Philosophiae Doctor - PhD / Tsetse flies (Glossina spp) are the biological vectors for Trypanosomes, the causative magents of Human African Trypanosomiasis (HAT). HAT is a debilitating disease that continues to present a major public health problem and a key factor limiting rural development in vast regions of tropical Africa. To augment vector control efforts, the International Glossina Genome Initiative (IGGI) was established in 2004 with the ultimate goal of generating a fully annotated whole genome sequence for Glossina morsitans. A working draft genome of Glossina morsitans was availed in 2011. In this thesis, transcriptional regulatory features in Glossina morsitans were analysed using the draft genome. A method for TSS identification in the newly sequenced Glossina morsitans genome was developed using TSS-seq tags sampled from two developmental stages of Glossina morsitans. High throughput next generation sequencing reads obtained from Glossina morsitans larvae and pupae were used to locate transcription start sites (TSS) in the Glossina morsitans genome. TSS-seq tag clusters, defined as a minimum number of reads at the 5’ predicted UTR or first coding exon, were used to define transcription start sites. A total of 3134 tag clusters were identified on the Glossina genome. Approximately 45.4% (1424) of the tag clusters mapped to the first coding exons or their proximal predicted 5’UTR regions and include 31 tag clusters that mapped to transposons. A total of 1101 (35.1%) tag clusters mapped outside the genic region and/or scaffolds without gene predictions and may correspond to previously un-annotated transcripts or noncoding RNA TSS. The core promoter regions were classified as narrow or broad based on the number of TSS positions within a TSS-seq cluster. Majority (95%) of the core promoters analysed in this study were of the broad type while only 5% were of the narrow type. Comparison of canonical core promoter motif occurences between random and bona fide core promoters showed that, generally, the number of motifs in biologically functional genomic windows in the true dataset exceeded those in the random dataset (p <= 0.00164, 0.00135, 0.00185 for the narrow, broad with peak and broad without peak categories respectively). Frequency of motif co-occurrence in core promoter was found to be fundamentally different across various initiation patterns. Narrow core promoters recorded higher frequency of the TATA-box and INR motifs and two-way motif co-occurrence showed that the TATA-box-INR pair is over-represented in the narrow category. Broad core promoters showed higher frequency of the BREd and MTE motifs and two-way motif co-occurrence showed that the MTE-DPE pair is over-represented in broad core promoters. TATA-less promoters account for 77% of the core promoters in this analysis. TATA-less core promoters showed a higher frequency of the MTE and INR motifs in contrast to observations in Drosophila where the DPE motif has been reported to occur frequently in TATA-less promoters. These motif combinations suggest their equal importance to transcription in their corresponding promoter classes in Glossina morsitans.

Transcription of the osteocalcin gene, which encodes a bone-specific 10 kDa protein, is controlled by the coordinated utilization of modularly organized basal and hormone-responsive enhancer elements. Activation of these sequences involves the interaction of specific transcription factors to these promoter elements. It is becoming increasingly accepted that nuclear architecture provides a basis for support of tightly regulated modulation of cell growth and tissue-specific transcription which is required for the onset and progression of differentiation. Thus packaging of DNA as chromatin can facilitate the cooperative interaction between activities of independent regulatory elements that contribute to the level of transcription. Here, we show that a specific nucleosomal organization supports the constitutive expression of the osteocalcin gene in ROS 17/2.8 rat osteosarcoma cells and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of this gene in normal diploid osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNase I hypersensitive sites (proximal: -170 to -70, and distal: -600 to -400) and a selective nucleosome positioning over the osteocalcin gene promoter are directly associated with developmentally stage-specific transcriptional activation in bone-derived cells. In addition, we found that chromatin hyperacetylation prevents a key transition in the chromatin structure which is required for the formation of the distal DNase I hypersensitive site. This transition involves the interaction of specific nuclear factors and is necessary for the subsequent ligand-dependent binding of the vitamin D receptor complex. Finally, we have established a requirement for sequences residing in the proximal region of the osteocalcin gene promoter for both formation of the proximal hypersensitive site and basal transcriptional activity. Our approach was to assay nuclease accessibility in ROS 17/2.8 cell lines stably transfected with promoter deletion constructs driving expression of a CAT reporter gene.

For a long time, 5' cap has been thought to be privilege only for eukaryotic organisms in form of 7-methylguanosine cap at the end of mRNA. This was changed only a few years ago. By using methods liquid chromatography and mass spectrometry a new molecule associated with RNA of Escherichia coli has been found. This molecule turned out to be nicotinamide adenine dinucleotide (NAD+ ) attached to 5' end of some small regulatory RNAs (sRNA). Later it has been shown, that RNA polymerase can attach NAD+ at 5' of RNA ab initio, meaning that RNA polymerase can utilize NAD+ as a substrate for transcription initiation. To some extent substrate for transcription initiation is chosen based on promoter sequence. Crucial requirement is presence of adenine at +1 position of DNA coding strand. This thesis focuses on promoter sequence requirements for transcription initiation with NAD+ . As a template for transcription four promoters with different modifications and their chimeras are used: RNA1, Pveg, lac UV5 and rrnB P1. Also, I tried to compare RNA polymerase from E. coli and B. subtilis in terms of transcription initiation substrate usage. Lastly, I describe here isolation of NudC, enzyme that cleaves NAD+ to nicotinamide mononucleotide (NMN) and adenosine monophosphate (AMP). NudC will be used for upcoming...

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The traditional use of antibiotics in aquaculture as growth promoters has been limited due to the negative effects caused by these drugs. As an alternative to the use of these drugs has been sought manipulation of the microbiota of the gastrointestinal tract of aquatic animals through the use of oligosaccharides and dietary fibers with prebiotic potential. Thus, this study aimed to apply different methodologies to obtain Dietary Fiber Concentrates (DFC) = mucilage (MG); pectin (PN) and β-glucan + manan (βG + M) and evaluate the prebiotic potential of these supplements in the diet of juvenile jundiá (Rhamdia quelen). The determination of the nutritional composition of the ingredients revealed that the predominant component in all DFCs were dietary fiber and insoluble fiber. The DFC that had higher extraction yield was βG + M (19.81 ± 8.54%), followed by pectin (14.54% ± 2.72), and mucilage (7.18 ± 1.54%). The composition of mucilage and pectin had a greater diversity of monosaccharides, since the βG+M consisted primarily of mannose (74.5%) and glucose (24.3%). The supplementation of DFC in jundiás diet was assessed for eight weeks through study of growth, body nutrient deposition, digestive enzymes, biochemical and metabolic parameters, responses to stress and immune and intestinal morphology. The jundiás supplemented with DFCs achieved higher growth than the control group and similar to animals supplemented with 5 g kg-1 commercial prebiotic (CP 5). Most somatic parameters and whole fish proximate composition were influenced by supplementation of DFCs. The supplementation of pectin promoted lower activity of digestive enzymes in relation the control group. The animals supplemented with DFC obtained positive changes in biochemical parameters. Furthermore, jundiás showed no response to application of the stressor, maintaining basal cortisol levels. The fish supplemented with DFCs had higher hepatic glycogen stores in relation the control group. Moreover, supplementation with DFCs increased the height of intestinal villi of jundiá. However, these values were lower for the animals of the group PC 5. For thickness of the epithelium this variable was higher in the control group compared to animals supplemented with β-glucan+Manana. / O uso tradicional de antibióticos na aquicultura como promotores de crescimento tem sido limitado em função dos efeitos negativos promovidos por estes medicamentos. Como alternativa ao uso destas drogas, tem se buscado a manipulação da microbiota do trato gastrointestinal dos animais aquáticos através da utilização de oligossacarídeos e de fibras alimentares com potencial prebiótico. Neste sentido, este estudo teve como objetivo aplicar metodologias para obtenção de diferentes Concentrados de Fibras Alimentares (CFAs) = Mucilagem (MG); Pectina (PN) e β-Glicana+Mananas (βG+M) e avaliar o potencial prebiótico destes suplementos na dieta de juvenis de jundiá (Rhamdia quelen). A determinação da composição nutricional dos ingredientes revelou que os componentes predominantes em todos os CFAs obtidos foram fibra alimentar total e fibra insolúvel. O CFA que apresentou maior rentabilidade de extração foi a βG+M (19,81%±8,54), seguida da Pectina (14,54%±2,72) e Mucilagem (7,18%±1,54). A composição da Mucilagem e Pectina obtiveram maior diversidade de monossacarídeos, já a βG+M consistiu basicamente de manose (74,5%) e glicose (24,3%). A suplementação dos CFAs na dieta de jundiás foi avaliada durante oito semanas, através de estudo de crescimento, deposição corporal de nutrientes, enzimas digestivas, parâmetros bioquímicos e metabólicos, resposta ao estresse e imunológica e morfometria intestinal. Os jundiás suplementados com os CFAs obtiveram crescimento superior em relação ao grupo controle e similar aos animais suplementados com 5 g kg-1 de prebiótico comercial (PC 5). A maioria dos parâmetros somáticos e de composição centesimal de peixe inteiro foram influenciados pela suplementação dos CFAs. A suplementação de Pectina promoveu menor atividade das enzimas digestivas em relação ao grupo controle. Os animais suplementados com os CFAs obtiveram alterações positivas nos parâmetros bioquímicos avaliados. Além disso, os jundiás não mostraram resposta à aplicação do agente estressor, mantendo os níveis de cortisol basal. Os peixes suplementados com os CFAs obtiveram maiores estoques de glicogênio hepático em relação ao grupo controle. Além do mais, a suplementação com os CFAs promoveu aumento na altura de vilos intestinais dos jundiás. Porém, estes valores foram menores em relação aos animais do grupo PC 5. Para espessura do epitélio (EE) esta variável foi maior no grupo Controle comparado aos animais suplementados com β- glicana + Manana.

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No. of bitstreams: 1 barros_tl_me_fmva.pdf: 11705024 bytes, checksum: de173cbfeccee2f8f7280c4571a2a53e (MD5) Previous issue date: 2017-11-07 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / 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 carcaça e o peso vivo. Apenas o grupo AC 5% apresentou aumento de bactérias lácticas no ceco. A cor do peito e a peroxidação lipídica da coxa não diferiram entre os grupos. Os grupos que receberam FAC apresentaram alta concentração sérica de albumina e proteínas totais, enquanto não houve diferença para as concentrações de globulina e a para a proporção albumina:globulina entre os grupos, assim como para as concentrações séricas de AST, ALT, GGT, creatinina e ácido úrico. O colesterol total foi menor nos animais alimentados com FAC, não diferindo do grupo CP. Os grupos CP, AC 5% e AC 7,5% apresentaram alta capacidade antioxidante total, mas sem diferença para capacidade oxidante total e peroxidação lipídica. Concluiu-se que a adição de FAC na dieta de frangos de corte melhorou o potencial antioxidante sem prejudicar o desempenho produtivo, a saúde e mantendo a população microbiana cecal e as características da carne. O uso do subproduto de acerola como um ingrediente alternativo na dieta de frangos de corte pode atender a demanda a substituição de AMD e AS na produção de frangos de corte e ainda colabora para a sustentabilidade. / 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 did not differ among the groups. Groups fed ACM had higher serum albumin and total protein, although there were no differences in globulin and albumin:globulin ratio among the experimental diets, as well as to serum concentration of AST, ALT, GGT, creatinine and uric acid. Total cholesterol was lower in the animals fed ACM, with no differences to PC. Groups PC, ACM 5% and ACM 7.5% had higher serum antioxidant activity but similar oxidant activity and lipid oxidation. It was concluded that the addition of ACM into broilers diets improved the birds antioxidant status without harming their performance and health and maintaining the caecal microflora and the meat characteristics. The use of acerola byproduct as an alternative ingredient in broilers feeding may reach the demand for the substitution of AGP and SA in poultry production and still collaborates to sustainability / FAPESP: 15/19448-7 / FAPESP: 15/25853-1 / FUNEP: 1106

Resumo: O fator de transcrição Oct-4 é bem conservado entre as espécies e é conhecido por ser expresso em embriões e células-tronco embrionárias (CTE), sendo um importante marcador da pluripotência. Recentemente, foi relatado que a combinação de Oct-4 com três outros fatores de transcrição Klf-4, c-Myc e Sox2 foram capazes de reprogramar células somáticas a um estado indiferenciado pluripotente, chamadas células-tronco pluripotentes induzidas ("células iPS"), as quais apresentam várias das mesmas propriedades das CTE incluindo a pluripotência, auto-renovação e proliferação. O objetivo desse estudo foi avaliar a funcionalidade do promotor Oct-4 de eqüino em CTE de murinos. Três vetores plasmidiais expressando GFP ("green fluorescent protein") sob o controle do promotor Oct-4 de equinos, camundongo e quatro vetores lentivirais, também contendo o gene reporter GFP e os promotores Oct-4 de equinos, camundongo e humanos, pLZ2-ecOCT-EGFP (meq) (sequência equivalente de camundongos), pLZ2-ecOCT-EGFP (heq) (sequência equivalente de humanos), pLZ2-mOCT-EGFP e pLZ2-hOCT-EGFP, respectivamente, foram construídos. Todos os vetores também contêm um sítio de resistência à blasticidina que permite a seleção das células estáveis e das células transduzidas. Essas construções plasmidiais foram verificadas se funcionavam eficientemente, bem como o efeito do promotor Oct-4 em transfectar transientes e estáveis CTE. As construções com promotor Oct-4 de camundongo, humano e eqüino (sequência análoga à de camundongo) produziram somente 6% de células GFP positivas com intensidade de fluorescência (IF) >1000 pela análise em citômetro de fluxo, enquanto que o plasmídeo contendo o promotor Oct-4 de eqüino (sequência equivalente à de humanos) produziu menos células GFP positivas (>3%) com IF >1000, quando... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The pluripotency transcription factor Oct-4 is well conserved among species and is known to be expressed in embryos and embryonic stem (ES) cells; it is being an important pluripotency marker. It was recently demonstrated that the combination of Oct-4 with three other factors Klf-4, c-myc and Sox2 were able to reprogram somatic cells to a pluripotent and undifferentiated state. These cells known as induced pluripotent stem (iPS) cells share several properties with ES cells including self-renewal, proliferation and pluripotency. The aim of this study was to assess the functionality of the horse Oct-4 promoter in mouse ES cells. Three plasmids vectors expressing GFP (green fluorescent protein) under the control of the horse, mouse and four lentivirus vectors also containing reporter gene GFP and horse, mouse and human promoters, pLZ2-ecOCT-EGFP (mouse sequence equivalent), pLZ2-ecOCT-EGFP (human sequence equivalent), pLZ2-mOCT-EGFP and pLZ2-hOCT-EGFP, respectively, were built. All these vectors also contain a blasticidin resistance cassette to allow selection of transfected stable cells and transduced cells. Afterwards, to assess the functionality of the Oct-4 promoter all plasmids were tranfected the into transient and stable mouse ES cells. Constructs with mouse, human and horse (mouse analog sequence) Oct-4 promoter produced only 6% GFP positive cells with fluorescence intensity (FI)>1000 by 20 FACs assay, while plasmid horse (human analog sequence) Oct-4 promoter produced less GFP positive cells (>3%) with FI>1000, when compared with the positive control and among groups. However, GFP expression was not present in stable cells, whereas there were Blasticidin-resistant colonies-forming from 6 days post-transfection. To optimize the system in mouse ES cells, pLZ2-mOCT-EGFP and pLZ2-hOCT-EGFP lentivectors, were tested as controls. It was used HIV-1-derived... (Complete abstract click electronic access below) / Orientadora: Gisele Zoccal Mingoti / Coorientador: Joaquim Mansano Garcia / Banca: César Roberto Esper / Banca: Flávio Vieira Meirelles / Banca: Áureo Evangelista Santana / Banca: Simone Cristina Méo Niciura / Doutor

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-12-13T14:00:15Z No. of bitstreams: 2 Tese - Regis Kamimura - 2013.pdf: 3024185 bytes, checksum: d75f4278deb066540dc7434aef6cf446 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-12-14T10:15:36Z (GMT) No. of bitstreams: 2 Tese - Regis Kamimura - 2013.pdf: 3024185 bytes, checksum: d75f4278deb066540dc7434aef6cf446 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-14T10:15:36Z (GMT). No. of bitstreams: 2 Tese - Regis Kamimura - 2013.pdf: 3024185 bytes, checksum: d75f4278deb066540dc7434aef6cf446 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-12-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Gut microbiota is changed by various factors, being the diet which has the highest influence, as well as the modulating additives of the microbiome. The purpose of this work was to evaluate the effects of growth promoting additives in the diet on the percentage of goblet cells with acid and neutral mucin in small intestine of pigs, the percent of gut cells in proliferation by the technique of immunohistochemistry by PCNA, to certify and quantify the presence of Paneth’s cells by specific staining and by immunohistochemistry, since that cell has a great importance in the immunity and in the gut microbiota. The experiment was conducted in a certified swine breeding farm (CSBF), applying the CRD (completely randomized design). Four hundred and eighty castrated male piglets were used, in five treatments, with ten piglets by cage, eight repetitions, and in the basal treatment 16 repetitions were used, being T1=basal diet, denominated negative control, T2=T1+antimicrobial agent, T3=T1+probiotic, Saccharomyces cerevisiae, T4=T1+prebiotic MOS, T5=T1+probiotic Saccharomyces cerevisiae + the prebiotic MOS. The diet was based on corn and soybean meal plus a supplement of minerals and vitamins. There were changes with higher increase in quantity of goblet cells, calculated by millimeter of gut mucosa with neutral mucus compared to those with acid mucus and to the negative control. There were no expressive changes for cells with acid mucus. The percentage of proliferating cells in small intestine by the immunehistochemistry technique for PCNA did not show significant changes and the presence of Paneth’s cells was confirmed by special staining and by immunehistochemistry. The quantity of Paneth’s cells of duodenum changed according to additives used, in comparison with the negative control which had a greater quantity. With the addition of antimicrobial agent there was a lesser quantity. With the prebiotics and synbiotics occurred milder reduction and this suggests that immunomodulation occurred. Besides promoting a better gut health due to a healthier and more appropriate microbiota to the rearing of piglets in the nursery phase, the addition of growth promoters in feeds favors significant gains and can be recommended, since the results are compensatory in productivity. / A microbiota intestinal é alterada por diversos fatores, sendo a dieta o que mais influência, assim como os aditivos moduladores do microbioma. Objetivou-se, neste estudo, avaliar os efeitos dos aditivos promotores de crescimento na dieta, sobre a porcentagem de células caliciformes com mucina ácida e neutra no intestino delgado de suínos, por modularem a microbiota e atuar na imunidade intestinal, avaliou a porcentagem de células intestinais em proliferação pela técnica de imunoistoquímica por PCNA, certificar e quantificar a presença de células de Paneth por coloração específica e por imunoistoquímica, relatos da literatura cita a ligação de probióticos com o epitélio intestinal via células de Paneth e caliciformes. O experimento foi conduzido em uma GRSC, aplicando o DIC. Utilizaram-se 480 leitões machos castrados, em cinco tratamentos, com dez leitões por gaiola, com oito repetições e no tratamento basal realizou-se 16 repetições. Sendo, T1=dieta basal, denominado de controle negativo, T2=T1+ antimicrobiano Avilamicina, T3=T1+probiótico Scchacaromyces cerevisae T4=T1+prebiótico MOS, T5=T1+probiótico Scchacaromyces cerevisae + o prebiótico MOS, foram abatidos com 65 dias de idade. A dieta foi à base de milho e farelo de soja mais um suplemento mineral e vitamínico. Ocorreram alterações com maior aumento na quantidade de células caliciformes com muco neutro comparativamente com aquelas de muco ácido e com o controle negativo. Não houve alterações expressivas para as células com muco ácido. A porcentagem de células no intestino delgado em proliferação pela técnica de imunoistoquímica para PCNA não apresentou alterações significativas, confirmou-se a presença de células de Paneth por colorações especiais e por imunoistoquímica. A quantidade de células de Paneth do duodeno alteraram-se em função dos aditivos utilizados, em comparação ao controle negativo que teve a maior quantidade, com a adição de antimicrobiano houve a menor quantidade, com prebiótico e simbiótico ocorreu redução mais branda, o que sugere ter ocorrido uma imunomodulação Os aditivos promotores de crescimento nas rações são tecnologias que favorecem ganhos significativos e podem ser recomendados, pois os resultados são compensatórios em produtividade. Além de promover uma melhor saúde intestinal em função de uma microbiota mais saudável e mais apta à criação de leitões na fase de creche

Um conjunto de quadros clínicos conhecidos genericamente por Leishmaniose, que representa um sério problema de Saúde Pública, tem como agentes etiológicos protozoários do gênero Leishmania. Em seu ciclo de vida, o parasita apresenta dois hospedeiros, um vertebrado e um invertebrado. O gênero Leishmania é dividido em dois subgêneros: Leishmania (Leishmania) e LeishmaniaViannia), de acordo com a posição ocupada pela forma promastigota no tubo digestivo do hospedeiro invertebrado. Genes que codificam RNA ribossômico são exclusivamente transcritos pela RNA polimerase I. A região promotora dessa polimerase é conhecida como espécie-específica. Estudos anteriores, baseados em ensaios de expressão transiente com construções nas quais a expressão do gene repórter CAT era dirigido por diferentes regiões do promotor mostraram que em Leishmania há um reconhecimento espécie-específico, mas que espécies filogeneticamente relacionadas reconhecem melhor o promotor do que a espécie homóloga. A caracterização estrutural da região promotora de RNA pol I de L. (V.) braziliensis possibilitou mapear o sítio de início de transcrição e definir a provável região promotora de RNA pol I desse organismo. Quando comparamos o 5´ ETS da região estudada, com a região correspondente das demais espécies do subgênero L. (Leishmania), notamos uma alta similaridade tanto no tamanho, quanto na seqüência de nucleotídeos. Já na região IGS os blocos de repetição apresentam tamanho similar, porém seqüências distintas, assim como a seqüência à montante do ETS. Não foi determinado o número de blocos de repetição presente em cada cístron de L.(V.) braziliensis. Os estudos de ligação de fatores nucleares à região central do promotor mostraram que houve um reconhecimento diferencial dessa região entre a espécie homóloga L. (V.) braziliensis e as espécies heterólogas L. (V.) guyanensis, pertencente ao mesmo subgênero e L. (L.) amazonenis. Os complexos protéicos associados a essa região central apresentaram maior afinidade com a espécie heteróloga L. (V.) guyanensis. O fato de ser o ensaio de CAT trabalhoso e demorado levou à busca de outro repórter que evidenciasse a funcionalidade da região promotora ao mesmo tempo em que permitisse a quantificação dessa expressão, de modo a medir a força do promotor. Neste trabalho iniciamos a padronização para utilizar GFP como gene repórter no estudo da funcionalidade de promotores. Foi possível mostrar que a GFP é detectável em microscopia confocal e que as células que expressam GFP podem ser quantificadas em FACS, no entanto, essas detecções só foram possíveis em parasitas selecionados por uma marca de seleção, ou seja, numa transfecção estável. Assim, substituir o gene repórter CAT pelo gene GFP foi inadequado para os ensaios de transfecção transiente, impedindo que fosse medida a atividade da região promotora de L. (V.) braziliensis em diferentes espécies. / Leishmaniasis is the generic name of a serious public health problem that is caused by protozoa of the genus Leishmania. In its lifecycle, the parasite has two hosts, a vertebrate and an invertebrate. The genus Leishmania is divided into two subgenera: Leishmania (Leishmania) and Leishmania (Vianna), according to the position occupied by the promastigotes form at the gut of the invertebrate host. Genes that encode ribosomal RNA are exclusively transcribed by RNA polymerase I. The promoter region of the RNA polymerase I is known to present a species-specific recognition. Previous studies, based on transient expression assays with constructions in which the expression of the reporter gene CAT was directed by different regions of the promoter showed that RNA pol I promoter in Leishmania is species-specific, but in phylogenetically related species the expression of the reporter gene is higher than in the homologous species. The structural characterization of the promoter region of RNA pol I of L. (V.) braziliensis enabled to map the site of initiation of transcription and to define the promoter region of RNA pol I. The comparison of the determined 5\' region of the ETS region, with the corresponding region of other species of the subgenus L. (Leishmania), showed a high similarity both in size, as in the sequence of nucleotides. However, the IGS region and the repetitive blocks of nucleotides have similar size but different sequences. The studies of binding of nuclear factors to the central region of the promoter showed that there was a recognition between the species counterpart L. (V.) braziliensis and the heterologous species L. (V.) guyanensis, belonging to the same subgenus and L. (L.) amazonensis, that belongs to another subgenus. The complex protein associated with this central region showed greater affinity with the heterologous species L. (V.) guyanensis. The fact that to quantify the CAT expression represents a laborious and time consuming test lead us to search for another reporter that could identified the functionality of the promoter region and at the same time allowed the quantification of expression in order to measure the strength of the promoter. In the present work, we began the standardization for the use of GFP gene as reporter in the study of the functionality of promoters. It was possible to show that GFP is detectable in confocal microscope and the cells that express GFP can be quantified in FACS, however, these findings were made possible only in parasites selected by the mark, ie in a stable transfection. So, the replacement of CAT by GFP reporter showed to be inadequate for testing promoters in transient transfection. This prevented the measure of the activity of the promoter region of L. (V.) braziliensis in different species.

L'étape initiale dans l'expression génique est la transcription de l'ADN génomique du gène en ARN. La transcription être initiée par l'assemblage d'ARN pol II autour du site de début de transcription, qui est également connues comme promoteurs. Cependant, la transcription est nécessite un autre gène distal des régions, des amplificateurs, qui sont augmenté ainsi la probabilité de la transcription. Amplificateurs et les promoteurs sont généralement définis par leur éloigné des sites d'initiation de la transcription et souvent distingués par les modifications des histones. Récemment, des études, il a été de plus en plus ont révélé de grandes similitudes entre les amplificateurs et les promoteurs. Les résultats antérieurs ont suggéré la possibilité que certains promoteurs de gènes peuvent afficher les fonctions activatrices. Cependant l'étendue de ce type de promoteurs et si elles fonctionnent réellement réglementé l'expression des gènes distales sont restés insaisissable.Mon projet est réalisé en vue de répondre à ces questions. En exploitant un essai amplificateurs reporter à haut débit, je démêler une partie sous-estimée du promoteur de base présentant une activité d'activateur, définie comme Epromoters. Ils présentent des propriétés distinctes par rapport à des amplificateurs et des promoteurs classiques distales, sont associés à la réponse au stress et d'interagir plus souvent avec d'autres promoteurs. En utilisant CRISPR complète / cas9 approche de suppression I a démontré que Epromoters sont généralement impliqués dans l'activation des gènes distales. Nos résultats identifient d'abord une nouvelle catégorie de promoteurs avec activité in vivo dans amplificateurs. / The initial step of gene expression is the transcription of genomic DNA of the gene into RNA. The transcription can only be initiated by the assembly of RNAPII machinery around transcription start site of a gene, known as core promoter. However, transcription also requires other gene-distal regulatory DNA regions, known as enhancers. Enhancers and promoters are traditionally distinguished by their histone modifications. Recently, there has been increasing number of studies revealing broad similarities between enhancers and promoters. Previous findings have suggested the possibility that some gene promoters display enhancer activity. However, the questions of how can we identify this type of promoter in genome-wide and whether they actually function to regulated the expression of distal genes are remained elusive.My project has carried out aiming to answer these above questions. Firstly, I have optimized the technique that has developed in the lab, named CapStarr-seq, which used as an approach to exploiting a high-throughput enhancer activity. Performing CapStarr-seq in human cell lines, I unraveled an underestimated proportion of promoter displaying enhancer activity, defined as Epromoters. They display distinct properties as compared to distal enhancers and classical promoters, are associated with stress response genes and interact more frequently with other promoters. Moreover, by using comprehensive CRISPR/Cas9 genomic deletion approach, I demonstrated that Epromoters are generally involved in the activation of distal genes. Taken together, our results first identify a new category of promoters with dual promoter and enhancer functions.

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