Les dinoflagellés jouent un rôle très important dans l’écologie des océans en y réalisant une grande partie de la production primaire, en formant une association symbiotique avec les coraux et en ayant la capacité de produire des fleurs d’algues potentiellement toxiques pour les communautés côtières humaines et animales. Malgré tout, la biologie moléculaire des dinoflagellés n’a que très peu été étudiée dans les dernières années, les connaissances de processus de base comme la régulation de la transcription y étant fortement limitées. Une tentative pour élucider ce mécanisme a été réalisée chez les dinoflagellés photosynthétiques Lingulodinium polyedrum et Amphidinium carterae. Une expérience d’induction de la transcription du gène de la Peridinin chlorophyll-a binding protein, le complexe majeur de collecte de lumière, a été réalisée par une baisse de l’intensité lumineuse et a montré une faible augmentation (moins de 2 fois) du transcrit à court et long terme. Des expériences de simple-hybride et de retard sur gel (EMSA) ont été faits pour identifier de potentielles interactions protéine-ADN dans la région intergénique du gène PCP organisé en tandem. Ces essais ont été infructueux pour identifier de telles protéines. Une analyse du transcriptome de L. polyedrum a été effectuée, montrant une importante sous-représentation de domaines de liaison à l’ADN classique (comme Heat-shock factor, bZIP ou Myb) et une surreprésentation du domaine d’origine bactérienne Cold shock en comparaison avec d’autres eucaryotes unicellulaires. Ce travail suggère que les mécanismes de régulation transcriptionnelle des dinoflagellés pourraient différer substantiellement de ceux des autres eucaryotes. / Dinoflagellates are an important part of the ocean’s ecology due to their large contribution to global carbon fixation, the symbiotic association they can make with corals and by their ability to form algal blooms potentially toxic for humans and animals in coastal communities. However, the molecular biology of dinoflagellates has been poorly studied in the past. Basic knowledge, such as regulation of gene expression, is severely limited. An attempt at deciphering basic gene regulation has been undertaken in the photosynthetic dinoflagellate Lingulodinium polyedrum and Amphidinium carterae using a reduction in available light intensity to induce the expression of the peridinin chlorophyll-a binding gene encoding the major light harvesting complex protein. A small increase in transcript abundance (less than 2 fold) was found in both short and long term experiments, yet neither yeast one-hybrid assays nor electrophoretic mobility shift assays (EMSA) showed any potential protein interactions with sequence derived from the intergenic spacer of the PCP tandem gene array. Interestingly, an analysis of the recently sequenced L. polyedrum transcriptome revealed an important under-representation of classic DNA-binding domains (such has Heat-shock factor, bZIP and Myb) and an over-representation of the bacterial cold-shock DNA-binding domain. This suggested that components of the transcription regulation machinery may be at least partially different in dinoflagellates.

The studies presented in this dissertation, Evolution And Divergence Of The Structural And Physical Properties Of DNA Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, pertain primarily to two key epigenetic regulators involved with the biological interpretation of methylated DNA marks. We provide insights into the emergence and evolution of the MBD2 and MBD3 and how those molecular entities influence heritable changes in gene activity. We further provide details regarding the mystery surrounding MBD3 function and the MBD2-mediated capacity of primitive animals to carry out methylation-specific epigenetic mechanisms. In chapter two, we describe the DNA binding properties of MBD2 and MBD3. This study provides information regarding previously unidentified MBD3 binding properties and potential biological function. In chapter three, we show that sponges demonstrate a MBD2-mediated capacity for binding methylated DNA sites, recruit NuRD components in vitro, and knockdown of MBD2 in the freshwater desmosponge, Ephydatia muelleri, promotes an abnormal growth phenotype.

Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.

No description available.

Peptídeos antimicrobianos (AMP) são importantes componentes do sistema imune de todos os organismos vivos. No plasma de Acanthoscurria rondoniae isolamos 15 AMP com similaridade com a hemocianina. As sete subunidades da hemocianina foram sequenciadas e sua estrutura tridimensional determinada. A rondonina, processada a partir de uma enzima do plasma em condições ácidas apresentou melhor atividade em pH ácido, sinergismo com gomesina, não citotóxico, não interagiu com membranas artificiais lipídicas, protegeu as células da infecção por vírus humanos de RNA e seu mecanismo de ação em leveduras está associado com material genético. Nossos resultados nos ajudam a entender porque aracnídeos sobreviveram por um longo tempo na escala evolutiva. E como as doenças infecciosas estão entre as principais causas de morte da população humana torna-se vital investir na busca de substâncias naturais ou sintéticas que exibam atividades antimicrobianas específicas e, acima de tudo, que as exerçam através de mecanismos de ação alternativos daqueles dos antibióticos disponíveis. / Antimicrobial peptides (AMP) are important components of the immune system of all living organisms. In the plasma of Acanthoscurria rondoniae we isolated 15 AMP with similarity to hemocyanin. The seven subunits hemocyanin were sequenced and determined its three-dimensional structure. The rondonin, processed from a plasma enzyme under acidic conditions showed best activity at acid pH, synergism with gomesin, non-cytotoxic, does not interacted with lipid artificial membrane, protected the cells from infection by human virus RNA and its mechanism of action in yeast is associated with genetic material. Our results help us understand why arachnids have survived for a long time on the evolutionary scale. And how infectious diseases are among the leading causes of death in human population becomes vital to invest in the search for natural or synthetic substances that exhibit specific antimicrobial activities and, above all, that engage through alternative mechanisms of action of these antibiotics available.

Caenorhabditis elegans optionally enter into a dauer diapause phase that results in a prolonged life in a semi-dormant state. Entry into and recovery from dauer diapause includes many physical changes in body structure, physiology, and gene expression. Entry into dauer diapause is regulated by several signaling pathways including transforming growth factor (TGF-β). Autophagy plays an important role in dauer formation and recover. During dauer transformation autophagy is up-regulated and may play a role in remodeling the molecular structure for long term survival during dauer diapause. This research helps determine the role of autophagy in dauer development and recovery mediated through the TGF-β signaling pathway. This research also determines in which tissue autophagy is necessary for dauer formation and recovery through TGF-β signaling. This research is shedding light on the function of autophagy in the TGF-β signaling pathway, both processes of which have been linked to tumorigenesis, heart disease and cancer. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

我們假設，將scleraxis (Scx)基因轉導入肌腱來源的幹細胞（TDSC），製成TDSC-Scx細胞系。TDSC-Scx會促進肌腱修復。本研究的目的在於探索Scx促進TDSC成肌腱分化的作用，以及TDSC-Scx對肌腱修復的促進作用。 / 使用慢病毒載體將Scx轉導入TDSCs，不含Scx的空載作為對照也轉導入TDSCs，用載體上帶有的抗性基因，殺稻瘟菌素對細胞進行篩選。分別建成TDSC-Scx和TDSC-Mock細胞系。Scx 的表達分別用定量PCR以及免疫螢光在mRNA和蛋白水準進行鑒定。TDSC-Scx成肌腱，成軟骨和成骨方向的分化能力用定量PCR檢驗。用大鼠臏腱視窗損傷模型進行了細胞移植試驗，測試TDSC-Scx對肌腱損傷的修復作用。實驗分為三組：（1）支架組，（2）空載體組，（3）Scx組。在細胞移植後的第二，四，八周，收集正在修復中的肌腱樣品，進行植入細胞的存留狀態，鈣化，組織學和生物力學測驗。 / TDSC-Scx比TDSC-Mock有更強的成肌腱分化能力。但是，在成軟骨-成骨分化方面，沒有結論。在動物試驗，植入的細胞在第二周仍然可見，但自第四周起，就不見了。在第八周，各組均有個別樣品輕微異位鈣化，但各組別間並無顯著差異。在早期，TDSC-Scx組比空載體組和支架組合得來更好的修復肌腱的能力。 / TDSC-Scx可能促進肌腱損傷的早期修復。 / We hypothesized that transduction of tendon-derived stem cell (TDSC) with scleraxis (Scx) might promote its tenogenic differentiation and promote better tendon repair compared to TDSC without Scx transduction. This study thus aimed to investigate the effect of Scx transduction on the tenogenic differentiation of TDSC and the effect of the resulting cell line in the promotion of tendon repair. / TDSCs were transduced with lentivirus-mediated Scx or empty vector and selected by blasticidin. The mRNA and protein expression of Scx were checked by qRT-PCR and Immuno fluorescence, respectively. The expression of different lineage markers were examined by qRT-PCR. A rat patellar tendon window injury model was used. The operated rats were divided into 3 groups: (1) scaffold-only group, (2) TDSC-Mock group and (3) TDSC-Scx group. At week 2, 4 and 8 post-transplantation, the repaired patellar tendon was harvested for ex vivo fluorescent imaging, vivaCT imaging, histology, or biomechanical test. / TDSC-Scx consistently showed higher expression of tendon-related markers compared to TDSC-Mock. However, the effect of Scx transduction on the expression of chondro-osteogenic markers was less conclusive. The transplanted TDSCs could be detected in the window wound at week 2 but not at week 4. Ectopic ossification was detected in some samples at week 8 but there was no difference among different groups. The TDSC-Scx group promoted early tendon repair histologically and biomechanically compared to the scaffold-only group and the TDSC-Mock group. / TDSC-Scx might be used for the promotion of early tendon repair. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tan, Chunlai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 66-70). / Abstracts also in Chinese. / Chapter Thesis/Assessment Committee --- p.i / Acknowledgment --- p.ii / Publication --- p.vi / Abstract --- p.vii / 摘要 --- p.viii / Chapter Chapter 1 --- Tendon injury and tendon tissue engineering --- p.1 / Chapter 1.1 --- Anatomy of tendon --- p.1 / Chapter 1.2 --- Epidemiology of tendon injury --- p.3 / Chapter 1.3 --- Process and problems of tendon healing --- p.4 / Chapter 1.4 --- Current treatment and cell-based therapy for tendon repair --- p.5 / Chapter 1.5 --- Transcriptional factor Scleraxis and tendon --- p.8 / Chapter 1.5.1 --- Helix-loop-helix (HLH) and bHLH proteins --- p.8 / Chapter 1.5.2 --- Scleraxis --- p.9 / Chapter 1.6 --- Research focus and implications --- p.11 / Chapter 1.7 --- Hypotheses and objectives of this study --- p.12 / Chapter 1.8 --- Clinical significance --- p.13 / Chapter Chapter 2 --- Materials and Methods --- p.14 / Chapter 2.1 --- Study Design --- p.14 / Chapter 2.2 --- Establishment of TDSC-Scx cell line --- p.15 / Chapter 2.2.1 --- Isolation of TDSC and cell culture --- p.15 / Chapter 2.2.2 --- Establishment of TDSC-Scx cell line --- p.17 / Chapter 2.2.2.1 --- Construction of plasmid --- p.17 / Chapter 2.2.2.2 --- Transfection --- p.23 / Chapter 2.2.2.3 --- Infection --- p.24 / Chapter 2.2.2.4 --- Selection --- p.24 / Chapter 2.2.2.5 --- Characterization of TDSC-Scx and TDSC-Mock --- p.25 / Chapter 2.2.2.5.1 --- qRT-PCR --- p.25 / Chapter 2.2.2.5.2 --- Immune-fluorescent (IF) --- p.26 / Chapter 2.2.2.6 --- Lineage marker expression --- p.26 / Chapter 2.2.3 --- Data analysis --- p.29 / Chapter 2.3 --- The effect of TDSC-Scx on healing in a patellar tendon window injury model --- p.30 / Chapter 2.3.1 --- Animal surgery --- p.30 / Chapter 2.3.2 --- Ex Vivo Fluorescence Imaging --- p.32 / Chapter 2.3.3 --- vivaCT --- p.33 / Chapter 2.3.4 --- Histology --- p.33 / Chapter 2.3.5 --- Biomechanical test --- p.35 / Chapter 2.3.6 --- Data analysis --- p.37 / Chapter Chapter 3 --- Results --- p.38 / Chapter 3.1 --- Generation of TDSC-Scx and TDSC-Mock cell lines --- p.38 / Chapter 3.1.1 --- Plasmid --- p.38 / Chapter 3.1.2 --- Cell morphology --- p.38 / Chapter 3.1.3 --- Expression of Scx --- p.38 / Chapter 3.1.4 --- Expression of chondro-/osteo-/tenogenic markers --- p.39 / Chapter 3.2 --- The healing effect of TDSC-Scx on a patella tendon window injury model --- p.40 / Chapter 3.2.1 --- The fate of transplanted cells --- p.40 / Chapter 3.2.2 --- Ossification --- p.40 / Chapter 3.2.3 --- Histology --- p.40 / Chapter 3.2.3.1 --- Fiber arrangement --- p.41 / Chapter 3.2.3.2 --- Cellularity --- p.41 / Chapter 3.2.3.3 --- Cell alignment --- p.42 / Chapter 3.2.3.4 --- Cell rounding --- p.42 / Chapter 3.2.3.5 --- Vascularity --- p.43 / Chapter 3.2.3.6 --- Fiber structure --- p.43 / Chapter 3.2.3.7 --- Hyaline degeneration --- p.43 / Chapter 3.2.3.8 --- Inflammation --- p.44 / Chapter 3.2.3.9 --- Ossification --- p.44 / Chapter 3.2.4 --- Biomechanical properties --- p.44 / Chapter Chapter 4 --- Discussion --- p.55 / Chapter 4.1 --- In vitro --- p.55 / Chapter 4.1.1 --- Scx transduction did not lead to morphological change in TDSCs --- p.55 / Chapter 4.1.2 --- Scx transduction led to higher expression of Scx mRNA --- p.55 / Chapter 4.1.3 --- TDSC-Scx expressed higher levels of tenogenic markers --- p.56 / Chapter 4.2 --- In vivo --- p.58 / Chapter 4.2.1 --- The fate of transplanted cells --- p.58 / Chapter 4.2.2 --- Ossification was vague --- p.58 / Chapter 4.2.3 --- TDSC-Scx promoted tendon healing --- p.58 / Chapter 4.2.4 --- Biomechanical properties --- p.59 / Chapter 4.2.5 --- Clinical consideration --- p.60 / Chapter 4.3 --- Similar studies --- p.61 / Chapter Chapter 5 --- Limitations --- p.63 / Chapter 5.1 --- Direct Scx protein expression and function information unavailable --- p.63 / Chapter 5.2 --- Differentiation assay --- p.64 / Chapter Chapter 6 --- Conclusion --- p.65 / Reference --- p.66 / Appendix --- p.71

Background & aims. DNA methylation induced tumor suppressor gene silencing plays an important role in carcinogenesis. By using methylation-sensitive representational difference analysis, we identified paired box gene 5 (PAX5) being methylated in human cancer. PAX5 locates at human chromosome 9p13.2 and encodes a 391 amino acids transcription factor. However, the role of PAX5 in gastric cancer is still unclear. Hence, we analyzed its epigenetic inactivation, biological functions, and clinical implications in gastric cancer. / Conclusions. Our results demonstrated that PAX5 promoter methylation directly mediates its transcriptional silence and commonly occurs in gastric cancer. PAX5 gene can act as a functional tumor suppressor in gastric carcinogenesis by playing an important role in suppression of cell proliferation, migration, invasion, and induction of cell apoptosis. Detection of methylated PAX5 may be utilized as a biomarker for the prognosis of gastric cancer patients. / Methods. Methylation status of PAX5 promoter in gastric cancer cell lines and clinical samples was evaluated by methylation specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS). The effects of PAX5 re-expression in cancer cell lines were determined in proliferation, cell cycle, apoptosis, migration and invasion assays. Its in vivo tumorigenicity was investigated by injecting cancer cells with PAX5 expression vector subcutaneously into the dorsal flank of nude mice. Chromosome Immunoprecipitation (ChIP) and cDNA expression array were performed to reveal the molecular mechanism of the biological function of PAX5. / Results. PAX5 was silenced or down-regulated in seven out of eight of gastric cancer cell lines examined. A significant down-regulation was also detected in paired gastric tumors compared with their adjacent non-cancer tissues (n = 18, P = 0.0196). In contrast, PAX5 is broadly expressed in all kinds of normal adult and fetal tissues. The gene expression of PAX5 in the gastric cancer cell line is closely linked to the promoter hypermethylation status. In addition, the expression levels could be restored by exposure to demethylating agents 5-aza-21-deoxycytidine. Re-expression of PAX5 in AGS, BGC823 and HCT116 cancer cells reduced colony formation (P < 0.01) and cell viability (P < 0.05), arrested cell cycle in G0/G1 phase (P = 0.0055), induced cell apoptosis (P < 0.05), repressed cell migration and invasion (P = 0.0218) in vitro. It also inhibited tumor growth in nude mice (P < 0.05). The molecular basis of its function were investigated by cDNA expression array and demonstrated that ectopic expression of PAX5 up-regulated tumor suppressor gene P53, anti-proliferation gene P21, pro-apoptosis gene BAX, anti-invasion gene MTSS1 and TIMP1; and down-regulated anti-apoptosis gene BCL2, cell cycle regulator cyclinD1, migration related gene MET and MMP1. ChIP assay indicated that P53 and MET are direct transcriptional target of PAX5. Moreover, PAX5 hypermethylation was detected in 90% (145 of 161) of primary gastric cancers compared with 16% (3 of 19) of non-cancer tissues (P < 0.0001). After a median follow-up period of 15.4 months, multivariate analysis revealed that gastric cancer patients with PAX5 methylation had a significant poor overall survival compared with the unmethylated cases (P = 0.0201). / Li, Xiaoxing. / Advisers: Hsiang Fu Kung; Jun Yu. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lo, Chun Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 71-80). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.iv / List of figures --- p.vi / Abbreviations --- p.vii / Contents --- p.viii / Chapter Chapter One --- General Introduction --- p.Page / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Human papillomavirus infection at the larynx --- p.2 / Chapter 1.2.1 --- Biology of human papillomavirus --- p.4 / Chapter 1.2.2 --- HPV E6 protein --- p.5 / Chapter 1.2.3 --- HPV E7 protein --- p.7 / Chapter 1.3 --- Apoptosis --- p.9 / Chapter 1.3.1 --- Apoptosis signaling pathways --- p.11 / Chapter 1.4 --- Transcription factor: Nuclear factor -kB --- p.14 / Chapter 1.4.1 --- Overview of the NF-kB signaling pathway --- p.14 / Chapter 1.4.2 --- Regulation of NF-kB signaling --- p.16 / Chapter 1.4.3 --- Roles of NF-kB in cancers --- p.19 / Chapter 1.5 --- Pteris semipinnata L extract: ent-11 -hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) --- p.21 / Chapter 1.6 --- Objectives --- p.22 / Chapter Chapter Two --- Materials and Methods / Chapter 2.1 --- Cell culture --- p.24 / Chapter 2.2 --- Cell proliferation analysis --- p.24 / Chapter 2.3 --- Western Blotting --- p.26 / Chapter 2.3.1 --- Total protein extraction --- p.26 / Chapter 2.3.2 --- Nuclear and cytoplasmic protein extraction --- p.26 / Chapter 2.3.3 --- Quantification of protein concentration --- p.27 / Chapter 2.3.4 --- Sodium dodecyl sulfate - polyacylamide gel electrophoresis (SDS-PAGE) and protein transfer --- p.28 / Chapter 2.3.5 --- Immunoblotting --- p.29 / Chapter 2.4 --- NF-kB Luciferase Assay --- p.29 / Chapter 2.5 --- Annexin V apoptosis assay --- p.31 / Chapter 2.6 --- mRNA expression analyses --- p.33 / Chapter 2.6.1 --- RNA extraction --- p.33 / Chapter 2.6.2 --- Reverse Transcription --- p.33 / Chapter 2.6.3 --- Polymerase Chain Reaction --- p.34 / Chapter 2.7 --- Antibodies --- p.35 / Chapter Chapter Three --- Results / Chapter 3.1 --- "Anti-proliferation effect of 5F on laryngeal cancer cells UMSCC11A, UMSCC12 and HEp-2 cells" --- p.36 / Chapter 3.2 --- Suppression by 5F in HEp-2 of mRNA and protein expression levels in HPV18 E7 while the expression level of HPV18 E6 was not altered --- p.38 / Chapter 3.3 --- Quantification of 5F-induced apoptosis in laryngeal cancer cells by Annexin V assay --- p.40 / Chapter 3.4 --- Morphological changes in laryngeal cancer cells induced by 5F --- p.41 / Chapter 3.5 --- "Cleavage of poly (ADP-ribose) polymerase (PARP) and pro-caspase-3 induced by 5F in UMSCC11 A, UMSCC12 and HEp-2 cell lines" --- p.47 / Chapter 3.6 --- "Down-regulation of TNF-α-induced NF-kB subunit p65 and p50 nuclear translocations in UMSCC11 A, UMSCC12 and HEp-2 by 5F" --- p.47 / Chapter 3.7 --- Dose-dependent inhibition of 5F on NF-kB transcriptional activity measured by luciferase assay --- p.53 / Chapter 3.8 --- Partial inhibition of TNF-α induced kBα degradation by 5F in UMSCC11A but not in UMSCC12 and HEp-2 --- p.56 / Chapter 3.9 --- Cell proliferation inhibition and apoptosis induction by Bay (11-7082) in laryngeal cancer cells --- p.56 / Chapter 3.10 --- Differential basal nuclear translocation of p65 and p50 in laryngeal cancer cell lines --- p.57 / Chapter 3.11 --- 5F regulated NF-kB target gene expression --- p.58 / Chapter Chapter Four --- Discussions --- p.64 / Reference --- p.71 / Appendix / Appendix 1 Map of pLuc- NF-kB plasmid --- p.81

Os projetos genoma têm fornecido uma grande quantidade de informação sobre a arquitetura gênica e sobre a configuração física de suas respectivas regiões flanqueadoras (RF). Estas RF contêm informações com o potencial de auxiliar na elucidação de vários processos biológicos, como os mecanismos de expressão gênica e de sua regulação. Estes mecanismos são de extrema importância para a compreensão do correto funcionamento dos organismos e das patologias que os afetam. Uma parte significativa dos mecanismos de controle de expressão gênica atuam na fase transcricional. Na base destes mecanismos está o recrutamento de proteínas que se ligam às regiões promotoras da transcrição, as quais são segmentos específicos de DNA que podem estar localizados tanto próximos à região de início da transcrição (TSS) quanto a centenas ou até a milhares de pares de bases dela. Essas proteínas compõem a maquinaria transcricional e podem ativar ou inibir o processo de transcrição. Experimentalmente, os segmentos regulatórios podem ser identificadas utilizando métodos complexos de biologia molecular, tais como SELEX, ChiP-ChiP, ChIP-Seq, dentre outros. Uma estratégia alternativa aos métodos experimentais é a utilização de metodologias computacionais. Análises computacionais tendem a ser mais rápidas, baratas e flexíveis do que protocolos experimentais, além de poderem ser utilizadas em larga escala. Atualmente, os métodos computacionais disponíveis necessitam de informações experimentais para a definição de padrões globais de preferências de sequências de DNA para a ligação de fatores de transcrição (TFBS, em inglês transcription factor binding sites). Entretanto, esses métodos apresentam uma elevada taxa de falso positivos e, por vezes, apresentam também taxas significativas de falso negativos, além de serem limitados ao estudo de fatores de transcrição de espécies bem conhecidas, o que diminui a área de aplicação dos mesmos. Diante deste cenário, o uso de métodos computacionais que não necessitem da informação referente aos sítios de ligação, bem como os que utilizem parâmetros mais robustos de detecção dos resultados, em detrimento dos escores de pontuação provindos de alinhamentos, podem acrescentar uma sensível melhoria ao processos de predição de regiões regulatórias. Neste projeto, foi desenvolvido um novo modelo computacional (TFBSAnalyzer) para análise e identificação de TFBS em elementos regulatórios, que utiliza técnicas de modelagem molecular para a construção de complexos entre um fator de transcrição ancorado a estruturas de DNA com sequências variáveis de bases e, através de cálculos termodinâmicos de entalpia de ligação, determina uma função de pontuação baseada na energia de ligação e realiza a predição de sítios de ligação ao DNA para o fator de transcrição em análise. Esta abordagem foi testada com três fatores de transcrição como sistemas-modelo, pertencentes à família dos receptores nucleares, a saber: o receptor de estrógeno ER-alfa (Estrogen Receptor Alpha), o receptor de ácido retinoico RAR-beta (Retinoid Acid Receptor Beta) e o receptor X retinóico RXR (Retinoid X Receptor). Os modelos previstos computacionalmente foram comparados aos dados experimentais disponíveis para estes receptores nucleares, os quais apresentaram as seguintes taxas de FP/FN: 10%/0 para RAR-beta e RXR, 21%/6% para ER-alfa. Também simulamos um experimento de ChIP-seq do ER-alfa no genoma humano, cujos genes selecionados foram submetidos a uma análise de enriquecimento de fatores de transcrição curados experimentalmente, que fazem sua regulação, revelando que o receptor de estrógeno está realmente envolvido no processo. Para mostrar a aplicabilidade geral de nosso método, nós modelamos a distribuição de energia de ligação para o receptor NHR-28 isoforma a de Caenorhabditis elegans com DNA . Obtivemos distribuições de energia semelhantes àquelas encontradas para os NRs modelos, portanto seria possível aplicar o método para buscar possíveis TFBSs para este receptor no genoma de C. elegans. Os dados gerados e as metodologias desenvolvidas neste projeto devem acrescentar uma sensível melhoria aos processos de predição de regiões regulatórias e consequentemente auxiliar no entendimento dos mecanismos envolvidos no processo de expressão gênica e de sua regulação. / The genome projects have provided a lot of information about the genetic architecture, as well as on the physical configuration of their flanking regions (FR). These FR have the potential to aid in the elucidation of many biological processes, such as the mechanisms involved in gene expression and its regulation. These mechanisms are extremely important for undeerstanfind the correct functioning of organisms as well as the pathologies that affect them. A significant part of the control mechanisms of gene expression act during transcription. On the basis of this mechanisms is the recruitment of proteins that bind to promoter regions of transcription, which are specific segments of DNA that can be located either near the transcription start site or at hundreds or even thousands of base pairs away. These proteins form the transcription machinery, which can activate or inhibit the transcription process. The regulatory segments can be identified experimentally using complex methods of molecular biology, such as SELEX, ChIP-chip, ChIP-seq, among others. An alternative strategy to these experimental methods is the use of computational methodologies for predicting regulatory regions. Computational analysis tend to be faster, cheaper and more flexible than the experimental protocols, and can be used on a larger scale. Currently, the available computational methods require information previously obtained from experiments in order to define global standards of preference of DNA-Binding sequences for transcription factors (TFBS - Transcription Factor Binding Sites). However, these methods have a high rate of false positives and sometimes also have significant rates of false negatives, besides being limited to the study of transcription factors of well-known species, which decreases their application area. In this scenario, the use of computational methods that do not require previous information concerning the binding sites and use more robust parameters of results detection, instead of alignment scores, may add significant improvement to the processes of predicting regulatory regions. In this project, we developed a new computational model TFBSAnalyzer) for analysis and identification of regulatory elements using molecular modeling techniques for the construction of complexes between a transcription factor bound to specific DNA structures with variable sequences of bases and, by means of thermodynamic calculations of bond enthalpy, provides a scoring function based on the binding energy and predicts the DNA binding sites for the transcription factor in analysis. This approach was tested initially with three transcription factors as models, belonging to the nuclear receptor family, namely estrogen receptor ER-alpha (Estrogen Receptor Alpha), the retinoic acid receptor RAR-beta (Retinoid Acid Receptor Beta) and the retinoic X receptor RXR (Retinoid X Receptor). The computationally predicted models were compared to experimental data available for these nuclear receptors, and presented the following rates of FP/FN: 10%/0 for RAR-beta and RXR, 21%/6% for ER-alpha. We also simulated an experiment of ChIP-seq with ER-alpha with the human genome, where the selected genes were subjected to a transcription factor enrichment analysis, with curated information, revealing that the estrogen receptor is indeed involved in their regulation. To show that our method has a general applicability, we modeled the binding energy distribution for the NHR-28 receptor, isoform a, from Caenorhabditis elegans. The energy distributions obtained were similar to the ones obtained for the model NR, so it would be possible to use the method and search for possible TFBS in the C. elegans genome. The data generated and the methodologies developed in this project should add a significant improvement to the prediction processes of regulatory regions and, consequently, help to understand the mechanisms involved in the gene expression process and its regulation.