Title: Construction of classifiers suitable for segmentation of clients Author: Bc. Jana Hricová Department: Department of Probability and Mathematical Statistics Supervisor: prof. RNDr. Jaromír Antoch, CSc., Department of Probability and Mathematical Statistics Abstract: The master thesis discusses methods that are a part of the data analy- sis, called classification. In the thesis are presented classification methods used to construct tree like classifiers suitable for customer segmentation. Core methodo- logy that is discussed in our thesis is CART (Classification and Regression Trees) and then methodologies around ensemble models that use historical data to cons- truct classification and regression forests, namely Bagging, Boosting, Arcing and Random Forest. Here described methods were applied to real data from the field of customer segmentation and also to simulated data, both processed with RStudio software. Keywords: classification, tree like classifiers, random forests

Os escorpiões estão entre os animais peçonhentos mais antigos da Terra, existindo por mais de 400 milhões de anos. As peçonhas desses animais contêm uma mistura complexa de proteínas e peptídeos capazes de interagir com alta complexidade no organismo de suas vítimas. No Brasil, o principal gênero de escorpião responsável pelos acidentes é o gênero Tityus, no qual estão inseridas as espécies T. serrulatus, T. bahiensis, T. obscurus e T. stigmurus. A peçonha do T. serrulatus é composta em sua maioria de peptídeos com ação em canais para sódio e potássio, mas possuem também enzimas como hialuronidases, proteases e outros compostos como hipotensinas, peptídeos antimicrobianos (PAMs) e peptídeos potenciadores de bradicinina. Muitos dos componentes encontrados nas peçonhas de escorpiões têm sido estudados por possuírem importantes atividades farmacológicas, como propriedades analgésicas, antimicrobianas, antitumorais e anti-inflamatórias. Todavia, sabe-se que há uma grande dificuldade na obtenção dessas substâncias em consequência do baixo rendimento dos componentes isolados a partir da peçonha. Com isso, atualmente, tem sido utilizada a expressão heteróloga de proteínas para viabilizar a obtenção de toxinas em quantidades suficientes para uso biotecnológico. Diante desse panorama, o presente trabalho teve como objetivo a expressão de dois peptídeos presentes na peçonha de Tityus serrulatus, bem como sua comparação com os peptídeos nativos com relação à sua estrutura e atividade funcional. As toxinas escolhidas foram a Ts8 e a Ts8-propeptídeo, também chamada de scorpine-like, que atuam em canais para potássio (KTxs). As sequências e os cDNAs moldes das toxinas expressas foram obtidos da biblioteca de cDNA da glândula de Tityus serrulatus construída em nosso laboratório. Os genes sintéticos contendo as toxinas de interesse foram transformados em células de Pichia pastoris da linhagem KM71H. A expressão das Ts8 e Ts8-propeptídeo recombinantes revelou a presença das toxinas, porém as mesmas sofreram degradação proteolítica por enzimas da P. pastoris. Dessa forma, foram realizadas as avaliações das expressões na presença de substrato como os casaminoácidos, bem como na diminuição do pH, temperatura e adição de inibidores de protease. Além disso, foi possível obter a rTs8, embora clivada, com atividade sobre canais para potássio. Novas etapas de purificação em colunas de troca iônica e fase reversa foram padronizadas para a obtenção das toxinas nativas a partir da peçonha de T. serrulatus. Adicionalmente, foi possível identificar a presença da Ts8 e Ts8-propeptídeo (scorpine-like) nativas por espectrometria de massas em uma das frações obtidas da cromatografia da peçonha. A adição de casaminoácidos foi favorável para a expressão das toxinas, porém não foi suficiente para minimizar a degradação proteolítica. Ensaios funcionais com os fragmentos das toxinas recombinantes e com as toxinas nativas demonstraram a liberação de citocinas como TNF-? e IL1-? em algumas das toxinas testadas. Além disso, as toxinas demonstraram inibir o crescimento da Pichia pastoris em teste antifúngico e não foram tóxicas para células de macrófagos alveolares nas concentrações testadas. Assim, esse trabalho contribuiu para demonstrar a atividade de enzimas proteolíticas na expressão em P.pastoris, avaliar as condições em que são liberadas, bem como demonstrar a atividade da rTs8 em ensaio eletrofisiológico e a atividade antifúngica das toxinas recombinantes e nativas. / Scorpions are among the oldest venomous animals on Earth, existing for over 400 million years. The venoms of these animals contain a complex mixture of proteins and peptides that can interact with high complexity with their victims. In Brazil, the main genus of scorpions responsible for accidents is Tityus genus, in which the species T. serrulatus, T. bahiensis, T. obscurus and T. stigmurus are inserted. T. serrulatus venom (Tsv) is composed mostly of peptides with action on sodium and potassium channels, but also have enzymes such as hyaluronidases, proteases and other compounds such as hypotensins, antimicrobial peptides (AMPs) and bradykinin potentiating peptides. Many of the components found in scorpion venoms have been studied for their important pharmacological activities, such as analgesic, antimicrobial, antitumor and anti-inflammatory properties. However, it is known that there is great difficulty in obtaining these substances because of the low yield of the isolated components from the venom. With this, the heterologous expression of proteins has been used to enable the production of toxins in sufficient amount for biotechnological purpose. In this panorama, the aim of this study is the expression of two peptides present in the venom of Tityus serrulatus, as well as their comparison with the native peptides in relation to their structure and functional activity. The toxins chosen were Ts8 and Ts8-propeptide, also called scorpine-like, acting on potassium channels (KTxs). Sequences and cDNAs templates of the expressed toxins were obtained from the cDNA library of the Tityus serrulatus gland constructed in our laboratory. Synthetic genes containing the toxins of interest were transformed into Pichia pastoris cells of strain KM71H. Expression of the recombinant Ts8 and Ts8-propeptide revealed the presence of the toxins, but they underwent proteolytic degradation by P. pastoris enzymes. Thus, we evaluate the expression in the presence of substrate as the casamino acids, as well as in the decrease of pH, temperature and addition of protease inhibitors. In addition, rTs8, although cleaved, could be obtained with potassium channel activity. New purification steps in ion exchange and reverse phase columns were standardized to obtain the native toxins from the venom of T. serrulatus. In addition, it was possible to identify the presence of native Ts8 and Ts8-propeptide (scorpine-like) by mass spectrometry in one of the fractions obtained from the venom chromatography. The addition of casamino acids was favourable for toxin expression, but was not sufficient to minimize proteolytic degradation. Functional assays with recombinant toxin fragments and native toxins have demonstrated the release of cytokines such as TNF-? and IL-1? in some of the toxins tested. In addition, the toxins were shown to inhibit the growth of Pichia pastoris in the antifungal test and were not toxic to alveolar macrophages cells at the concentrations tested. Thus, this work contributed to demonstrate the activity of proteolytic enzymes in P.pastoris expression, to evaluate the conditions under which they are released, as well as to demonstrate the activity of rTs8 in the electrophysiological assay and the antifungal activity of recombinant and native toxins

La O-fucosylation, catalysée par Pofut1, est une glycosylation rare qui consiste en l’ajout d’un fucose O-lié sur la sérine ou la thréonine d’une séquence consensus (C2X4(S/T)C3), portée par un domaine EGF-like (ELD) d’une glycoprotéine membranaire ou sécrétée. Notre analyse de la lignée murine Pofut1cax/cax, hypomorphe pour le gène Pofut1, a révélé une hypertrophie musculaire post-natale associée à une diminution du pool de cellules satellites. Ce phénotype est en partie associé à un défaut d’interaction entre les récepteurs NOTCH hypo-O-fucosylés des myoblastes dérivés de cellulessatellites (MDCS) et leurs ligands DSL, ce qui aboutit à une plus faible activation de la signalisation Notch. D’autres protéines potentiellement impliquées dans la myogenèse peuvent également être la cible de POFUT1. C’est notamment le cas de la protéine Wnt inhibitory factor 1 (WIF1), qui dispose de cinq ELDs, dont deux sont potentiellement aptes à recevoir un O-fucose (ELDs III et V). Par une approche phylogénétique, nous avons montré la conservation de ces deux sites de O-fucosylation et de deux sites de N-glycosylation chez la plupart des bilatériens. Nos expériences démontrent l’occupationde tous ces sites, excepté le site de O-fucosylation de l’ELD V, chez la protéine WIF1 murine. La capacité de l’ELD III, produit de manière isolée, à recevoir un fucose O-lié a été démontrée après O-fucosylation in vitro, par l’association de cycloaddition azide-alcyne assistée au cuivre (CuAAC) et de spectrométrie de masse en mode MRM. Cette nouvelle approche expérimentale a par la suite été standardisée et sa sensibilité évaluée en comparant deux autres ELDs (ELDs 12 et 26 de NOTCH1) connus pour être O-fucosylés mais présentant des affinités différentes pour POFUT1. De façonsurprenante, l’ELD V de WIF1 ne peut être O-fucosylé, probablement en raison d’un clash stérique entre cet ELD et POFUT1, prévenant ainsi leur interaction. L’analyse de la protéine WIF1 entière a confirmé les résultats obtenus sur les ELDs isolés et démontre l’occupation des deux sites de N-glycosylation. Enfin, nos résultats montrent également l’importance de ces deux N-glycanes, mais également celle du O-fucose de l’ELD III, pour une sécrétion optimale de la protéine WIF1 murine. / The, Pofut1-catalyzed O-fucosylation, is a rare glycosylation which consists of the addition of an O-linked fucose to the serine or threonine of a consensus sequence (C2X4(S/T)C3), carried by an EGF-like domain (ELD) of a membrane or secreted glycoprotein. Our analysis of the murine line Pofut1cax/cax, hypomorphic for the Pofut1 gene, revealed post-natal muscle hypertrophy associated with a decrease in the satellite cell pool. This phenotype was partly associated with a lack of interaction between hypo-O-fucosylated NOTCH receptors of satellite cell-derived myoblasts (SCDM) and their DSL ligands, which resulted in a lower activation of Notch signaling. Other proteins potentially involved in myogenesis may also be the target of POFUT1. This is indeed the case for the protein Wnt inhibitory factor 1 (WIF1), which has five ELDs, whose only two are potentially able to receive an O-fucose (ELDs III and V). Using a phylogenetic approach, we showed in most bilaterians that these two O-fucosylation sites and two N-glycosylation sites were conserved. Our experiments showed theoccupation of all these sites, except for the O-fucosylation site of murine WIF1 protein ELD V. The ability of the ELD III, produced as an isolated protein, to receive O-linked fucose was demonstrated after an in vitro O-fucosylation by combination of copper-catalysed azide-alkyne cycloaddition (CuAAC) and MRM-mass spectrometry. This new experimental approach was then standardized and its sensitivity was evaluated by comparing two other ELDs (NOTCH1 ELDs 12 and 26) known to beO-fucosylated but with different affinities for POFUT1. Surprisingly, WIF1's ELD V could not be O-fucosylated, probably due to a steric clash between this ELD and POFUT1, thus preventing their interaction. The analysis of the full-length WIF1 protein confirmed our results obtained with isolated ELDs and demonstrated the occupation of the two N-glycosylation sites. Finally, our results also showed the importance of these two N-glycans, but also the importance of ELD III’s O-fucose, foroptimal secretion of the murine WIF1 protein.

Um papel importante foi atribuído ao receptor toll-like 4 (TLR4) no desenvolvimento da placa aterosclerótica. O TLR4 foi primeiramente descrito como um receptor para bactérias gram-negativas; posteriormente foi demonstrado que sua expressão está aumentada em placas ateroscleróticas e que pacientes que possuem um polimorfismo disfuncional do TLR4 são menos suscetíveis ao desenvolvimento dessa doença. Portanto, o objetivo desse estudo foi o de investigar, em um modelo experimental de aterosclerose, a influência da deleção do TLR4 na formação e morfologia da placa aterosclerótica, no perfil lipídico e em marcadores inflamatórios. Camundongos duplo knockout (DKO), deficientes no receptor de LDL e TLR4, foram gerados cruzando-se camundongos deficientes para o receptor de LDL (LDLrKO) com camundongos deficientes para o TLR4 (TLR4KO). Todos os grupos receberam dieta rica em gordura e colesterol por 12 semanas. As concentrações plasmáticas de colesterol e triglicérides foram medidas por ensaio colorimétrico. Cortes seriados da raiz aórtica foram corados com Oil red O e as áreas de lesão quantificadas por analisador de imagens. O colágeno foi medido por coloração de picrossirius. A formação de nitrotirosina e expressão de CD40L, MMP9 e iNOS nas placas foram feitas por imunohistoquímica. As comparações foram feitas por ANOVA com pós teste de Student Newman-Keuls. Os dados foram expressos como média ± EPM. Camundongos DKO desenvolveram placas menores que camundongos LDLrKO (117.6 ±1.4 vs 198.8 ± 3.3 104m2). Camundongos TLR4KO não formaram placa. As placas dos camundongos DKO apresentaram menor núcleo lipídico que as dos LDLrKO (76.2± 13.2 vs 161.7 ± 2.9 104m2). O colágeno ao redor do núcleo lipídico é maior nos camundongos DKO do que nos LDLrKO (24.9 ± 1.8 vs 16.5 ± 2.5 % da placa). A distribuição do colágeno nos camundongos DKO ocorre principalmente ao redor da placa, de forma mais organizada, enquanto que nos LDLrKO onde sua distribuição é mais difusa. As placas dos camundongos DKO apresentaram menor expressão de CD40L e iNOS do que as dos LDLrKO (13.1 ± 0.7 vs 18.5 ± 2.5 AU e 7.7 ± 0.9 vs 10.2 ± 0.4 AU, respectivamente). A expressão de MMP9 foi menor nas placas dos camundongos DKO do que as dos LDLrKO (2.99 ± 0.3 vs 1.99 ± 0.2 AU). A marcação para nitrotirosina foi maior nos camundongos LDLrKO quando comparada com as dos grupos DKO e TLR4KO (142.89 ± 208.5, 77.16 ± 227.7 e 71.73 ± 95.9 10m2, respectivamente). Todos esses resultados sugerem que o processo inflamatório é menor na ausência do TLR4. As concentrações plasmáticas de colesterol não foram diferentes entre os grupos LDLrKO e DKO mas os camundongos LDLrKO apresentaram concentrações plasmáticas de triglicérides maiores do que os camundongos DKO após a dieta (265.2 ± 27.6 vs 150.5 ± 8.8 mg/dL). O receptor toll-like 4 influencia na estrutura e formação da placa aterosclerótica independentemente dos níveis séricos de colesterol / A crucial role has been suggested for toll-like receptor 4 (TLR4) in atherosclerotic plaque formation and development. TLR4 was described primarily, as a receptor for gram-negative bacteria lipopolisacharide; later it was showed that its expression is increased in atherosclerotic plaques and patients that carries a TLR4 dysfunctional polymorphism are less susceptible to development of this disease. Therefore, the aim of this study was to investigate, in an experimental model of atherosclerosis, the influence of TLR4 deletion in atherosclerotic plaque formation and morphology, cholesterol profile and inflammatory markers. Double knockout mice (DKO), deficient in LDL receptor and TLR4, were generated by breeding LDL receptor knockout mice (LDLrKO) with TLR4 knockout mice (TLR4KO). All three experimental groups, LDLrKO, TLR4KO and DKO were fed a high fat-cholesterol diet for 12 weeks. Plasma cholesterol and triacylglicerol concentrations were measured by colorimetric assay. Cross sections of aortic sinus were stained with Oil red O and lesion areas were quantified by an image analyzer. Collagen content was measured by picrossirius staining. We also measured nitrotyrosine formation, CD40L, MMP9 and iNOS expression by immunohistochemistry. Comparisons were made by ANOVA followed by Student-Newman-Keuls post- test. Data are mean ± SEM. DKO mice developed smaller plaques than LDLrKO mice (117.6 ±1.4 vs 198.8 ± 3.3 104m2). TLR4KO mice developed no plaque. Plaques from DKO mice have also a smaller lipid core than the ones from LDLrKO mice (76.2± 13.2 vs 161.7 ± 2.9 104m2). Collagen content around the lipid core is higher in DKO mice compared to LDLrKO mice (24.9 ± 1.8 vs 16.5 ± 2.5 % of the whole plaque). Interestingly, collagen distribution in DKO mice seems to occur mainly on the plaque periphery, in a more organized manner, while in LDLrKO mice it is fuzzier, being present also inside the plaque. Plaques from DKO present lower expression of CD40L and iNOS than LDLrKO mice (13.1 ± 0.7 vs 18.5 ± 2.5 AU and 7.7 ± 0.9 vs 10.2 ± 0.4 AU, respectively). MMP9 expression is lower in DKO mice as compared to LDLrKO mice (2.99 ± 0.3 vs 1.99 ± 0.2 AU). Nitrotyrosine staining was higher in LDLrKO mice as compared to DKO and TLR4KO groups (142. 89 ± 208.5, 77.16 ± 227.7 and 71.73 ± 95.9 10m2, respectively). All together, these findings suggest that inflammatory process is milder in the absence of TLR4. Serum cholesterol were not different between LDLrKO and DKO mice but LDLrKO presented higher triacylglicerol serum levels after 12 weeks on high fat high cholesterol diet as compared to DKO mice (265.2 ± 27.6 vs 150.5 ± 8.8). Toll like receptor 4 influences atherosclerotic plaque formation and structure independently from serum cholesterol levels

Os fibroblastos são atualmente considerados componentes ativos da resposta imune porque estas células expressam receptores do tipo Toll (TLRs), são capazes de reconhecer padrões moleculares associados a patógenos e mediar a produção de citocinas e quimiocinas durante a inflamação. A resposta imune inata do hospedeiro a lipopolissacarídeos (LPS) de Porphyromonas gingivalis é incomum, já que diferentes estudos relataram que este LPS pode ser um agonista para TLR2 e um antagonista ou agonista para TLR4. A sinalização por TLRs envolve proteínas adaptadoras, como MyD88 e TRAM, que são necessárias para a transdução do sinal até o núcleo para que ocorra a transcrição de RNAm para os mediadores da inflamação. O objetivo deste estudo foi investigar e comparar se a sinalização por meio de TLR2 ou TLR4 poderia afetar a produção de Interleucina (IL)-6, IL-8 e CXCL12 em fibroblastos humanos gengivais (HGF) e fibroblastos humanos de ligamento periodontal (HPLF). Objetivamos também comparar a participação das moléculas adaptadoras MyD88 e TRAM na expressão do RNAm dos mesmos alvos. Material e Métodos: Após silenciamento mediado por RNA de interferência de TLR2, TLR4, MyD88 ou TRAM, confirmado por RT-qPCR, HGF e HPLF, provenientes de três dadores voluntários, foram estimulados com LPS de Porphyromonas gingivalis ou com dois agonistas sintéticos de TLR2, Pam2CSK4 e Pam3CSK4, por 6 horas. A expressão do RNAm e das proteínas IL-6, IL-8, e CXCL12 foram avaliados por qRT-PCR e ELISA, respectivamente. Resultados: A expressão do RNAm de TLR2 foi regulada em HGF, mas não em HPLF por todos os estímulos. O silenciamento de TLR2 diminuiu IL-6 e IL-8 em resposta ao LPS de P. gingivalis, Pam2CSK4 e Pam3CSK4 de maneira semelhante, em ambas as subpopulações de fibroblastos (p<0,05). Por outro lado, a produção de CXCL12 permaneceu inalterada pelo silenciamento de TLR2 ou TLR4. No caso do silenciamento de MyD88 e TRAM, em ambos os subtipos de fibroblastos, o RNAm para os mesmos alvos também teve sua expressão diminuída (p<0,05). Já a expressão constitutiva de CXCL12 foi aumentada com o silenciamento de MyD88 ou TRAM (p<0,05). Conclusão: Estes resultados sugerem que a sinalização por meio de TLR2, por fibroblastos, as células residentes mais numerosas em gengiva e ligamento periodontal, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal, mas não interfere nos níveis de CXCL12, uma quimiocina importante no processo de reparação. Concluímos também que o silenciamento de MyD88 ou TRAM é capaz de diminuir o aumento da transcrição gênica de IL-6 e IL-8 provocado por LPS de P. gingivalis, embora a expressão constitutiva de CXCL12 seja regulada positivamente. / Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen associated molecular patterns and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for TLR2 and an antagonist or agonist for TLR4. TLRs signaling pathway involves adaptor proteins, like MyD88 and TRAM, which are crucial for signal transduction to the nucleus and mRNA expression of inflammatory mediators. This study investigated and compared whether signaling through TLR2 or TLR4 could affect the production of IL-6, IL-8 and CXCL12 in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). The role of MyD88 and TRAM on the mRNA expression of the same targets were also evaluated. Methods: After small interfering RNA-mediated silencing of TLR2, TLR4, MyD88 or TRAM, confirmed by RT-qPCR, HGF and HPLF from three volunteer donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein production were evaluated by RT-qPCR and ELISA, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, Pam2CSK4 and Pam3CSK4 in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. For MyD88 or TRAM silencing, IL-6 and IL-8 mRNA were also decreased, in both fibroblasts subtypes. However CXCL12 mRNA constitutive expression was increased by siMyD88 or siTRAM. Conclusion: These results suggest that signaling through TLR2 by fibroblasts, the most numerous resident cells in gingiva and periodontal ligament, may control the production of IL-6 and IL-8, which in turn contribute to periodontal pathogenesis, but does not interfere with CXCL12 levels, an important chemokine in the repair process. Also, MyD88 or TRAM knockdown may decrease the IL-6 and IL-8 LPS-induced upregulation and increase the constitutive CXCL12 mRNA.

Introdução: Um dos primeiros sistemas de defesa contra os microrganismos é a via dos receptores Toll-like (TLRs). A ativação destes receptores leva à síntese de citocinas, dando início à resposta imune inata. Em modelos animais, o TLR2, TLR4 e TLR9 parecem estar relacionados com o reconhecimento de antígenos de Leishmania. A relação entre TLRs e leishmânia pode ser um mecanismo chave no desenvolvimento da doença ou no controle da mesma. Até o momento não existem estudos de TLRs na leishmaniose cutânea humana. Objetivo: Determinar o padrão de expressão e as células associadas com o TLR2, TLR4 e TLR9 na leishmaniose cutânea. O objetivo secundário é correlacionar a quantidade de TLRs com a quantidade de citocinas e células inflamatórias na pele. Métodos: Cem biópsias de pacientes com leishmaniose cutânea causadas por Leishmania (V.) braziliensis foram selecionadas inicialmente. Apenas os casos confirmados (presença de amastigotas no raspado, teste de Montenegro positivo, imunoistoquímica com presença de antígenos de Leishmania e reação em cadeia da polimerase com DNA de Leishmania (V.) braziliensis foram incluídos. Um grupo controle de pele normal foi incluído para comparação (quatro casos). A expressão de TLR2, TLR4 e TLR9 foi determinada por técnica imunoistoquímica, da mesma forma que os fenótipos celulares (células NK, macrófagos, células dendríticas, células CD4 e CD8) e citocinas (IL-1, IL-6, IL-12, TNF-alfa, IFN-gama). Dupla-marcação foi realizada para identificar as células que expressaram os TLRs analisados. Análise semi-quantitativa foi utilizada para avaliação da expressão de TLRs na epiderme, enquanto na derme foi realizada análise quantitativa. O nível de significância foi estabelecido com p<0,05. Resultados: Doze casos preencheram os critérios de inclusão. Os pacientes eram todos masculinos, com lesões apenas em membros inferiores e mediana de idade de 23 anos [16-47]. A expressão de TLR2, TLR4 e TLR9 na epiderme da pele normal foi alta. Quando comparados com pele normal, tanto TLR4 quanto TLR2 mostraram menor expressão no epitélio dos pacientes com leishmaniose e não houve expressão de TLR9. A média de células expressando TLR2 na derme foi de 136,36±82,46 células/mm2, ao passo que a média de células expressando TLR4 foi de 3,21±4,11 células/mm2. A contagem de TLR9 foi de 86,15±88,36 células/mm2 predominando em áreas de formação de granulomas. A regressão linear não demonstrou relação entre a contagem de células marcadas ou citocinas com TLR2 ou TLR4. O aumento proporcional da expressão de TLR9 relacionou-se com maior expressão de IL-12 e IL-4 (p < 0,05). A dupla marcação demonstrou que os macrófagos expressaram TLR2. A dupla marcação não mostrou expressão de TLR2 nas células dendríticas e nas células NK. Conclusão: A leishmaniose cutânea localizada associa-se com a presença de TLR2, TLR4 e TLR9. No epitélio a expressão de TLR2 e TLR4 em pacientes com leishmaniose está diminuída em relação aos pacientes controles. A expressão do TLR2 na derme é estatisticamente maior que a de TLR4 e TLR9, a qual é expressa pelos macrófagos. A expressão de TLR9 ocorre principalmente nas áreas de granulomas havendo relação com a expressão de IL-12 e IL-4 / Introduction: One of the first systems of defense against microorganisms is the Toll-like receptors (TLRs) pathway. The activation of these receptors promotes the cytokine synthesis, initiating the innate immune response. In animal models, TLR2, TLR4 and TLR9 appear to be related to the recognition of antigens of Leishmania. The relationship between TLRs and Leishmania can be a key mechanism in the development of the disease or it control. Until now, there are not studies about TLRs in human cutaneous leishmaniasis. Objective: To determine the expression pattern and the cells associated with TLR2, TLR4 and TLR9 in cutaneous leishmaniasis. The secondary objective is to correlate the amount of TLRs with the amount of cytokines and inflammatory cells. Methods: One hundred biopsies from patients with cutaneous leishmaniasis caused by Leishmania (V.) braziliensis were initially selected. Only confirmed cases of cutaneous leishmaniasis were included in the analysis (presence of amastigotes in the scraping, positive Montenegro test, immunohistochemistry with the presence of Leishmania antigens and polymerase chain reaction with DNA from Leishmania (V.) braziliensis. A control group (4 cases) of normal skin was included for comparison. The expression of TLR2, TLR4 and TLR9 was determined by immunohistochemistry, as well as cell phenotypes (NK cells, macrophages, dendritic cells, CD4 and CD8) and cytokines (IL-1, IL-6, IL-12, TNF-alpha, IFN-gamma). Double-staining was used to determine the cells expressing TLRs. Semi-quantitative analysis was used for evaluation of the expression of TLRs in the epidermis. Quantitative analysis was performed to evaluate the expression in the dermis. The level of significance was defined as p <0.05. Results: 12 cases fulfilled inclusion criteria. The patients were all male, with lesions in lower limbs and median age of 23 years [16-47]. The expression of TLR2 and TLR4 in the epidermis of normal skin was high. When compared with normal skin, TLR2 and TLR4 showed lower expression in the epidermis. There was no expression of TLR9 in the epidermis in cases of cutaneous leishmaniasis and normal skin. The mean number of cells expressing TLR2 in the dermis was 136.36±82.46 cells/mm2, while the average of cells expressing TLR4 was 3.21±4.11 cells/mm2. The count of TLR9 was 86.15±88.36 cells/mm2, and it was found mainly in the areas of granuloma formation. Linear regression showed no relationship between the number of labeled cells or cytokines with TLR2 or TLR4. There was an association between TLR9 and two cytokines (IL-12 and IL-4). This correlation suggested that the proportional increase in the expression of TLR9 was related to greater expression of IL-12 and IL-4 (p<0.05). The double staining showed that macrophages and endothelial cells expressed TLR2. The double staining showed no expression of TLR2 in dendritic cells and NK cells. Conclusion: The localized cutaneous leishmaniasis associated with the presence of TLR2, TLR4 and TLR9. The expression of TLR2 in the dermis was statistically greater than that of TLR4 and TLR9, which is expressed by macrophages. The expression of TLR9 occurs primarily in the areas of granulomas was associated with the expression of IL-12 and IL-4

Zhou, Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 89-107). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

O gênero Pestivirus, pertencente à família Flaviviridae, é constituído por espécies virais de importância na saúde animal no mundo todo, as quais podem afetar a economia dos países de forma impactante. São reconhecidas quatro espécies pelo Comitê Internacional de Taxonomia Viral (ICTV): vírus da peste suína clássica (Classical Swine Fever Virus – CSFV), vírus da doença da fronteira (Border Disease Virus- BDV), vírus da diarreia viral bovina tipo 1 (Bovine Viral Diarrhea Virus 1- BVDV-1) e 2 (BVDV-2). Algumas das espécies deste gênero- CSFV e BVDV- são de notificação obrigatória na Organização Mundial de Saúde Animal (OIE), causando sanções econômicas importantes quando presentes. Recentemente, possíveis novas espécies vêm sendo caracterizadas, porém ainda não foram reconhecidas como espécies do gênero Pestivirus. Com o objetivo de gerar mais informações acerca da diversidade genética de pestivírus no país, o presente trabalho descreve os genomas completos e a caracterização genômica e filogenética de uma cepa de BVDV-1 subtipo i e duas de vírus ‘HoBi’-like. Os genomas completos foram obtidos através de sequenciamento de nova geração; as anotações, predição da poliproteína viral e dos sítios de clivagem foram feitos através do software Geneious, e a análise filogenética foi realizada através do software MEGA 6. O BVDV-1 subtipo i foi pela primeira vez isolado no Brasil, sendo também a primeira descrição de genoma completo deste subtipo de BVDV. Também foi descrito o genoma completo de duas cepas de vírus ‘HoBi’-like isolados no Brasil, além da caracterização de outras cepas de ‘HoBi’-like disponíveis em bancos de dados. Os dados moleculares destes isolados foram comparados com aqueles das demais espécies do gênero Pestivirus, e estas informações deverão auxiliar na futura classificação deste como espécie. Os resultados apresentados na dissertação adicionam conhecimento sobre a diversidade genética de BVDV-1 no Brasil além de informações acerca do vírus ‘HoBi’-like, reforçando esta espécie ainda não reconhecida como um novo membro do gênero Pestivirus, os ‘HoBi’-like vírus. / The genus Pestivirus, within the family Flaviviridae, includes species that are important pathogens affecting animal health that can cause impacting losses in the economy worldwide. According to the International Committee on Taxonomy of Virus (ICTV), there are four recognized species in this genus: Classical swine fever virus (CSFV), Bovine Viral Diarrhea Virus1 and 2 (BVDV -1, BVDV-2), and Border disease virus (BDV). Some of the species within this genus - CSFV and BVDV- are notifiable to the World Organization for Animal Health (OIE), and can cause exportation barriers or sanctions. Other putative new species have been characterized recently, but remain officially unrecognized. In order to generate data about the genetic diversity of pestivirus in Brazil, this study describes complete genomes and the genomic and phylogenetic characterization of an isolate of BVDV-1i and two isolates of ‘HoBi’-like virus. Complete genomes were sequenced through Next Generation Sequencing; genome annotations, polyprotein prediction and identification of cleavage sites were performed with software Geneious, and phylogenetic analysis with software MEGA 6. BVDV-1 subtype i was found in Brazil for the first time, and this is the first complete genome ever characterized for this subtype. Two strains of ‘HoBi-like’ virus isolated in Brazil were also described and characterized together with other ‘HoBi’-like strains available in databases. The molecular data obtained for these isolates were compared to those of other Pestivirus species. These data can help in future classification of these ‘HoBi’-like strains as a new recognized species. The knowledge on genetic diversity and the characterization of pestiviruses can contribute with surveillance programs and with appropriate animal health measures to control these viral diseases.

近年來，腸促胰島素類藥物胰高血糖素樣肽－1受體（GLP-1R）激動劑（如liraglutide，exenatide）和二肽基肽酶-4（DPP-4）抑制劑（如sitagliptin，vildagliptin）在2型糖尿病治療中得到廣泛應用。然而，2型糖尿病患者中腸促胰島素效應嚴重受損。研究表明，2型糖尿病患者的腸促胰島素激素（GLP-1和GIP）分泌並不顯著降低，因此腸促胰島素效應受損主要是由於2型糖尿病患者中β細胞對腸促胰島素激素反應性降低。GIP在2型糖尿病患者中刺激胰島素分泌的功能幾乎完全消失。相比較，GLP-1刺激胰島素分泌功能在2型糖尿病患者中部分保留。2型糖尿病中腸促胰島素效應受損的具體機制目前仍不清楚。本論文主要從2型糖尿病患者的腸促胰島素效應受損的機理及對腸促胰島素類藥物療效的影響上進行相關研究。 / 我們較早的研究發現高血糖能降低胰島β細胞GLP-1R受體的表達，從而損傷胰島β細胞GLP-1R信號通路是2型糖尿中腸促胰島素受損的部分原因。由於高血脂和高血糖都是糖尿病的主要特徵，我進一步研究了高血脂對β細胞的腸促胰島素信號通路的影響。體外實驗表明，棕櫚酸能降低胰島β細胞中GLP-1R的表達，並且抑制了GLP-1刺激的cAMP產生及CREB的磷酸化；在β細胞中外源性表達GLP-1R能部分恢復棕櫚酸損傷的GLP-1刺激cAMP產生及CREB的磷酸化。此外，在db/db小鼠和HFD誘導的肥胖及糖尿病小鼠模型中，降脂藥bezafibrate和niacin 能顯著提高腸促胰島素類藥物sitagliptin 和exendin-4的降糖效果，並且伴隨著對胰島形態結構的改善以及增加胰島β細胞質量。 / 另一方面，2型糖尿病患者胰島β細胞的功能和品質持續性的減少。其中慢性的高血糖和高血脂是兩個主要因素。臨床研究發現sitagliptin的降糖效果隨著糖尿病持續的時間增加而降低，而具體機理並不清楚。我們以前研究發現高血糖損傷胰島β細胞GLP-1R的表達及其信號通路是2型糖尿病腸促胰島素效應受損的重要原因。我進一步探討了exendin-4在STZ-HFD 誘導的較輕程度糖尿病（MH）小鼠（相對較輕的高血糖以及β細胞質量減少）和重度糖尿病(SH)小鼠（嚴重高血糖以及β細胞質量減少）的治療效果。研究發現， exendin-4只在MH小鼠中顯著降低血糖，改善糖耐量，恢復正常胰島結構及增加β細胞質量。而在兩組小鼠中exendin-4 都能降低體重，增加胰腺重量，但對食都沒影響。儘管exendin-4能顯著降低MH小鼠中胰島素耐量實驗葡萄糖水平，但HORM-IR無顯著差異。此外，exendin-4處理對胰島素刺激的肝臟及肌肉組織中磷酸化AKT和GSK水準在兩組小鼠中都無明顯改變。 / 總之，我的研究強調了血糖血脂的控制在2型糖尿病患者中的重要作用。我也發現高血糖、高血脂導致2型糖尿病患者β細胞功能持續受損的同時也損傷了GLP-1R信號通路，以至腸促胰島素類藥物療效的降低。我們的研究對腸促胰島素類藥物在2型糖尿病患者中的合理使用提供了重要資訊。 / Incretin-based drugs, such as glucagon-like peptide-1 (GLP-1) receptor agonists (e.g. liraglutide and exenatide) and dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g. sitagliptin and vildagliptin), which inhibit degrading intact GLP-1, have been a novel therapeutics for the treatment of type 2 diabetes. Type 2 diabetes mellitus (T2DM) is associated with reduced incretin effects. The underlying mechanism, however, is not well understood. / Our previous studies showed that hyperglycemia downregulates glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) which potentially contributes to the impaired incretin responses in cells. Whereas the effects of hyperlipidemia, another common clinical feature of T2DM, on GLP-1 response is largely unknown. In this study, I investigated the effects of free fatty acids (FFA) on incretin receptor signalings, and examined the glucose-lowering efficacy of incretin-based drugs in combination with lipid-lowering agents. I found that palmitate treatment decreased GLP-1R expression in rodent insulinoma cell lines, which was associated with impaired GLP-1 stimulated cAMP production and phosphorylation of CREB. Over-expression of GLP-1R restored the cAMP production and the phosphorylation of CREB. Treatment with bezafibrate or niacin in combination with des-fluoro-sitagliptin or exendin-4 produced more robust glycemic control associated with improved pancreatic islet morphology and islet cell function in db/db mice and HFD-fed mice. / On the other hand, chronic hyperglycemia and hyperlipidemia can cause a progressive deterioration in pancreatic beta-cell function and mass in patients with type 2 diabetes mellitus. It has been reported that the efficacy of incretin-based therapeutics is attenuated with the duration of diabetes. In our previous study, we have shown that hyperglycemia downregulates GLP-1 receptor which in turn may contribute to the impaired incretin effect in type 2 diabetes. In this study, I further investigated the efficacy of GLP-1 based drug exendin-4 in a rodent model of type 2 diabetes with different degrees of hyperglycemia and reduction of beta cell mass. I found that in moderate hyperglycemia (MH) group but not in severe hyperglycemia (SH) group, exendin-4 treatment significantly reduced fed glucose levels and plasma lipid profiles, improved glucose tolerance and glucose stimulated insulin secretion, and was associated with restored islets morphology and increased beta cell mass. Exendin-4 significantly decreased body weight gain and increased pancreatic mass both in MH and SH group. Although glucose levels were significantly reduced in MH group with exentin-4 treatment during insulin tolerance test, exendin-4 had no effects on HORM-IR, food intake, and insulin stimulated p-AKT/p-GSK in liver and muscle in both MH and SH group. / In summary, my findings highlight the importance of comprehensive lipid and glycemic control in type 2 diabetes mellitus. I found that factors including hyperglycemia and hyperlipidemia that cause progressive decline in beta cell failure impaired beta cell GLP-1R signaling as well as the efficacy of incretin-based therapies. These results add to our knowledge regarding the mechanism of incretin-based therapy in improving glycemic control in type 2 diabetic patients and provide new insights in designing treatment strategies including incretin-based therapy for type 2 diabetic patients. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Kang, Zhanfang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 103-123). / Abstract also in Chinese. / 論文摘要 --- p.viii / Impaired Incretin Effects in Type 2 Diabetes: Mechanism and Therapeutic Implication --- p.1 / DECLARATION --- p.i / ACKNOWLEGEMENTS --- p.ii / ABBREVIATIONS --- p.iii / PUBLICATIONS AND AWARDS --- p.v / Chapter 1 --- Abstract --- p.vi / Chapter 2 --- Chapter : Introduction --- p.1 / Chapter 2.1 --- The history of incretin discovery --- p.1 / Chapter 2.2 --- The incretin hormones:GLP-1 and GIP --- p.1 / Chapter 2.3 --- Gene structure and regulation of incretin hormone gene expression --- p.2 / Chapter 2.3.1 --- Gene structure and regulation of GLP-1 gene expression --- p.2 / Chapter 2.3.2 --- Gene structure and regulation of GIP gene expression --- p.5 / Chapter 2.4 --- Secretion and metabolism of GLP-1 and GIP --- p.5 / Chapter 2.4.1 --- Regulation of GLP-1 and GIP secretion --- p.5 / Chapter 2.4.2 --- Degradation of GLP-1 and GIP by DPP-4 enzyme --- p.8 / Chapter 2.5 --- GLP-1 and GIP receptor --- p.10 / Chapter 2.6 --- biological actions of GLP-1 and GIP --- p.11 / Chapter 2.6.1 --- Actions of GLP-1 in peripheral tissues --- p.12 / Chapter 2.6.2 --- Actions of GIP in peripheral tissues --- p.17 / Chapter 2.7 --- Impaired incretin effect in type 2 diabetes patients --- p.17 / Chapter 2.7.1 --- Secretion of incretin hormones in patients with type 2 diabetes --- p.18 / Chapter 2.7.2 --- Impaired the responsiveness to GLP-1 and GIP in pancreatic beta cells --- p.19 / Chapter 2.8 --- Incretin-based drugs --- p.19 / Chapter 2.9 --- Type 2 diabetes and beta cell failure --- p.21 / Chapter 2.9.1 --- Natural history of type 2 diabetes --- p.21 / Chapter 2.9.2 --- Decline of beta cell function and mass in type 2 diabetes --- p.22 / Chapter 2.9.3 --- Factors for progressive loss of beta cell function and mass --- p.24 / Chapter 3 --- Chapter: Pharmacological reduction of free fatty acids restores the efficacy of incretin-based therapies in diabetic mouse models through beta cell GLP-1 receptor signalings --- p.30 / Chapter 3.1 --- Summary --- p.30 / Chapter 3.2 --- Introduction --- p.32 / Chapter 3.3 --- Materials and Methods --- p.35 / Chapter 3.3.1 --- Chemicals and Reagents --- p.35 / Chapter 3.3.2 --- Preparation of 8 mM sodium palmitate solution with 10.5% BSA --- p.35 / Chapter 3.3.3 --- Construct of an adenoviral vector for expressing mouse GLP-1R --- p.36 / Chapter 3.3.4 --- Cell culture and treatment --- p.37 / Chapter 3.3.5 --- RNA extraction and quantitative RT-PCR --- p.37 / Chapter 3.3.6 --- Analysis of phosphorylation of CREB --- p.38 / Chapter 3.3.7 --- Measurement of insulin secretion --- p.39 / Chapter 3.3.8 --- RT-PCR analysis of mouse GLP-1R expression --- p.39 / Chapter 3.3.9 --- Measurement of cAMP production --- p.40 / Chapter 3.3.10 --- Animals and experimental protocols --- p.40 / Chapter 3.3.11 --- Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) --- p.41 / Chapter 3.3.12 --- Acute glucose-lowering actions of Ex-4 and D-GIP in db/db Mice --- p.41 / Chapter 3.3.13 --- Lipid levels measurement --- p.42 / Chapter 3.3.14 --- Histological analysis --- p.42 / Chapter 3.3.15 --- Statistical analysis --- p.42 / Chapter 3.4 --- Results --- p.43 / Chapter 3.4.1 --- Reduced expression of GLP-1R in palmitate-treated b cells and islets of hyperlipedemic db/db mice. --- p.43 / Chapter 3.4.2 --- Palmitate impairs GLP-1 stimulated cAMP production and p-CREB in rodent insulinoma cell lines --- p.45 / Chapter 3.4.3 --- Palmitate reduced GLP-1 and GIP stimulated insulin secretion in rat INS-1E cells --- p.46 / Chapter 3.4.4 --- Mouse GLP-1R mRNA is expressed in rat INS-1E cells after infected with Ad-GLP-1R --- p.47 / Chapter 3.4.5 --- Expression of exogenous GLP-1R restores GLP-1 stimulated cAMP production and p-CREB in palmitate-treated rodent insulinoma cell lines --- p.48 / Chapter 3.4.6 --- Lipid lowering enhances the efficacy of DPP-4 inhibitor des-fluoro-sitagliptin in db/db mice --- p.50 / Chapter 3.4.7 --- Lipid-lowering enhances the efficacy of DPP-4 inhibitor des-flouro-sitagliptin in HFD-fed mice --- p.56 / Chapter 3.4.8 --- Lipid lowering enhances the efficacy of an agonist to GLP-1R (Ex-4) but not to GIPR (D-GIP) in db/db mice --- p.59 / Chapter 3.5 --- Discussion --- p.67 / Chapter 4 --- Chapter : Further study on the impairment of incretin effect by hyperglycemia in a rodent model of type 2 diabetes --- p.71 / Chapter 4.1 --- Summary --- p.71 / Chapter 4.2 --- Introduction --- p.73 / Chapter 4.3 --- Materials and Methods --- p.76 / Chapter 4.3.1 --- Animals and treatment --- p.76 / Chapter 4.3.2 --- Oral glucose tolerance test and insulin tolerance test --- p.76 / Chapter 4.3.3 --- Plasma parameters --- p.77 / Chapter 4.3.4 --- Histological staining and quantification of beta cell mass --- p.77 / Chapter 4.3.5 --- Insulin signaling analysis --- p.78 / Chapter 4.3.6 --- Western blot analysis --- p.78 / Chapter 4.3.7 --- Statistical analysis --- p.79 / Chapter 4.4 --- Results --- p.80 / Chapter 4.4.1 --- Exendin-4 reduced fed glucose levels in MH mice but not in SH mice --- p.80 / Chapter 4.4.2 --- Exendin-4 reduced body weight gain and did not affect food intake in both MH mice and SH mice --- p.81 / Chapter 4.4.3 --- Exendin-4 improved glucose tolerance and glucose stimulated insulin secretion in MH mice but not in SH mice --- p.83 / Chapter 4.4.4 --- Effects of exendin-4 on insulin sensitivity in MH and SH mice --- p.84 / Chapter 4.4.5 --- Effects of exendin-4 on lipid profiles in MH and SH mice --- p.86 / Chapter 4.4.6 --- Effects of exendin-4 on tissues weight in MH and SH mice --- p.87 / Chapter 4.4.7 --- Pancreatic islet morphology and analysis of beta cell mass --- p.88 / Chapter 4.4.8 --- Exendin-4 had no significant effects on insulin signaling pathway in liver and muscle --- p.91 / Chapter 4.5 --- Discussion --- p.94 / Chapter 5 --- Chapter : Summary --- p.100 / Chapter 6 --- References --- p.103

Plastids are organelles responsible for several essential aspects for plant development, like photosynthesis, nitrogen assimilation and synthesis of several compounds of secondary metabolism. Chloroplasts differentiation and activity are highly regulated by light, and several proteins and mechanisms involved in these processes have been characterized. The GOLDEN 2-LIKE (GLK) transcription factors controls the expression of several genes related to photosynthesis, plastid biogenesis and maintenance. Solanum lycopersicum genome harbors two copies of this gene, SlGLK1 and SlGLK2 and, although they are functionally redundant, their expression pattern is different, once SlGLK1 predominates in leaves, while only SlGLK2 is expressed in fruit, precisely at the pedicel region. During tomato domestication, selection for varieties that ripened evenly resulted in the fixation of uniform ripening mutation (Slglk2) in most cultivated varieties, resulting in alterations in fruit metabolic composition. In this context, the objective of this work was to functionally characterize SlGLK2 gene aiming to understand in which way phytochrome mediated light and phytohormones, particularly auxins and cytokinins, regulates this gene expression, and how SlGLK2 presence affects fruit nutritional quality. To achieve this, a detailed transcriptional profile of SlGLK2 was performed in fruits of wild plants, Slglk2 mutant and plants deficient for light perception or hormonal signaling. The effect of SlGLK2 over nutritional quality was evaluated by characterizing carbon and vitamin E metabolism. Additionally, reporter protein GUS activity was quantified in transgenic plants that express uidA gene under control of promoters responsive to cytokinins or auxins in SlGLK2 or Slglk2 genotypes, to analyze if hormonal activity is affected by SlGLK2 presence. Finally, in order to verify if the presence of SlGLK2 is sufficient to reverse the chlorotic phenotype of the mutant aurea, promoting the differentiation and plastidial maturation even in the absence of functional phytochromes, transgenic lines were generated by overexpressing the SlGLK2 gene on aurea-Slglk2 genetic background. The integrated data analysis allowed us to conclude that the content of soluble sugars and vitamin E correlate with the expression of SlGLK2, that the expression of SlGLK2 is repressed by auxins, that SlGLK2 positively participates in the signaling of cytokinins, and that its overexpression partially reverts the phenotype of the aurea-Slglk2 mutant fruits. The results obtained in this work contributes to a better understanding of the regulatory network that interconnects SlGLK2 gene, phytohormones and light, promoting the plastidial activity and consequently, determining the nutritional quality of the tomato fruit, an important component of the human diet / Os plastídios são organelas responsáveis por diversos aspectos essenciais do desenvolvimento das plantas como a fotossíntese, assimilação de nitrogênio e síntese de diversos compostos do metabolismo secundário. A diferenciação e atividade dos cloroplastos são altamente reguladas pela luz, e diversas proteínas e mecanismos envolvidos nestes processos têm sido caracterizados. Os fatores de transcrição GOLDEN 2-LIKE (GLKs) controlam a expressão de diversos genes relacionados à fotossíntese, biogênese e manutenção plastidial. Solanum lycopersicum possui duas cópias desses genes, SlGLK1 e SlGLK2 e, embora sejam funcionalmente redundantes, seu padrão de expressão é diferente, uma vez que SlGLK1 predomina nas folhas ao passo que SlGLK2 é expresso apenas nos frutos, mais precisamente na região pedicelar. Durante o processo de domesticação do tomateiro, a seleção de variedades de amadurecimento uniforme resultou na fixação da mutação uniform ripening (Slglk2) na maioria das variedades cultivadas, resultando em mudanças na composição metabólica dos frutos. Neste contexto, este trabalho teve como objetivo geral caracterizar funcionalmente o gene SlGLK2 visando compreender de que forma a luz (mediada por fitocromos) e os fitormônios (particularmente citocininas e auxinas), regulam a expressão deste gene e como a presença de SlGLK2 afeta a qualidade nutricional dos frutos. Para isso, foi realizado um detalhado perfil transcricional de SlGLK2 em frutos de plantas selvagens, Slglk2 mutantes e deficientes para a percepção luminosa e para a sinalização hormonal. O efeito de SlGLK2 sobre a qualidade nutricional foi avaliado caracterizando o metabolismo de carbono e de vitamina E. Adicionalmente, foi quantificada a atividade da proteína repórter GUS em plantas transgênicas que expressam o gene uidA sob controle de promotores responsivos a citocininas ou auxinas em plantas com genótipo SlGLK2 ou Slglk2 para analisar se a atividade hormonal é afetada pela presença de SlGLK2. Finalmente, com o intuito de verificar se a presença de SlGLK2 é suficiente para reverter o fenótipo clorótico do mutante aurea, promovendo a diferenciação e maturação plastidial mesmo na ausência de fitocromos funcionais, foram geradas linhagens transgênicas sobreexpressando o gene SlGLK2 em fundo genético aurea-Slglk2. A Análise dos resultados permitiu concluir que o conteúdo de açúcares solúveis e vitamina E correlacionam com a expressão de SlGLK2, que a expressão de SlGLK2 é reprimida por auxinas, que SlGLK2 participa positivamente da sinalização de citocininas, e que a sua sobreexpressão reverte, parcialmente, o fenótipo dos frutos da mutante aurea-Slglk2. Os resultados obtidos nos levam a uma melhor compreensão da rede regulatória que interconecta o gene SlGLK2, os fitormônios e a luz promovendo a atividade plastidial e, por consequência, determinando a qualidade nutricional dos frutos de tomateiro, importante componente da dieta humana