El establecimiento de un reservorio latente del virus del HIV en células T CD4+ es la mayor barrera contra la erradicación de esta enfermedad. Para lograr su erradicación, se necesitaría combinar la terapia antirretroviral HAART con drogas capaces de reactivar los virus durmientes. El objetivo principal de este estudio es entender cómo se establece la latencia tras la integración del virus en el genoma, con el propósito de identificar factores involucrados que pudieran ser dianas de una nueva terapia. Hemos generado una librería de clones que contienen un minigenoma latente del virus que expresa GFP como reportero cuando se reactiva. Esta librería permite estudiar la posible relación existente entre estado de la cromatina en el lugar de integración y actividad del promotor. También hemos estudiado la implicación de la interferencia transcripcional en el establecimiento de la latencia en los clones cuya integración del HIV ha tenido lugar en genes transcripcionalmente activos. Para investigar el mecanismo de represión durante la latencia, se han llevado a cabo depleciones de factores relacionados con el reensamblaje de la cromatina y proteínas relacionadas con represión transcripcional. Finalmente, hemos buscado drogas que puedan reactivar el virus latente como posible terapia a combinar con la terapia antiretroviral. / The establishment of a latent HIV reservoir in CD4+ T cells is the main barrier to prevent the eradication of the virus and converts its infection in a chronic disease. To achieve its eradication, it would be needed to combine HAART with drugs able to reactivate the dormant viruses. The main objective of this study is to understand how latency is established after proviral integration into the genome, with the aim of identifying factors involved that could be targeted by new therapeutic approaches. We have generated a library of clones containing a latent HIV minigenome that expresses GFP as a reporter only when reactivated. This library allows the study of the relationship between the chromatin state at the site of integration and HIV promoter activity. We have also studied the implication of transcriptional interference in the establishment of latency in those clones where HIV has integrated in transcriptionally active genes. To further investigate the mechanism of transcriptional repression in latency we have performed knockdowns of known chromatin reassembly factors and repression-related proteins by using shRNA expression. Finally we have searched drugs that can reactivate the latent HIV as a possible therapy to combine with HAART.

The genome sequencing projects have generated a wealth of genomic data and the analysis of this data has provided many interesting findings. However, genome wide analysis of bacteria for promoters has lagged behind, because it has been difficult to accurately predict the promoters with so much background noise that are found in bacterial genomes. One approach to overcome this problem is to predict phylogenetically conserved promoters across multiple genomes of different bacteria, thus filtering out many of the false positives, which are predicted by the current methods. However, there are no programmes capable of doing this. Therefore, the work presented in this thesis has developed a position weight matrix (PWM) based programme called Multiscan that predicts conserved promoters across multiple bacterial genomes. Since Chlamydia is one of the most sequenced bacterial genera and has a high level of conservation of genes and large-scale conservation of gene order between species, Multiscan was developed and tested on Chlamydia. When Multiscan analysed a genome wide dataset of equivalent non-coding regions (NCRs) upstream of genes, from Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia caviae for σ66 promoters that are phylogenetically conserved, Multiscan predicted 42 promoters. Since only one of the 42 promoters predicted by Multiscan had previously available biological data to confirm its prediction, an additional subset of 10 of the remaining 41 σ66 promoters were analysed in C. trachomatis by mapping the 5' end of the transcripts. The primer extension assay synthesised cDNA products of the correct length for seven of the 10 genes chosen. When the performance of Multiscan was compared to one of the accepted method for genome wide prediction of promoters in bacteria, the &quotstandard PWM method", Multiscan predicted 32 more promoters than the &quotstandard PWM method" in Chlamydia. Furthermore, the promoters predicted by Multiscan were up to three more mismatches from the Escherichia coli σ70 consensus sequence than the promoters predicted by the standard PWM method. Although Multiscan predicted 42 promoters that were well conserved across the three chlamydial species, the analysis was unable to identify the 14 known σ66 promoters in C. trachomatis. These promoters were missed (1) because they were dissimilar to the E. coli σ70 consensus sequence and/or (2) because the promoters were poorly conserved across the three chlamydial species. To address the second possibility, the 14 false negatives were analysed by another phylogenetic footprinting method. Fourteen sets of equivalent NCRs located upstream of the homologous genes from the three chlamydiae were aligned with the computer programme Clustal W and the alignment analysed &quotby eye" for evidence of phylogenetic footprints containing the 14 false negatives. The analysis identified that seven of the 14 false negatives were poorly conserved across the chlamydial species. Analysis of two of the seven promoters that could not be footprinted, the promoters of ltuA and ltuB, by mapping the transcriptional start sites in C. caviae, confirmed their poor conservation across C. trachomatis and C. caviae. This analysis showed that substantial differences exist in chlamydial σ66 promoters from equivalent NCRs upstream of genes. This study has developed a new computer programme for genome wide prediction of promoters that are phylogenetically conserved and has shown the value of this programme by identifying seven new well conserved promoters and seven candidate poorly conserved promoters in Chlamydia.

Among women in the 45 to 64 age group, over half of the recorded deaths are from cancer, breast cancer being the most common. Just over 30% off all deaths in New Zealand women is caused by breast cancer. Treatment of cancer is difficult, not only due to the physiological and immunological similarities between a cancer cell and a normal cell, but also due to the high cardiotoxicity of many treatments, and also the problems related with the development of resistance. Approximately 40% of the cancer cells treated with the chemotherapy drug doxorubicin will become resistant to treatment. Drug efficacy is strongly associated with the proliferation status of a cell, as cancer cells divide rapidly, this can often be the defining factor between effective treatments or the development of resistance. Central to this proliferation status is an enzyme known as topoisomerase IIa. This essential enzyme is expressed in all cells and is required to relieve the torsional stress in DNA that is created during normal cellular processes. A number of commonly used anti-cancer drugs have been found to target topoisomerase IIa in cancer cells and significantly, during the development of drug resistance levels of topoisomerase IIa enzyme have been found to be reduced in some cell lines and tumours. There are a number of factors that can modulate the amount of topoisomerase IIa enzyme found in a cell, and one of the ways to understand this is to examine the regulation of the topoisomerase IIa gene, most importantly the proteins that interact with the promoter region to direct transcription. The human topoisomerase IIa promoter has been found to be regulated by a number of transcription factors that can bind to their cognate sequences. The introduction of mutations within specific sequences of the topoisomerase IIa promoter has enabled the identification of a key regulatory region within the promoter, a sequence of DNA that encompasses both the ICB1 and GC1 regulatory elements. Transcription factor NF-Y is found to bind to ICB1 element, whereas transcription factors Sp1 and Sp3 have been found to associate with the GC element. However this region of the promoter was also found to bind a fourth uncharacterised component. This research aims to further define the protein components that are found to bind to this important ICB1/GC1 regulatory region and distinguish the protein-protein and protein-DNA interactions that are important for the regulation of the human topoisomerase IIa promoter.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-140).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study aimed to evaluate the effect of inclusion of concentrated dietary fiber as prebiotic agent in diets on metabolic, immune responses, performance parameters, deposition of nutrients and production of digestive enzymes of juvenile silver catfish. Concentrates were prepared from dietary fiber citrus pulp, biomass of yeast brewery and grain included in linseed and mixed diets followed a diet containing the prebiotic commercial mananoligossacarids base Actigen® and control treatment without added prebiotic agent. For 50 days, 600 juvenile silver catfish with average initial weight of 3.54±0.53 g were kept in a water recirculation system with two biological filters, settling box, heating and 20 tanks with a capacity of 230 liters. Were randomly assigned to 30 fish per experimental unit, which were fed the experimental diets, three times a day (8:00, 13:00 and 17:00) to apparent satiation. At the end of the experiment the animals were subjected to biometrics were collected blood, liver, mucous, intestine and data length and weight, beyond a sample of fish. The experimental design was a randomized, with five treatments and four replications, the data were subjected to analysis of variance and means were compared by Tukey test (P<0.05). Cholesterol levels, total protein, globulin and mucoprotein were higher in animals fed yeast autolysate and linseed fiber in the diet. A higher amount of liver glycogen in fish fed with control diet and Actigen®, the liver protein content was higher (P<0.05) in a diet containing linseed fiber. Fish fed diets containing yeast autolysate and linseed fiber were superior (P<0.05) to the other treatments tested, as well as higher crude protein values and deposited body fat. Animals fed diets containing citrus pulp showed lower performance and nutrient deposition. The yield of body, digestive indices and production of digestive enzymes were not affected by the tested treatments. The yeast autolysate and linseed fibers provide a prebiotic effect when added to diets for juvenile silver catfish, since they benefit the immune system and provide improved performance and deposition of nutrients by the animal. / Este trabalho teve como objetivo avaliar o efeito da inclusão de fibra alimentar concentrada como agente prebiótico em dietas sob as respostas metabólicas, imunológicas, parâmetros de desempenho, deposição de nutrientes e produção de enzimas digestivas de juvenis de jundiá. Foram avaliados concentrados de fibra alimentar preparados a partir da polpa cítrica, biomassa de levedura de cervejaria e grão de linhaça e incluídos em dietas mistas, além de uma dieta contendo o prebiótico comercial a base de mananoligossacarídeos Actigen® e uma dieta controle sem adição de agente prebiótico. Durante 50 dias, 600 juvenis de jundiá com peso médio inicial de 3,54±0,53g foram mantidos em um sistema de recirculação de água dotado de dois filtros biológicos, caixa de decantação, reservatório de água, aquecimento e 20 tanques com capacidade de 230 litros. Distribuiu-se ao acaso 30 peixes por unidade experimental, os quais receberam as dietas experimentais, três vezes ao dia (8:00, 13:00 e 17:00 horas) até a saciedade aparente. Ao final do experimento os animais foram submetidos a biometria onde coletou-se sangue, fígado, muco e intestino dos peixes, além de dados de peso e comprimento e uma amostra de peixes. O delineamento experimental o inteiramente casualizado, com cinco tratamentos e quatro repetições, os dados foram submetidos a análise de variância e as médias comparadas pelo teste de Tukey (P<0,05). Os níveis de colesterol, proteína total, globulina e mucoproteína, foram superiores nos animais alimentados com autolisado de levedura e fibra de linhaça na dieta. Observou-se maior quantidade de glicogênio hepático nos peixes alimentados com dieta controle e Actigen®, o conteúdo de proteína hepática foi superior (P<0,05) para os animais que receberam dieta contendo fibra de linhaça. Os peixes alimentados com dietas contendo autolisado de levedura e linhaça fibra apresentaram desempenho superior (P<0,05) aos demais tratamentos testados, bem como maiores valores de proteína bruta e gordura corporal depositada. Animais alimentados com dietas contendo polpa cítrica mostraram menor desempenho e deposição de nutrientes. O rendimento de carcaça, índices digestivos e produção de enzimas digestivas não foram afetados pelos tratamentos testados. O autolisado de levedura e a fibra de linhaça proporcionam efeito prebiótico quando adicionados a dietas para juvenis de jundiá, uma vez que beneficiam o sistema imune e proporcionam maior desempenho e deposição de nutrientes pelos animais.

Oocyte activation deficiency (OAD) is an infertile condition observed in patients who have experienced recurrent total fertilisation failure (TFF) following intracytoplasmic sperm injection treatment. This condition was considered to be an idiopathic factor for a long time but strong clinical evidence now suggests that dysfunctional forms of phospholipase C zeta (PLC&zeta;) may be predominant causative factors for OAD. Genetic contribution has played a role in patients suspected of having OAD, as four PLC&zeta; exonic mutations have been discovered and characterised as being the cause of infertility. In this study, a novel nonsense mutation, PLC&zeta;K322Stop, was identified in the PLC&zeta; XY-linker region of Patient LR. This variant results in the truncation of approximately half of PLC&zeta;, therefore was non-functional when activity was tested. Patient LR, which also exhibited a previously reported mutation, PLC&zeta;H233L, may suggest that the patient is sub-fertile, as opposed to being infertile, as initially expected. Although research has purely focused upon the coding regions of PLC&zeta;, it was obvious that our knowledge of PLC&zeta; regulatory elements remain very limited. Next generation sequencing (NGS) was therefore employed to detect variants in the non-coding regions of PLC&zeta;, promoter and introns, which may have resulted in the observed phenotypic diversity of PLC&zeta; expression in fertile and infertile patients. As a result of mapping failure, an alternative approach was considered to identify variants within human PLC&zeta;, and this involved using the single nucleotide polymorphism (SNP) database. Over 2500 SNPs were localised in the intronic regions of PLC&zeta; and thus, it could be speculated that these variants may help elucidate the wide variation of PLC&zeta; expression reported. Additionally, two particular patients with TFF (79 and 107) were investigated in this study to identify an association with PLC&zeta; and their infertile state. For Patient 79, multiple PLC&zeta; immunofluorescence analysis was performed and a significant improvement in PLC&zeta; expression was observed one year after his first investigation. This may have been the result of an external factor, which influenced protein expression. As for Patient 107, a novel substitution mutation, PLC&zeta;V193E, was identified and was predicted to affect PLC&zeta; stability and folding. There is global interest to create a safer and alternative OAD therapy, namely a human recombinant PLC&zeta; protein (hrPLC&zeta;). The first method, using a bacterial cell line resulted in successful purification and identification but the product proved to be inactive following mouse oocyte microinjection. The second method involved production of a mammalian-expressed hrPLC&zeta;, which was successfully purified and identified but due to time restrictions, could not be tested for functionality. Concurrently, the findings in this thesis have reinforced the association between PLC&zeta; and OAD, and provided improved options for the diagnosis and treatment of OAD.

Orientador: Elisa Helena Giglio Ponsano / coorientador: Manoel Garcia Neto / Banca: Paulo César Ciarlini / Banca: Elizabeth Santin / Resumo: O subproduto de acerola é uma rica fonte de compostos bioativos que apresentam alta atividade antioxidante. No presente estudo, o desempenho produtivo, a população microbiana cecal, as características da carne, os parâmetros bioquímicos e a atividade antioxidante e oxidante sérica de frangos de corte alimentados com ração adicionada de farelo de subproduto de acerola (FAC), como um ingrediente alternativo, foram comparados com os mesmos parâmetros de frangos de corte alimentandos sem adição de FAC mas com agente melhorador de desempenho (AMD) e antioxidante sintético (AS). As dietas experimentais foram: controle positivo (CP), contendo 0,007% de sulfato de colistina 8% (AMD) e 0,01% de butilhidroxitolueno (BHT) (AS), controle negativo (CN), sem AMD, AS ou FAC, dieta com 5% de FAC (AC 5%) e dieta com 7,5% de FAC (AC 7,5%). Cento e sessenta pintinhos (Cobb 500) foram vacinados com a vacina Livacox T, via ocular e distribuídos aleatoriamente em 16 boxes, com 4 repetições por tratamento, contendo 10 aves em cada. Os animais foram. Parâmetros produtivos foram mensurados semanalmente até 42 dias de idade, quando os animais foram abatidos e a carne, o sangue e o conteúdo cecal foram coletados para as análises de cor/rancidez oxidativa, a contagem da população bacteriana cecal, os parâmetros bioquímicos e o status oxidante/antioxidante sérico. Não houve diferenças para ganho de peso, consumo de ração e conversão alimentar entre os diferentes grupos, assim como para os rendimentos de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Acerola byproducts are a rich source of bioactive compounds with high antioxidant activity. In the present study, productive performance, bacterial caecal population, meat characteristics, biochemical parameters and serum antioxidant status of broilers fed acerola byproduct (ACM) as an alternative ingredient were compared with the same parameters of broilers fed diets with no ACM but with antimicrobial growth promoters (AGP) and synthetic antioxidant (SA). The experimental diets comprised: positive control (PC), containing 0.007% colistin sulfate 8% (AGP) and 0.01% butylated hydroxytoluene (BHT) (SA) and no ACM; negative control (NC), without AGP, AS or ACM; diet with 5% ACM (AC 5%); and diet with 7.5% ACM (AC 7.5%). One hundred sixty one day old Cobb 500 male chicks were vaccinated with Livacox T via ocular and randomly distributed into 16 pens. Four repetitions were performed, with ten birds per pen. used in the experiment. Productive parameters were measured weekly until day 42, when the broilers were slaughtered and the meat, the blood and the caecal contents were collected for the analyses of oxidative rancidity and color of the meat, serum oxidant/antioxidant status and caecal bacterial population counts. There were no differences among the treatments regarding to feed intake, body weight gain and feed conversion, as well as to dressing percent and live weight. Only ACM at 5% caused an increase in the caecal lactic bacteria count. Breast color and thigh lipid rancicity ... (Complete abstract click electronic access below) / Mestre

Magister Scientiae - MSc / OVARIAN cancer research focuses on answering important questions related to the disease, determining whether new approaches are feasible to contribute towards improving current treatments or discovering new ones. This study focused on the transcriptional regulation of genes that have been implicated in ovarian cancer, based on the occurrences of single nucleotide polymorphisms (SNPs) within transcription factor binding sites (TFBSs). Through the application of several in silico tools, databases and custom programs, this research aimed to contribute toward the identification of potentially bio-medically important genes or SNPs for pre-diagnosis and subsequent treatment planning of ovarian cancer. A total of 379 candidate genes that have been experimentally associated with ovarian cancer were analyzed. This led to the identification of 121 SNPs that were found to coincide with putative TFBSs potentially influencing a total of 57 transcription factors that would normally bind to these TFBSs. These SNPs with potential phenotypic effect were then evaluated among several population groups, defined by the International HapMap consortium resulting in the identification of three SNPs present in five or more of the eleven population groups that have been sampled. / South Africa