Os microrganismos associados à cárie são, em sua maioria, microrganismos acidogênicos e acidúricos, anaeróbios estritos e facultativos. A presença de fungos é associada à microbiota de cárie radicular, sendo a espécie fúngica mais relacionada a Candida albicans. Embora estudos de cultivo e de análise de DNA comprovem a presença de fungos na microbiota associada a lesões de cárie radicular, demonstrando um gradiente crescente de colonização com a progressão da doença, pouco se sabe sobre o papel que estes microrganismos desempenham no processo de doença. O objetivo deste trabalho foi estudar o papel de Candida albicans na cárie radicular, através da análise de transcriptoma de biofilmes naturais de superfícies radiculares hígidas (n=10, SRS) e de lesão de cárie radicular ativa (n=9, RC). Foi avaliada a expressão diferencial de genes de Candida albicans, as funções específicas e vias metabólicas associadas a este microrganismo. O RNA total microbiano foi extraído e o mRNA isolado e sequenciado na plataforma Illumina Hi-Seq2500. Foram formados pool (grupamentos) das amostras com valores inferiores a 30ng/RNA para a construção de bibliotecas genômicas. Os dados gerados pelo sequenciamento de RNA-Seq foram compilados em uma tabela de contagem (reads) e mapeados com o genoma de referência (C. albicans SC5314) utilizando o software CLC Genomics Workbench 7.5.1. Para o cálculo do nível de expressão gênica os dados foram normalizados com o algoritmo DESeq. A expressão diferencial foi calculada com binomial negativa e False Discovery Rate (FDR<0,05). Os genes com maior expressão em RC e em SRS foram analisados pela mediana relativa de expressão (RME; Relative Median Expression) e expressão diferencial, assim como as vias metabólicas associadas a genes de virulência e metabolismo de açúcares. Dois genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) apresentaram expressão diferencial significativa em superfície radicular hígida (SRS) e tem suas funções relacionadas a formação de biofilme. Enquanto que em superfície radicular cariada (RC) sete genes (UTP20, FDR=0.018; ITR1, FDR=0.036; DHN6, FDR=0.046; CaO19.7197, FDR=0.046; CaO19.7838, FDR=0.046; STT4, FDR=0.046; GUT1, FDR=0.046) apresentaram expressão diferencial significativa e tem suas funções relacionadas a atividade metabólica, transporte de açúcares, tolerância ao estresse, invasão e regulação de pH. Candida albicans é um microrganismo importante no desenvolvimento da doença cárie radicular. / The microbiota associated with root caries must be acidogenic and aciduric. S. mutans, Lactobacillus, Actinomyces, Veilonella, Bifidobacterium, and other bacteria play important roles in root caries biofilm. Yeasts are also present on carious and non-carious root biofilms, being the specie Candida albicans the most prevalent yeast found in root biofilms. Although the presence of Candida albicans is stablished in the literature, and there are an increasing gradient of Candida species. colonization with caries progression, the role of this microorganism in root caries has not being totally elucidated. The aim of this study is to analyse the role of Candida albicans in root caries thought a transcriptomic analysis of biofilms of sound root surfaces (n=10, SRS) and root caries lesions (n=9, RC) using a high-throughput sequencing of cDNA (RNA-Seq). The differential expression of genes of Candida albicans SC5314, the specific functions and pathways associated with the gene expression of the present study were investigated. The total bacterial RNA was extracted and the mRNA was isolated (Illumina Hi-Seq2500). Samples with low RNA concentration (less than 30ng/RNA) were pooled, yielding a final sample size of SRS=10 and RC=9. Sequence reads were compiled in a count table and mapped to C. albicans SC5314 genome of reference, using the software CLC Genomics Workbench 7.5.1. Gene expression was calculated in the algorithm DESeq, and the differential expression was calculated with binomial negative (Log2FoldChange) and False Discovery Rate (FDR<0,05). The genes with higher expression in RC and SRS were analysed by the Relative Median Expression (RME), and the virulence factors and pathways and sugar metabolization related with Candida albicans pathogenicity in root caries were analysed. Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) are up-regulated in Sound Root Surface (SRS) have their functions related to biofilm formation and seven (UTP20, FDR=0.018; ITR1, FDR=0.036; DHN6, FDR=0.046; CaO19.7197, FDR=0.046; CaO19.7838, FDR=0.046; STT4, FDR=0.046; GUT1, FDR=0.046) are up-regulated in biofilm of carious dentin (RC) have functions related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation.

Orientadora: Profa. Dra. Ana Carolina Quirino Simões / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Engenharia Biomédica, 2016. / A leptospirose é uma zoonose classificada como doença tropical negligenciada, responsável por problemas sérios de saúde pública. A doença é causada por uma bactéria patogênica do gênero Leptospira e ordem Spirochaetales, sendo que a última agrupa tanto espécies patogênicas (complexo interrogans) quanto saprofíticas (complexo biflexa). Sete genomas de leptospiras foram sequenciados, entre eles os de duas espécies causadoras de leptospirose. Um estudo comparativo aprofundado entre os genomas das espécies patogênicas e saprófitas pode elucidar quais são os genes exclusivos a cada espécie e envolvidos na patogenicidade e defesa das leptospiras. Estudos anteriores realizados por nosso grupo mostraram que a espécie saprófita, Leptospira biflexa, possui genes exclusivos para resposta ao estresse oxidativo, indicando a sua aquisição por evento de transferência horizontal. Por outro lado, a espécie patogênica Leptospira interrogans também apresenta padrão particular de expressão para este tipo de situação. A resposta ao estresse oxidativo é uma forma de defesa da bactéria ao sistema imunológico do hospedeiro, ou seja, uma forma de resposta importante para a compreensão da patogenicidade destas bactérias. Faz-se necessário entender as diferenças entre as duas espécies tanto para uma resposta deste tipo, mas também para outras relacionadas à patogenicidade da bactéria em questão. A presente dissertação apresenta a comparação entre os genomas das leptospiras já sequenciadas, focando nos genes de stress oxidativo, reparo de DNA e de mecanismos de virulência e patogenicidade, bem como estuda a estrutura e dinâmica molecular proteica de dois reguladores importantes, OxyR e lexA, em Leptospira biflexa. Adicionalmente, ela contempla a analise de genes diferencialmente expressos obtidos por RNA-seq em Leptospira biflexa em um experimento de série temporal após a exposição a raios ultravioleta. / Leptospirosis is an important zoonosis classified as a neglected tropical disease, responsible for serious public health problems resulting in costs to the economy. The disease is caused by pathogenic bacteria of the Leptospira genus and Spirochaetales order, which comprehends both pathogenic species (interrogans complex) and saprophytic ones (biflexa complex). Seven species of leptospira had their genomes completely sequenced, including the genomes of two major pathogenic species responsible for leptospirosis disease. A comparative study of the genomes of pathogenic and saprophytic species can elucidate which are the unique genes to each species and important for pathogenicity and defense of leptospira. Previous studies by our group showed that the saprophytic species, Leptospira biflexa, has unique genes for oxidative stress response, indicating its acquisition by horizontal transfer event. Furthermore, the pathogenic Leptospira interrogans species also presents particular expression pattern for this kind of situation. The oxidative stress response is a form of protection of the bacteria to the immune system of the host organism, an important form of response for understanding the pathogenicity of these bacteria. Thus it is necessary to understand the differences between the two species for such response and also for other related pathogenic bacteria in question. This dissertation aims to compare the genomes of leptospira already sequenced, focusing on oxidative stress genes, DNA repair and virulence and pathogenicity mechanisms and study the structure and protein molecular dynamics of two important regulators, OxyR and lexA in Leptospira biflexa. Additionally, the study includes the analysis of differentially expressed genes obtained by RNA-seq in Leptospira biflexa in a time series experiment after exposure to ultraviolet rays.

Over 90% of neurons in the adult CNS of Drosophila are born from neuronal stem cells (neuroblasts) during the post-embryonic phase of neurogenesis. Most of the post-embryonic neurons derive from type I neuroblasts, which undergo repeated asymmetric divisions to produce a series of ganglion mother cells (GMCs). Each GMC then divides once resulting in two neurons, the “A” (Notch-on) and “B” (Notch-off) daughters. The respective daughter neurons of each type then constitute the A and B hemilineages for that neuroblast. 33 postembryonic hemilineages contribute neurons to each thoracic hemisegment, and these immature neurons arrest their development at a similar stage until metamorphosis. These arrested neuroblast lineages are uniquely identifiable by morphology. Access to a large pool of clonally-related and morphologically similar neurons makes this system tractable to RNA-seq analysis, since one can genetically label and isolate many cells per animal, which are predicted to share similar gene expression profiles. Our primary focus is to examine hemilineages with similar targets (e.g. leg neuropil) to identify genes that are required to establish and maintain hemilineage identity early in development. Given that activating these hemilineage neurons as a group drives distinct behaviors and that they form morphologically coherent structural units during development, we hypothesized that these hemilineages should express patterns of genes that are: 1) distinct from other hemilineages and 2) characteristic of individual hemilineages. We have used hemilineage-specific GAL4 lines to isolate hemilineages for RNA-seq analysis, ultimately gathering data for 11 of the 33 hemilineages as well as for some larger populations of neurons. We found that, in addition to combinatorial patterns of genes specifying the hemilineage neurons, there are some genes that are expressed by only a single hemilineage within the ventral nervous system (VNS). Most hemilineages display unique expression of certain transcription factors (TFs) and axon guidance genes. We collected data for two pairs of sibling hemilineages (lineage 1 and lineage 12) in order to identify differences between the A and B hemilineages derived from a common neuroblast. While A neurons display greater overall transcriptional diversity than B neurons, sibling hemilineages share very similar expression profiles. Comparing the gene expression between immature and mature larval neurons revealed that mature neurons express many genes not expressed in immature neurons, such as neuropeptide signaling genes and many neurotransmitter and ion channel genes associated with mature neuron function. Birth order also appears to dictate many differences in expression profile. Late-born immature neurons are typified by a period of transient Notch-related gene expression that is absent from early-born neurons. We are characterizing the function of many differentially expressed genes in particular hemilineages.

Circadian clocks are internal timekeepers that allow organisms to anticipate and exploit predictable daily changes in their environment, aiding survival. Clock-driven rhythms, such as asexual spore development (conidiation) in Neurospora crassa, show temperature compensated periodicity that persists in constant conditions and can be reset by environmental time cues. This ability of circadian clocks to maintain a constant period and phase of behaviour over a range of temperatures is important, and whilst much of the machinery making up the circadian clock is known, the mechanism that underpins temperature compensation is not well understood. Further, it is unknown how the clock can control conidiation in the face of changing temperatures. To investigate possible mechanisms underlying temperature compensation, I first explored how compensation may arise within the central clock machinery using a comprehensive dynamic model of the Neurospora crassa circadian clock. This clock incorporates key components of the clock, and I introduced known temperature-sensitive component changes based on experimental observations. This analysis indicated that temperature-dependent changes in the binding of CK-1a to the FRQ-FRH complex may be pivotal in the temperature compensation mechanism. Previous work has highlighted the importance of the blue-light photoreceptor VIVID (VVD), as VVD knockout strains show a temperature-dependent delay in the phase of peak conidiation. Next I explored this potential role using a theoretical output model. By incorporating regulation of this pathway by VVD, I found that VVD may contribute to phase control by increasing expression of genes or proteins that peak early on in the output pathway. RNA-Seq experiments were carried out to assess the contribution of VVD to the overall transcriptomic profile of Neurospora. The analysis highlighted several key genes through which VVD may regulate the conidiation pathway, including the clock-controlled genes eas and ccg-9, which both show temperature- and strain-dependent changes in expression patterns over the time course of conidiation. In conclusion, VVD may indeed have an important role in the temperature compensation of output pathways, though further work is needed to assess the specific
contributions of genes highlighted by my RNA-Seq analysis to the compensatory mechanism.

abstract: Damage to the central nervous system due to spinal cord or traumatic brain injury, as well as degenerative musculoskeletal disorders such as arthritis, drastically impact the quality of life. Regeneration of complex structures is quite limited in mammals, though other vertebrates possess this ability. Lizards are the most closely related organism to humans that can regenerate de novo skeletal muscle, hyaline cartilage, spinal cord, vasculature, and skin. Progress in studying the cellular and molecular mechanisms of lizard regeneration has previously been limited by a lack of genomic resources. Building on the release of the genome of the green anole, <i>Anolis carolinensis</i>, we developed a second generation, robust RNA-Seq-based genome annotation, and performed the first transcriptomic analysis of tail regeneration in this species. In order to investigate gene expression in regenerating tissue, we performed whole transcriptome and microRNA transcriptome analysis of regenerating tail tip and base and associated tissues, identifying key genetic targets in the regenerative process. These studies have identified components of a genetic program for regeneration in the lizard that includes both developmental and adult repair mechanisms shared with mammals, indicating value in the translation of these findings to future regenerative therapies. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Submitted by Mariana Gatto (marianagatto11@hotmail.com) on 2018-05-21T17:47:50Z No. of bitstreams: 1 Tese Mariana Gatto.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-22T14:28:44Z (GMT) No. of bitstreams: 1 gatto_m_dr_bot.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) / Made available in DSpace on 2018-05-22T14:28:44Z (GMT). No. of bitstreams: 1 gatto_m_dr_bot.pdf: 3235524 bytes, checksum: b7c9938cd744aff0ff8a8ee5f858831e (MD5) Previous issue date: 2018-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A leishmaniose visceral (LV) é uma doença negligenciada causada por Leishmania donovani na Índia e África ou Leishmania infantum na Europa e América Latina. O desenvolvimento de resposta imune eficaz e subsequente eliminação destes patógenos, requer o reconhecimento inicial da Leishmania, o qual é intermediado por receptores de reconhecimento padrão expressos por células da imunidade inata, entre eles os receptores de ligação a nucleotídeo (NLRs). Alguns NLRs ativam uma plataforma de proteínas, os inflamassomas, responsáveis pela ativação da caspase-1 e maturação de IL-1β e IL-18 e outra classe de NLRs, chamada NODs, ativam vias que culminam na ativação de NF-κB e produção de mediadores inflamatórios. O envolvimento desses receptores na LV ainda é pouco elucidado. Mesmo diante dos mecanismos de defesa do hospedeiro, esses parasitas conseguem sobreviver dentro dos macrófagos utilizando várias estratégias para escapar da resposta imune. Para um melhor entendimento dos mecanismos imunes envolvidos na LV, caracterizamos o perfil transcricional e a formação de inflamassomas e NODsomas de células THP-1 infectadas com L. infantum. Os resultados mostram que a L. infantum não induziu produção de TNF-α, IL-6 e IL-1β e nem ativação de caspase-1 após 8, 24 e 48 horas de infecção. Além disso, a infecção resultou em padrão de expressão gênica similar às células sem estímulo e distinto de células estimuladas com LPS, indicando que os parasitas entram nas células de forma mais silenciosa. Após a infecção houve aumento da expressão de alguns genes como ACTG1, ACTB, CD36 e DUSPs relacionados com vias de motilidade celular e regulação de MAPKs. Os genes CSF1 e CDC20 foram dois dos 30 mais expressos após infecção e estão relacionados com ciclo celular e diferenciação de macrófagos para um perfil anti-inflamatório. O gene GBP1, associado com ativação de inflamassomas, foi sub expresso após a infecção. Além disso, infecção com L. infantum resultou na expressão de poucos genes relacionados com a via dos NLRs, destacando-se entre esses o TNFAIP3 e IL1RN, referentes à modulação negativa dessa via. Os resultados obtidos indicam que a L. infantum entra nas células THP-1 de forma mais silenciosa, desativa vias inflamatórias, entre essas a via de receptores NLRs e evita a montagem de uma resposta imunológica efetora. Provavelmente o parasita usa esses recursos como mecanismos adicionais de escape para garantir sua sobrevivência dentro das células. / Visceral leishmaniasis (VL) is a neglected infectious disease caused by Leishmania donovani in India and Africa or Leishmania infantum in Europe and Latin America. The development of an effective immune response and subsequent elimination of these pathogens requires the initial recognition of the Leishmania that is mediated by pattern recognition receptors expressed in innate immunity cells, such as nucleotide-binding receptors (NLRs). Some NLRs activate a multiprotein platform named inflammasomes, responsible for the activation of caspase-1 and consequent maturation of IL-1β and IL-18; and another class of NLRs, the NODs, activate pathways that trigger NF-κB activation and production of inflammatory mediators. The involvement of NLRs in LV is poorly elucidated. Even in the presence of host defense mechanisms, these parasites can survive within the macrophages by employing successful strategies to escape from immune response. For a better understanding of the immune mechanisms involved in LV, we characterized the transcriptomic profiling and assembly of inflammasomes and NODsomas during infection with L. infantum in THP-1 cells. The results show that L. infantum did not induce the production of TNF-α, IL-6 and IL-1β nor activation of caspase-1 after 8, 24 and 48 hours of infection. In addition, the infection resulted in a pattern of gene expression similar to the non-stimulated cells and distinct from LPS-stimulated cells, indicating that the parasites enter inside cells in a more silent way. After infection, there was increased expression of some genes, such as ACTG1, ACTB, CD36 and DUSPs related to cellular motility and regulation of MAPKs pathways. The CSF1 and CDC20 genes were two of the 30 most expressed after infection and were related to cell cycle pathway and macrophage differentiation to an anti-inflammatory profile. The GBP1 gene, associated with inflammasome activation, was downregulated after infection. In addition, infection with L. infantum resulted in the expression of few genes related to the NLRs pathway, such as TNFAIP3 and IL1RN that are related to down modulation of this pathway. The results indicate that L. infantum enters inside the THP-1 cells more quietly, deactivates inflammatory pathways, including the NLR receptor pathway, and avoids the assembly of an effector immune response. Probably the parasite uses these strategies as additional escape mechanism to ensure its survival within host cells.

Os microrganismos associados à cárie são, em sua maioria, microrganismos acidogênicos e acidúricos, anaeróbios estritos e facultativos. A presença de fungos é associada à microbiota de cárie radicular, sendo a espécie fúngica mais relacionada a Candida albicans. Embora estudos de cultivo e de análise de DNA comprovem a presença de fungos na microbiota associada a lesões de cárie radicular, demonstrando um gradiente crescente de colonização com a progressão da doença, pouco se sabe sobre o papel que estes microrganismos desempenham no processo de doença. O objetivo deste trabalho foi estudar o papel de Candida albicans na cárie radicular, através da análise de transcriptoma de biofilmes naturais de superfícies radiculares hígidas (n=10, SRS) e de lesão de cárie radicular ativa (n=9, RC). Foi avaliada a expressão diferencial de genes de Candida albicans, as funções específicas e vias metabólicas associadas a este microrganismo. O RNA total microbiano foi extraído e o mRNA isolado e sequenciado na plataforma Illumina Hi-Seq2500. Foram formados pool (grupamentos) das amostras com valores inferiores a 30ng/RNA para a construção de bibliotecas genômicas. Os dados gerados pelo sequenciamento de RNA-Seq foram compilados em uma tabela de contagem (reads) e mapeados com o genoma de referência (C. albicans SC5314) utilizando o software CLC Genomics Workbench 7.5.1. Para o cálculo do nível de expressão gênica os dados foram normalizados com o algoritmo DESeq. A expressão diferencial foi calculada com binomial negativa e False Discovery Rate (FDR<0,05). Os genes com maior expressão em RC e em SRS foram analisados pela mediana relativa de expressão (RME; Relative Median Expression) e expressão diferencial, assim como as vias metabólicas associadas a genes de virulência e metabolismo de açúcares. Dois genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) apresentaram expressão diferencial significativa em superfície radicular hígida (SRS) e tem suas funções relacionadas a formação de biofilme. Enquanto que em superfície radicular cariada (RC) sete genes (UTP20, FDR=0.018; ITR1, FDR=0.036; DHN6, FDR=0.046; CaO19.7197, FDR=0.046; CaO19.7838, FDR=0.046; STT4, FDR=0.046; GUT1, FDR=0.046) apresentaram expressão diferencial significativa e tem suas funções relacionadas a atividade metabólica, transporte de açúcares, tolerância ao estresse, invasão e regulação de pH. Candida albicans é um microrganismo importante no desenvolvimento da doença cárie radicular. / The microbiota associated with root caries must be acidogenic and aciduric. S. mutans, Lactobacillus, Actinomyces, Veilonella, Bifidobacterium, and other bacteria play important roles in root caries biofilm. Yeasts are also present on carious and non-carious root biofilms, being the specie Candida albicans the most prevalent yeast found in root biofilms. Although the presence of Candida albicans is stablished in the literature, and there are an increasing gradient of Candida species. colonization with caries progression, the role of this microorganism in root caries has not being totally elucidated. The aim of this study is to analyse the role of Candida albicans in root caries thought a transcriptomic analysis of biofilms of sound root surfaces (n=10, SRS) and root caries lesions (n=9, RC) using a high-throughput sequencing of cDNA (RNA-Seq). The differential expression of genes of Candida albicans SC5314, the specific functions and pathways associated with the gene expression of the present study were investigated. The total bacterial RNA was extracted and the mRNA was isolated (Illumina Hi-Seq2500). Samples with low RNA concentration (less than 30ng/RNA) were pooled, yielding a final sample size of SRS=10 and RC=9. Sequence reads were compiled in a count table and mapped to C. albicans SC5314 genome of reference, using the software CLC Genomics Workbench 7.5.1. Gene expression was calculated in the algorithm DESeq, and the differential expression was calculated with binomial negative (Log2FoldChange) and False Discovery Rate (FDR<0,05). The genes with higher expression in RC and SRS were analysed by the Relative Median Expression (RME), and the virulence factors and pathways and sugar metabolization related with Candida albicans pathogenicity in root caries were analysed. Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) are up-regulated in Sound Root Surface (SRS) have their functions related to biofilm formation and seven (UTP20, FDR=0.018; ITR1, FDR=0.036; DHN6, FDR=0.046; CaO19.7197, FDR=0.046; CaO19.7838, FDR=0.046; STT4, FDR=0.046; GUT1, FDR=0.046) are up-regulated in biofilm of carious dentin (RC) have functions related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation.

abstract: Well-established model systems exist in four out of the seven major classes of vertebrates. These include the mouse, chicken, frog and zebrafish. Noticeably missing from this list is a reptilian model organism for comparative studies between the vertebrates and for studies of biological processes unique to reptiles. To help fill in this gap the green anole lizard, Anolis carolinensis, is being adapted as a model organism. Despite the recent release of the complete genomic sequence of the A. carolinensis, the lizard lacks some resources to aid researchers in their studies. Particularly, the lack of transcriptomic resources for lizard has made it difficult to identify genes complete with alternative splice forms and untranslated regions (UTRs). As part of this work the genome annotation for A. carolinensis was improved through next generation sequencing and assembly of the transcriptomes from 14 different adult and embryonic tissues. This revised annotation of the lizard will improve comparative studies between vertebrates, as well as studies within A. carolinensis itself, by providing more accurate gene models, which provide the bases for molecular studies. To demonstrate the utility of the improved annotations and reptilian model organism, the developmental process of somitogenesis in the lizard was analyzed and compared with other vertebrates. This study identified several key features both divergent and convergent between the vertebrates, which was not previously known before analysis of a reptilian model organism. The improved genome annotations have also allowed for molecular studies of tail regeneration in the lizard. With the annotation of 3' UTR sequences and next generation sequencing, it is now possible to do expressional studies of miRNA and predict their mRNA target transcripts at genomic scale. Through next generation small RNA sequencing and subsequent analysis, several differentially expressed miRNAs were identified in the regenerating tail, suggesting miRNA may play a key role in regulating this process in lizards. Through miRNA target prediction several key biological pathways were identified as potentially under the regulation of miRNAs during tail regeneration. In total, this work has both helped advance A. carolinensis as model system and displayed the utility of a reptilian model system. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

Filariasis and onchocerciasis are debilitating diseases affecting 120 million people globally. The massive socio-economic impact of these diseases energized the international community to declare a goal of eliminating filariasis 2020. This resulted in a dramatic increase in the efforts to eliminate filariasis and onchocerciasis, employing a strategy of mass drug administration (MDA). However, these programs rely upon the small arsenal of drugs. This leaves these programs vulnerable to failure in the face of developing resistance and local intolerance to the current drug regimens. Thus, new drugs against these infections are critically needed. A homologue of the ecdysone receptor (EcR), a master regulator of development in insects, has been identified in B. malayi. The potential of the EcR as a drug target has been underscored by work in the agricultural industry, where insecticides targeting the ecdysone developmental pathway are effective and non-toxic to non-target species. As the EcR is absent in humans, it represents an attractive potential chemotherapeutic target. The first study investigates the hypothesis that the ecdysone receptor controls the embryogenesis and molting in the filarial parasite. In-vitro embryogram and in-vivo phenotypic studies were conducted to delineate the effect of 20-hydroxyecdysone on the Brugia malayi parasites. The results suggest that the hormone accelerates embryogenesis and causes precocious molts, resulting in the death of the parasite. Further, transcriptomic and proteomic analysis of the ecdysone treated worms provided evidence that the up-regulated genes participate in embryogenesis. Based upon the validation of the ecdysone receptor as a potential drug target, subsequent studies focused on the development of a drug discovery model to screen for agonists and antagonists of the B. malayi ecdysone receptor. A stable cell line was created to aid the high throughput screening to rapidly identity agonist and antagonist compounds. A total of 7 agonists and 2 antagonists were identified. A homology model of the BmEcR ligand-binding domain was created as an alternate method for virtual screening of small molecules as well as to study the ligand-receptor interactions. The hits identified with the assay were docked in the active site of the BmEcR homology model providing an excellent correspondence of data between the molecular assay and the virtual screening method.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2018-10-04T13:32:15Z No. of bitstreams: 2 Dissertação - Lucas França Fagundes - 2017.pdf: 2859645 bytes, checksum: a3a69c2ef467d243d394e4ee270a7680 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-05T14:14:11Z (GMT) No. of bitstreams: 2 Dissertação - Lucas França Fagundes - 2017.pdf: 2859645 bytes, checksum: a3a69c2ef467d243d394e4ee270a7680 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-10-05T14:14:11Z (GMT). No. of bitstreams: 2 Dissertação - Lucas França Fagundes - 2017.pdf: 2859645 bytes, checksum: a3a69c2ef467d243d394e4ee270a7680 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-12-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Due in most part to the rapid growth and adaptability of the Eucalyptus in Brazil, the country's forestry sector is one of the most competitive of the world. Drought is one of the main obstacles of the culture, which has been expanding to the country’s interior regions such as the Cerrado biome. The objective of this work was to identify genes and biological processes involved in the responses of the Eucalyptus roots to drought. The experiment was conducted in a completely randomized design with 2 treatments (control and deficit) x 3 root RNA gathering time (3, 6 and 12h after start of treatment) and three biological replicates (commercial clones 1528, I144, VM1). The plants grew in hydroponics for 50 days, before the experiment start by using PEG8000 to induce water deficit (osmotic potential of -0.9 MPa). The root RNA of the seedlings was extracted using the RNeasy Plant Mini® kit (Quiagen). RNA samples were sequenced on the HiSeq 2000 platform (Illumina), yielding approximately 626 million 50 bp sequences in single end mode. After quality control, the sequences were aligned in the reference genome of Eucalyptus grandis. After counting reads aligned on each gene, differential expression analyses were performed with edgeR package of R/Bioconductor. A total of 6169 differentially expressed genes (DEG) (FDR ≤ 5% and Log2 FoldChange > |2|) were identified. Of these, 3350 were up-regulated and 2819 were repressed under osmotic stress, considering all the time-series evaluations. At three hours after treatment the highest number of DEG was obtained, with 3150, followed by six hours (2494 DEG) and 12h (525 DEG). Functional enrichment analysis was performed with the Fisher exact test, by software Blast2GO, with the DEG associated with their respective Gene Ontology (GO) terms. As a total of 90 GO terms, enriched and non-redundant, was verified that our results indicate a rapid initial response of Eucalyptus roots induced by PEG 8000 in hydroponics. The most remarkable characteristics of this response under hydroponic conditions are: increased energy consumption via carbohydrate catabolism, activation of the glycolytic pathway, repression of the respiratory chain in the mitochondria, increase of the fermentation pathway, partial execution of the citric acid cycle and repression of the primary metabolism (DNA replication and RNA biosynthesis, development and growth). These enriched functional terms point to the predominance of hypoxia stress, induced by the high viscosity of PEG in hydroponics, as compared to the osmotic stress. / Devido em grande parte ao rápido crescimento e adaptabilidade do Eucalyptus no Brasil, o setor florestal do país é um dos mais competitivos do mundo. O déficit hídrico é um dos principais entraves atuais da cultura, que vem se expandindo para as regiões interioranas do país, como o Cerrado. O presente trabalho teve como objetivos identificar os genes e os processos biológicos relacionados à resposta das raízes do Eucalyptus ao déficit hídrico. O experimento foi conduzido em delineamento inteiramente casualizados com 2 tratamentos (controle e déficit) x 3 tempos de coleta (3, 6 e 12h após início do tratamento) e três réplicas biológicas (clones comerciais 1528, I144, VM1). As plantas cresceram em hidroponia por 50 dias, quando foi iniciado o experimento de déficit hídrico induzido com PEG8000 (potencial osmótico de -0,9 MPa). O RNA das raízes das mudas foi extraído utilizando o kit RNeasy Plant Mini® (Quiagen). As amostras de RNA foram sequenciadas na plataforma HiSeq 2000 (Illumina), produzindo aproximadamente 626 milhões de sequências de 50 pb no modo single end. Após controle qualidade, as sequências foram alinhadas no genoma de referência de Eucalyptus grandis. Após contagem das sequências alinhadas em cada gene, realizou-se análises de expressão diferencial com o pacote edgeR do R/Bioconductor. Foram identificados 6169 genes diferencialmente expressos (GDE) (FDR ≤ 5% e Log2 FoldChange > |2|). Desses, 3350 foram ativados e 2819 reprimidos sob estresse osmótico, considerando todos os tempos avaliados. O tempo de três horas foi o que mais identificou GDE, com 3150, seguido pelo de seis (2494 GDE) e 12h (525 GDE). A análise de enriquecimento funcional foi realizada com o teste exato de Fisher, com os GDE associados aos seus respectivos termos Gene Ontology (GO), por meio do aplicativo Blast2GO. Como total de 90 termos GO, enriquecidos não redundantes, foi verificado que nossos resultados apontam para uma rápida resposta inicial das raízes do Eucalyptus induzidas pelo PEG 8000 em hidroponia. As características mais marcantes dessa resposta, em condições de hidroponia, são: o aumento do consumo energético via catabolismo de carboidratos, ativação da via glicolítica, repressão da cadeia respiratória nas mitocôndrias, aumento da via de fermentação, execução parcial do ciclo do acido cítrico e repressão do metabolismo primário (replicação do DNA e biossíntese de RNA, desenvolvimento e crescimento). Os termos enriquecidos apontam para uma predominância do estresse de hipóxia, induzido pela alta viscosidade do PEG em hidroponia, em comparação ao estresse hídrico.