O glioblastoma multiforme (GBM) é um tipo de câncer grave que acomete o sistema nervoso central (SNC), e a sobrevida dos pacientes é de aproximadamente 12 meses. O tratamento com o quimioterápico paclitaxel (PTX) reduz o GBM experimental e humano. No entanto, sua utilização é limitada pelas reações adversas graves que acarreta. A nanoemulsão rica em colesterol (LDE), a qual mimetiza a lipoproteína de baixa densidade, tem sido empregada como um sistema de entrega de fármacos eficiente em alguns casos de tumores. No presente trabalho visou-se avaliar a eficácia do oleato de PTX (OPTX), um derivado mais lipofílico do que o PTX, associado a LDE (LDE-OPTX) em ensaios in vitro e in vivo. Inicialmente, células tumorais da linhagem de glioblastoma murino GL261 foram incubadas com PTX em solução ou com LDE-OPTX, nas concentrações de 1 ou 10 µM. Os resultados obtidos mostraram que o tratamento in vitro com PTX e o LDE-OPTX causa toxicidade in vitro em células GL261 pela redução da proliferação e indução de apoptose, e que ainda reduz a secreção de MCP-1 (proteína quimiotáxica de monócitos). Os ensaios in vivo mostraram a toxicidade intensa do PTX comercial, uma vez que os animais com GBM não sobreviveram ao tratamento com 75mg/Kg, i.p., a cada 3 dias, e foram a óbito a partir do oitavo dia de tratamento. Diferentemente, os animais tratados com a mesma dose de LDE-OPTX sobreviveram ao tratamento, sem sinais de toxicidade, mas os dados obtidos mostraram que este protocolo de tratamento não foi eficaz para redução do volume tumoral. Assim, os animais com GBM passaram a ser tratados com doses diárias, i.p., de 15 mg/kg de PTX ou de 75mg/Kg de LDE-OPTX. Os resultados obtidos mostraram a ineficácia e eficácia dos tratamentos com PTX e LDE-OPTX, respectivamente, em reduzir o GBM; no entanto os animais tratados com LDE-OPTX apresentaram redução no peso corporal e no número de linfócitos circulantes. Em conjunto, os dados obtidos mostram a habilidade de preparação LDE-OPTX causar toxicidade in vitro nas células GL261 e sua eficácia terapêutica em dose elevada, em reduzir o GBM em modelo murino. / Glioblastoma multiforme (GBM) is a type of severe cancer that affects the central nervous system, and patient survival is about12 months. The treatment with the chemotherapeutic paclitaxel (PTX) reduces the experimental and human GBM, however, their use is limited by side effects. The lipid nanoemulsion (LDE), that is mimetic to low density protein, has been employed as an efficient drug nanocarrier to treat cancer. Therefore, the present study aimed to assess the effectiveness of the oleate PTX (OPTX), a more lipophilic derivative of PTX, associated to (LDE), in in vitro and in vivo studies. Initially, glioblastoma murine strain GL261 was incubated with commercial PTX solution or LDE-OPTX at concentrations of 1 or 10 µM. Data obtained showed that treatment with PTX or LDE-OPTX caused in vitro toxicity to GL261 cells, by reducing the proliferation and inducing apoptosis. Moreover, both treatments reduced the secretion of monocyte chemotacticprotein-1 (MCP-1). In vivo experiments showed the severe toxicity of commercial PTX, as mice with GBM did not survive to the treatment with 75mg/kg, i.p., each 3 days, and died after 8 days of treatment. Conversely, animals treated with the same schedule of treatment with LDE-OPTX survived until the end of treatment, without any toxicity signal. Nevertheless, the treatment was not effective to reduce the GBM volume. Hence, other sets of animals with GBM were treated with daily i.p. dose of 15mg/kg of PTX or 75mg/kg of LDE-OPTX. Data obtained showed the inefficacy and efficacy of PTX and LDE-OPTX treatments, respectively, to reduce the volume of GBM. Nevertheless, mice treated with LDE-OPTX lost weight and lower number of circulating lymphocytes. Together, our data show the ability of LDE-OPTX treatment cause in vitro toxicity on GL261 cells e the in vivo therapeutic efficacy of higher doses on GBM murine model.

Antes de se iniciar os estudos clínicos de uma nova droga, é necessário realizar uma bateria de testes de segurança, para avaliar o risco humano. Os radiofármacos como qualquer outra nova droga, devem ser testados levando em conta sua especificidade, duração de tratamento e principalmente a toxicidade de ambas as partes, a molécula não marcada e a sua radioatividade em si, além das impurezas provindas da radiólise. Órgãos regulatórios como o Food and Drug Administration-EUA (FDA) e a Agência de Medicina Européia (EMEA), estabelecem guias para a regulamentação de produção e pesquisas de radiofármacos, No Brasil a produção de radiofármacos não era regulamentada até o final de 2009, quando foram estabelecidas pela Agência Nacional de Vigilância Sanitária (ANVISA) as resoluções nº 63, que visa as Boas Práticas de Fabricação de Radiofármacos e a nº 64 que visa o registro do radiofámaco. Para a obtenção do registro de radiofármacos são necessárias a comprovação da qualidade, segurança, eficácia e especificidade do medicamento. Para a segurança dos radiofármacos devem ser apresentados estudos de toxicidade aguda, subaguda e crônica como também a toxicidade reprodutiva, mutagênica e carcinogênica. Hoje o IPEN-CNEN/SP produz um dos radiofámacos mais importantes da medicina nuclear, o 18F-FDG, que é utilizado em muitas aplicações clínicas, em particular no diagnóstico e estadiamento de tumores. O objetivo deste trabalho foi avaliar a toxicidade sistêmica (aguda/subaguda) do radiofármaco 18F- FDG em um sistema teste in vivo, conforme preconiza a RDC nº 64, que servirá de modelo para os protocolos de toxicidade dos radiofármacos produzidos no IPEN. Os ensaios realizados foram: os testes de toxicidade aguda e de toxicidade subaguda, estudos de biodistribuição do 18F-FDG, ensaio cometa e toxicidade reprodutiva. Na toxicidade aguda, ratos sadios foram injetados com 18F- FDG e observados durante 14 dias enquanto na toxicidade subaguda os animais foram observados durante 28 dias. Os resultados não mostraram nenhuma evidência de toxicidade na exposição ao 18F-FDG na toxicidade aguda e na subaguda. A biodistribuição demonstrou resultados semelhantes aos da literatura, onde a bexiga é o órgão que mais recebe radiação. O ensaio cometa mostrou que a radiação do radiofármaco não foi significativa para gerar danos no DNA. Na toxicidade reprodutiva, casais de ratos expostos ao 18F-FDG geraram filhotes completamente normais e saudáveis. Por fim, o 18F-FDG não evidenciou nenhuma toxicidade. / Before starting clinical trials of a new drug, it is necessary to perform a battery of safety tests for assessing human risk. Radiopharmaceuticals like any new drug must be tested taking into account its specificity, duration of treatment and especially the toxicity of both parties, the unlabeled molecule and its radionuclide, apart from impurities emanating from radiolysis. Regulatory agencies like the Food and Drug Administration - USA (FDA) and the European Medicine Agency (EMEA), establish guidelines for the regulation of production and research of radiopharmaceuticals. In Brazil the production of radiopharmaceuticals was not regulated until the end of 2009, when were established by the National Agency for Sanitary Surveillance (ANVISA) resolutions No. 63, which refers to the Good Manufacturing Practices of Radiopharmaceuticals and No. 64 which seeks the registration of record radiopharmaceuticals. To obtain registration of radiopharmaceuticals are necessary to prove the quality, safety, efficacy and specificity of the drug . For the safety of radiopharmaceuticals must be presented studies of acute toxicity, subacute and chronic toxicity as well as reproductive, mutagenic and carcinogenic. Nowadays IPEN-CNEN/SP produces one of the most important radiopharmaceutical of nuclear medicine, the 18F-FDG, which is used in many clinical applications, particularly in the diagnosis and staging of tumors. The objective of this study was to evaluate the systemic toxicity (acute/ subacute) radiopharmaceutical 18F-FDG in an in vivo test system, as recommended by the RDC No. 64, which will serve as a model for protocols toxicity of radiopharmaceuticals produced at IPEN . The following tests were performed: tests of acute and subacute toxicity, biodistribution studies of 18F-FDG, comet assay and reproductive toxicity. In acute toxicity, healthy rats were injected with 18F-FDG and observed for 14 days while in subacute toxicity animals were observed for 28 days. The results showed no evidence of toxicity at exposure 18F-FDG in acute and subacute toxicity. The biodistribution showed similar results to the literature, where the bladder is the organ that receives the most radiation. The comet assay showed that the radiation from the radiopharmaceutical was not significant to generate DNA damage. In reproductive toxicity in coupled rats exposed to 18F-FDG generated completely normal and healthy puppies. Finally, the 18F-FDG did not show any toxicity.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T08:03:49Z (GMT). No. of bitstreams: 1 FontesJardim_DenisLeonardo_D.pdf: 4065886 bytes, checksum: 09542135435393259e6847f6bad0b542 (MD5) Previous issue date: 2014 / Resumo: Tradicionalmente, o modelo de desenvolvimento de drogas em onco-hematologia utiliza-se de estudos de fase I como a primeira etapa no desenvolvimento clínico de uma droga. Nesses estudos, procura-se estabelecer a dose recomendada de novos agentes e seu perfil de segurança inicial, sendo as toxicidades o principal desfecho a ser monitorado. Posteriormente, estudos de registro são conduzidos para a aprovação de novas medicações por agências regulatórias. A maioria das medicações foi historicamente desenvolvida para uma população não-selecionada de um subtipo tumoral. No entanto, recentemente, o conhecimento em onco-hematologia vem sofrendo profundas mudanças, principalmente com o advento de novas técnicas de biologia molecular que permitem um melhor conhecimento da biologia das neoplasias. Com isso, é crescente a percepção de que existem subpopulações de pacientes com alterações moleculares específicas que demandam estratégias direcionadas; também é crescente o número de novas drogas de alvo molecular em desenvolvimento. Nesse contexto, é pouco sabido se o modelo tradicional de desenvolvimento de drogas, historicamente utilizado para medicações citotóxicas e para populações não selecionadas, estaria adequado frente essas novas demandas. Neste projeto de doutorado, avaliamos a correlação dos resultados de dose e toxicidade dos estudos de fase I com desfechos semelhantes nos estudos futuros de registro de novas medicações aprovadas pelo Food and Drug Administration (FDA) em onco-hematologia, com ênfase na comparação entre drogas citotóxicas tradicionais e drogas de alvo molecular. Determinamos que em 73% das comparações os estudos de registro adotaram uma dose entre 80% e 100% daquela recomendada pelos estudos de fase I, sendo que para drogas de alvo molecular os estudos clínicos iniciais foram menos preditores da dose futuramente aprovada dos agentes. Setenta por cento das toxicidades clinicamente relevantes em estudos de registro foram ao menos citadas em estudos de fase I, havendo uma correlação positiva com um maior número de pacientes incluídos em estudos iniciais, até cerca de 60 pacientes. Apesar das diferenças entre as populações de um estudo de fase I e de registro, a análise mostrou que um dos objetivos dos estudos de fase I foi cumprido, que seria de garantir a segurança de pacientes através da determinação de dose. Também buscamos comparar a eficácia em termos de controle de neoplasias entre drogas desenvolvidas com o conceito de terapia personalizada em relação àquelas para uma população não selecionada. Através da metodologia de meta-análise, demonstramos que em estudos de registro randomizados utilizados pelo FDA, terapias consideradas personalizadas proporcionaram maiores chances de respostas antitumorais e prolongamento do tempo até progressão de doença. A análise de todos os estudos de registro (randomizados e não randomizados) também mostrou que a terapia personalizada foi um fator independente relacionado ao aumento de sobrevida global dos pacientes. Os resultados obtidos sugerem que, frente as recentes modificações no entendimento de neoplasias e desenvolvimento de drogas, o modelo tradicional de determinação de doses em estudos de fase I necessita de adaptações, principalmente para drogas de alvo molecular, e que a incorporação do conceito de terapia personalizada é benéfica e segura para pacientes / Abstract: Traditionally, phase I trials are the first step in the clinical development model of new drugs in hematology-oncology. Phase I trials aim to establish the recommended dose of new agents for further development and their initial safety profile. Toxicities are the main outcome in these studies. Subsequently, registration trials are conduct to be the basis for drug approval by regulatory agencies, including the Food and Drug Administration (FDA). The majority of new drugs were developed for an unselect histologic type of cancer. Nevertheless, new technologies led to advances in the comprehension of the molecular basis of cancers. There is a growing evidence suggesting that specific molecular subtypes of cancers need specific strategies to target their genetic drivers. Additionally, the availability of new targeted therapies is increasing. In this context, it is not known how the traditional drug development model originally developed for cytotoxic drugs and for an unselect cancer population would perform. In this PhD thesis, we compared dosing and toxicity information from phase I trials with registration trials that led to approval of new drugs for cancer treatment by the FDA, with special attention to the sub-analysis comparing targeted versus cytotoxic agents. In 73% of matched trials (phase I trial and the respective registration trial), the dose from the registration trial was within 20% of the dose recommended by the phase I. In a multivariable analysis, phase I trials of targeted agents were less predictive of the final approved dose when compared to cytotoxic agents. Of the 530 clinically relevant toxicities in later trials, 70% were described in the respective phase I. A significant relationship between increasing the number of patients in phase I (up to 60) and the ability to describe future clinically relevant toxicities was observed. Despite several differences in design and cancer population when compared to registration trials, phase I trials were able to assure safety of patients based on its dose recommendation, which is one of the major objectives of these trials. We also compared efficacy outcomes between anticancer drugs developed under a personalized strategy (biomarker-driven treatment) with drugs developed for an unselected population. In randomized registration trials (using a random-effects meta-analysis model), personalized therapy arms were associated with a higher relative response rate ratio (compared to their corresponding control arms), prolongation of progression-free survival (PFS) and trend towards longer overall survival (OS) in comparison to non-personalized trials. Analysis of experimental arms in all registration trials (randomized and non-randomized) demonstrated that personalized therapy was an independent predictor of better response rates, PFS and OS. Treatment-related mortality rate was similar for personalized and non-personalized trials. Our results suggests that the traditional dose determination model from phase I trials needs further adaptations, especially for new molecularly targeted therapies. In addition, a personalized therapy approach for development of new agents was safe and associated with improved efficacy outcomes. We believe that our results will help to implement strategies for the development of new agents for the treatment of hematologic and solid cancers / Doutorado / Clinica Medica / Doutor em Clínica Médica

Orientador: Gisela de Aragão Umbuzeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia / Made available in DSpace on 2018-08-20T00:20:58Z (GMT). No. of bitstreams: 1 Vacchi_FrancineInforcato_M.pdf: 2178218 bytes, checksum: c13ca17191dbaab46671272dc416c546 (MD5) Previous issue date: 2012 / Resumo: Cerca de 70% dos corantes utilizados em indústrias têxteis são corantes do tipo azo, que se caracterizam pelo grupo -N=N- ligado a sistemas aromáticos, sendo que a função azo inclui os principais tipos de corantes. A eliminação da cor no efluente é um grande desafio para o setor têxtil, já que, aproximadamente, 15% da produção mundial de corantes são descartados para o meio ambiente durante sua produção, processamento e aplicação. O corante comercial Disperse Red 1 utilizado neste trabalho é composto por seis corantes diferentes e surfactante, porém o corante principal em sua composição é o Disperse Red 1 (N-Ethyl-N-(2-hydroxyethyl)-4-(4- nitrophenylazo) aniline; CAS number 2872-52-8) com 60% em massa. O corante comercial foi submetido ao processo de cloração com gás cloro, simulando as condições utilizadas em Estações de Tratamento de Efluentes. A toxicidade do corante comercial CI Disperse Red 1 e do subproduto clorado foi avaliada em testes agudos com Ceriodapnhia dubia, Ceriodaphnia silvestrii, Daphnia similis, Daphnia magna, Hydra attenuata, e em testes crônicos com Pseudokirchneriella subcapitata, Ceriodapnhia dubia, e Hydra attenuata. A mutagenicidade foi avaliada com ensaio Salmonella/microssoma com as linhagens TA98, TA100 e YG1041. D. similis foi o organismo mais sensível ao corante comercial; e H. attenuata foi mais sensível ao subproduto clorado. A CE50 obtida para D. similis para o corante comercial é similar ao corante puro, mostrando que a toxicidade do corante comercial é devido ao Disperse Red 1. O subproduto clorado foi menos tóxico do que o corante para todos os organismos testados, com exceção de H. attenuata. Porém, o subproduto clorado foi mais mutagênico do que o corante para todas as linhagens testadas. Mais estudos são necessários para compreender os mecanismos envolvidos na toxicidade destes corantes, considerando sua alta toxicidade para organismos aquáticos, a fim de fornecer informações para a elaboração de corantes ambientalmente corretos. Os resultados obtidos neste trabalho podem fornecer informações úteis para a derivação de critérios para qualidade da água para esse corante / Abstract: About 70% of dyes used in textiles industries are of type azo. These days 15% of the dye world production is discharged into the environment during its production, processing and application. Effluent that contains dyes must have their color removed in order to be in compliance with environmental regulations, but sometimes the treatment can lead to more toxic compounds. The commercial azo dye Disperse Red 1 used in this study is composed of six different dye constituents and surfactant; however the main dye of the commercial product is the Disperse Red 1 (N-Ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo) aniline; CAS number 2872-52-8). Commercial dye samples were treated with chlorine gas, in order to simulate the conditions used in effluent treatment plant. The ecotoxicity of the commercial dye and chlorinated byproduct was evaluated in acute tests with Ceriodaphnia dubia, Ceriodaphnia silvestrii, Daphnia similis, Daphnia magna, and Hydra attenuata as well as in chronic toxicity tests with Pseudokirchneriella subcapitata, Ceriodapnhia dubia and Hydra attenuata. Mutagenicity was evaluated using Salmonella/microsome assay with TA98, TA100 and YG1041 strains. The commercial dye Disperse Red 1 was similarly toxic to P. subcapitata, C. dubia and D. similis, even with different endpoints. The chlorinated byproduct was less toxic to all organisms tested than the dye, except for H. attenuata. The toxicity of the commercial dye is due to Disperse Red 1 itself and the surfactant does not seem to contribute to the toxicity, at least to D. similis. The chlorinated byproduct was more mutagenic than the commercial dye. More studies are necessary to address the mechanisms involved in the toxicity of this azo dye considering its high toxicity to aquatic organisms in order to provide information for the design of more environmental friendly dye products. The results obtained in this work can provide useful information for the derivation of water quality criteria for this dye / Mestrado / Tecnologia e Inovação / Mestre em Tecnologia

Polycyclic aromatic compounds are ubiquitously distributed pollutants in the aquatic and terrestrial environment containing harmful properties on creatures such as carcinogenicity, teratogenicity and toxicity, but are not so well analyzed yet. In the present study, the embryotoxic and teratogenic effects of selected hydroxylated and methylated PACs on the embryonal development of Danio rerio as an aquatic model organism were analyzed with the Fish Embryo Toxicity Test (FET) and the Tail Length Test (TLT) to obtain information on the toxic and teratogenic impact of the tested PACs on the environment. Two of the five tested PACs, 9-MA and DMBA, showed embryotoxic respectively teratogenic effects on the embryonal development of the zebrafish. The embryotoxicity of 9-MA was indicated in the high mortality rate of the exposed zebrafish embryos, whereas the teratogenic effect of DMBA was revealed in the emergence of sub-lethal malformations during the embryonal development such as a shortened tail length, tail curvatures, tail tip deformity or the formation of edema on the yolk sac and pericard as well as abnormal heartbeat and blood circulation. The high mortality rate of the zebrafish embryos exposed to 9-MA did not increase over the exposure time of 96h, which suggests that the chorion of the zebrafish egg could not protect the embryo at all against the strong embryotoxic effect of 9-MA. The sub-lethal malformations of the zebrafish embryos exposed to DMBA could be induced to the metabolic activation of AhR-agonist DMBA through the AhR-pathway or the accumulation of the neurotransmitter Acetylcholine due to the inhibitory function of DMBA on the ACh-Esterase, which caused a neuromuscular system defect or uncontrolled contractions of the axis musculature. Further research, may focus on the mode of action of PACs such as 9-MA and DMBA and their impact on organisms in order to take reasonable precautions to avoid or to diminish the uptake of PACs from the environment.

Ce travail porte sur l'étude du polyglyoxylate d'ethyle (PGEt) : sa synthèse, sa caractérisation et sa dégradation. Le PGEt a été obtenu par polymérisation anionique. Les conditions optimales font intervenir un amorçage par NE3 dans le CH2Cl2 à une température inférieure à -20°C. L'existence d'une température plafond (Tp = 310K pour [M]0 = 1M) nécessite l'utilisation d'agents de terminaison (phényl isocyanate ou bromure de 2-bromo-2-méthyl propionyle) afin d'obtenir des PGEt stables. La dégradation par hydrolyse in vitro du PGEt a été étudiée par RMN 1H, CES... Le mécanisme fait intervenir des coupures de chaines et des hydrolyses des esters. Les produits ultimes de dégradation identifiés sont l'éthanol et l'hydrate d'acide glyoxylique. Le caractère biodégradable du PGEt a été confirmé par respirométrie. Des études préliminaires de toxicité (sur des hématies, le nématode C. Elegans, et la croissance des plantes) n'ont pas révélé d'effet nocif du PGEt et de ses produits de dégradation. / This work focuses on the study of poly(ethyl glyoxylate) (PRtG), from its synthesis and its characterization to its degradation to some biological applications. The chosen conditions for PEtG's synthesis were initiated by an anionic way (NEt3) in presence of CH2Cl2. Because the ceiling temperature of the monomer (EtG) is low (7°C), it is necessary to block the hydroxyl ended groups generated in situ. The use of PhNCO leads to stables PEtG. The PEtG can be used as a macroinitiator of atom transfer radical polymerization (ATRP if it is wisely done. The copolymerization of styrene is then controlled. The degradation by in vitro hydrolysis of PEtG mainly led to ethanol and glyoxylic acid hydrate release. The biodegradable character of PGEt was assessed by a respirometric test and a preliminary study of toxicity (on red blood cells, worms and plants) revealed no significant effect at the concentrations generally used.

Les infections par cytomégalovirus sont un problème majeur en transplantation rénale du fait de l’augmentation du risque de perte de greffon et de l’augmentation de la morbi-mortalité des patients. Toutefois la mise en place d’un traitement prophylactique par ganciclovir a significativement fait diminuer l’incidence de ces infections. Cette efficacité est toutefois limitée par une importante hématotoxicité notamment des neutropénies. La survenue de cet évènement indésirable conduit à une réduction des doses voire à un arrêt du traitement, favorisant ainsi l’émergence de résistances virales. Ces résistances sont un problème grandissant chez les personnes transplantées du fait du manque de protocole de prise en charge de celles-ci. Dans ce contexte notre objectif était de mieux comprendre la survenue et le mécanisme de cette toxicité. Dans un premier temps nous avons étudié le métabolisme intracellulaire du ganciclovir chez des patients. Nous avons remarqué qu’il y a une forte corrélation entre l’exposition à la forme active du ganciclovir et la diminution du nombre de neutrophiles au 3ème mois de traitement. Nous avons par la suite étudié l’impact de variations génétiques sur des transporteurs. Nous avons remarqué qu’un polymorphisme était fortement associé à une diminution du nombre de neutrophiles et qu’il entrainait également une augmentation de la concentration intracellulaire de ganciclovir à l’aide d’un modèle in vitro. Cette thèse fournit de nouveaux outils d’exploration du métabolisme et de l’accumulation intracellulaire du ganciclovir qui pourraient être utiles pour la prévention de la survenue de neutropénies sous ganciclovir. / Cytomegalovirus infection is a major issue in transplant patients as it affects the graft survival and contributes to patients’ morbi-mortality. The implementation of ganciclovir prophylaxis has significantly decreased its incidence, however GCV frequently induces neutropenia. This adverse effect leads to a decrease in the ganciclovir dose or to a discontinuation of the therapy, thereby favoring viral resistance. Resistance to ganciclovir is a growing problem in solid organ transplantion because of the lack of proper data to support treatment decisions when it is encountered. In this context we aim at better understanding the factors involved in this toxicity. First we explored the intracellular metabolism of ganciclovir in patients’ white blood cells. We found that the active form of ganciclovir is associated with neutrophil toxicity at month 3 of treatment. Then we explored the effect of targeted polymorphisms among transporter genes in two cohorts of renal transplant patients. We found that a single nucleotide polymorphism is strongly associated with a decrease in the neutrophil count and in ganciclovir intracellular accumulation. This thesis provides relevant tools for a deeper exploration of ganciclovir intracellular metabolism and accumulation which might be useful for the prevention of ganciclovir induced neutropenia.

Many severe clinical conditions encountered in Africa involve the effects of iron overload on diseases (AIDS, TB etc) and vital organs (e.g., liver). To intervene successfully in the HIV pandemic, knowledge of AIDS pathogenesis and factors that stimulate or inhibit viral replication are crucial. Iron overload is believed to cause serious damage during HIV infection. Indeed, it is believed that the metal is required by infected cells to synthesize viral particles. The consequences of excess iron in vivo include the stimulation of microorganism growth, an increase in oxidative stress and an impairment of immune system function. Iron chelation have been reported to modulate some of these effects. In a project designed to assess the effect of in vitro iron overload (also synonymously referred to as iron loading) on HIV infection, cells (HIV-infected and controls) were directly loaded with iron and desferrioxamine (DFO, an iron chelator) respectively or combined. We then performed experiments to investigate the effects of these chemicals on host cell defenses and viral replication. Effective iron loading of all cell lines used was confirmed by emission spectroscopy. Through viability assays using tetrazolium salts, flow cytometric analysis of apoptosis and necrosis using Annexin-V, and specialised ELISAS; the effect of iron loading on host cell viability, survival or death and cytokine production was studied. The effect of iron loading on virus infectivity was also investigated by looking at core protein (p24) levels and reverse transcriptase (RT) activity. Prior to the start of the bioassays, several dyes were compared during identical procedures to find an effective and consistently functional dye assay for the assessment of cell growth/viability or proliferation. Viability assays provided a qualitative picture of events while flow cytometric analysis allowed us to compare viability with specific types of cell death (apoptosis/necrosis). Excess iron in the form of 500ƒÝM FeSO4-7H2O in addition to serum iron was found to be non-toxic to cells alone but detrimental to HIV-infected cells. Equimolar amounts of DFO inhibited cell growth, cytokine production and viral replication. Our results indicate that Fe loading stimulates viral replication. Iron chelation on the other hand decreased HIV replication suggesting a possible area for further therapy research using iron chelators in situations of iron loading in the presence of HIV/AIDS. / Dr. Debra Meyer

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