II proposes a targeted observing strategy for data assimilation, which uses the influence matrix diagnostic to target errors in chosen state variables. This strategy is applied in observing system experiments, in which synthetic electron density observations are assimilated with the LETKF into the Thermosphere-Ionosphere- Electrodynamics Global Circulation Model (TIEGCM) during a geomagnetic storm. Results show that assimilating targeted electron density observations yields on average about 60%–80% reduction in electron density error within a 600 km radius of the observed location, compared to 15% reduction obtained with randomly placed vertical profiles. Topic III proposes a methodology to account for systematic model bias arising ifrom errors in parametrized solar and magnetospheric inputs. This strategy is ap- plied with the TIEGCM during a geomagnetic storm, and is used to estimate the spatiotemporal variations of bias in electron density predictions during the transitionary phases of the geomagnetic storm. Results show that this strategy reduces error in 1-hour predictions of electron density by about 35% and 30% in polar regions during the main and relaxation phases of the geomagnetic storm, respectively. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics 2018

Le virus de l’hépatite C (VHC) est, encore aujourd’hui, un problème de santé mondiale majeur entraînant dans certains cas des cirrhoses et des hépatocarcinomes. De nombreux efforts ont été fournis depuis les années 80 afin de développer un traitement efficace et sûr de cette infection touchant les hépatocytes. Le traitement interféron/ribavirine, utilisé dans les années 2000, a aujourd’hui été remplacé par des thérapies utilisant des agents antiviraux directs, beaucoup plus efficaces. Ces traitements restent cependant perfectibles notamment du fait de certains effets secondaires, de leur coût élevé et de potentielles interactions médicamenteuses avec d’autres composés thérapeutiques. L’équipe de glycochimie du Laboratoire de Chimie Biologique s’est intéressée à la synthèse de C-nucléosides analogues de la ribavirine. Parmi-eux, un composé, le SRO91, s’est révélé efficace contre des réplicons du VHC et présente une faible toxicité. Dans le but d’améliorer l’activité anti-VHC du SRO91, deux axes ont été développés dans ce projet : l’adressage vers les cellules du foie, et l’amélioration de la pénétration cellulaire. Un premier conjugué entre un galactoside et SRO91 a ainsi été synthétisé, afin de profiter de la forte interaction du galactose avec les récepteurs aux asialoglycoprotéines, principalement exprimés à la surface des hépatocytes. Afin d’améliorer sa pénétration cellulaire, le nucléoside a également été conjugué à des peptides de fusion du VHC. Ces séquences peptidiques très hydrophobes sont capables de s’insérer dans la membrane cellulaire et de permettre la fusion. Trois peptides ont été sélectionnés en se basant sur la littérature : HCV3 (VFLVG), HCV6 (YVGDLSGSVFL) et HCV7 (SWHINRTALNSNDS), synthétisés par SPPS puis conjugués au nucléoside ou à un fluorophore. L’activité membranotropique des peptides sur des liposomes a alors été étudiée par calorimétrie (DSC et ITC), spectrofluorescence et microscopie à épifluorescence. Ces études ont ainsi permis de montrer que, parmi les séquences sélectionnées, HCV7 semble montrer la meilleure activité en pénétration membranaire alors que HCV6 s’est révélé être la séquence la plus fusogénique. / Hepatitis C virus (HCV) is a global healthcare issue responsible for cirrhosis and hepatocarcinoma. Strong efforts have been made since the 80’s to develop efficient and safe treatments for this liver infection. Hence, the treatment based on interferon/ribavirin, developed in 2002, has been replaced by much more efficient therapies involving direct-acting antivirals. However, the different side-effects, high cost and possible drug-drug interactions make room for improvements to this treatment. In the Laboratoire de Chimie Biologique, several C-nucleosides, analogs of ribavirin have been developed. Among them, one compound, named SRO91, seems effective against HCV replicons with low toxicity. This thesis work focused on improving SRO91 anti-HCV activity by implementing a targeting strategy and enhancing cell-penetration. We built our targeting strategy on the strong interaction between galactose and asialoglycoprotein receptors. Thus, a SRO91-galactose conjugate was synthesized, in order to address the antiviral to hepatocytes. To enhance cell-penetration we conjugated our nucleoside to HCV fusion peptides, since these highly hydrophobic sequences are able to anchor in cell membranes, leading to fusion. Three peptides were selected based on the literature: HCV3 (VFLVG), HCV6 (YVGDLSGSVFL) and HCV7 (SWHINRTALNSNDS), synthesized by SPPS and conjugated to SRO91 or a fluorescent tag. Several techniques were used to study the membranotropic activity of theses sequences on liposomes as membrane models, including calorimetry (DSC and ITC), spectrofluorescence and epifluorescence microscopy. Thus, among the selected peptides, HCV7 seems to be the more potent as a membrane-penetrating agent but HCV6 shows the best fusogenic activity.

[pt] O surgimento da World Wide Web representou uma nova oportunidade de publicidade, disponível para qualquer empresa: A possibilidade de exposição global para uma grande audiência a um custo extremamente pequeno. Como conseqüência disso, surgiu toda uma nova indústria oferecendo serviços relacionados à publicidade de busca, na qual uma empresa anunciante paga por uma posição de destaque em listas de anúncios. A fim de manter a credibilidade e a participação de mercado do serviço que os veicula - por exemplo, uma máquina de busca - os anúncios devem ser exibidos apenas para os usuários que se interessem por eles, no que se chama de Publicidade Direcionada. Em virtude disso, surge a necessidade de se utilizar um sistema de recomendação que seja capaz de escolher que anúncios exibir para quais usuários. Nos sistemas de recomendação baseados em filtragem colaborativa, as preferências de outros usuários são utilizadas como atributos para um sistema de aprendizado, pois estas podem ser bastante detalhadas, gerando recomendações não só para os itens mais populares como também para nichos de itens. Neste trabalho, é desenvolvido um sistema de recomendação de anúncios que aplica Filtragem Colaborativa baseada em fatoração de matrizes ao problema de predição do Click- Through Rate, uma métrica em Publicidade Direcionada que expressa a relevância de um anúncio para os usuários que buscam por uma determinada palavra- chave. A fim de validar o método proposto de predição do Click-Through Rate, realizamos vários experimentos em um conjunto de dados sintéticos. Adicionalmente, o trabalho contribui para o projeto do LearnAds, um framework de recomendação de anúncios baseado em Aprendizado de Máquina. / [en] The emergence of the World Wide Web represented a new advertising opportunity available to any company: The possibility of global exposure to a large audience at a very small cost. As a result, a whole new industry has emerged by offering services related to search advertising, in which an advertiser pays for a prominent position in lists of ads. In order to maintain the credibility and market share of the service that conveys them - for example, a search engine - such ads must be displayed only to users who are interested in them, on what is called Targeted Advertising. Therefore, those services need to use a recommendation system that can choose which ads show to which users. Recommendation systems based on collaborative filtering use the preferences of other users as features to a learning system, since such preferences can be quite detailed, generating recommendations not only for the most popular items but also to item niches. In this work, we develop an ads recommendation system that applies Collaborative Filtering based on matrix factorization to the problem of predicting the Click-Through Rate, a Targeted Advertising metric that expresses the relevance of a particular ad for the users searching for a specific keyword. In order to validate the proposed method of Click-Through Rate prediction, we carry out several experiments on a synthetic data set. Additionally, the work contributes to the design of LearnAds, a framework for ads recommendation systems based on Machine Learning.