Orientador: Ney Lemke / Banca: Scheila de Ávila e Silva / Banca: José Luiz Rybarczyk Filho / Resumo: A transcrição realizada pela RNA polimerase (RNAP) é um processo cuidadosamente controlado no desenvolvimento e na manutenção das funções vitais dos organismos. O desenvolvimento de novas técnicas e equipamentos para seu estudo, como as técnicas de pinça ótica ou magnética, a microscopia de força atômica e a fiuorescência de molécula única, complementaram os resultados dos estudos bioquímicos tradicionais e nos levaram a um maior entendimento do processo. A ocorrência de "pausas" em sítios específicos durante o alongamento, já observadas na década de 80, passou a ser estudada com maior interesse devido a sua importância biológica: acredita-se que essas pausas assegurem que a transcrição e a tradução ocorram simultaneamente em bactérias, permitam o dobramento correto das estruturas secundárias e terciárias do R A, facilitem a ligação de reguladores de alongamento e precedam a etapa de terminação transcricional. Modelos teóricos baseados na estabilidade termodinâmica do complexo de alongamento transcricional foram bem sucedidos na previsão da cinética do alongamento. Seus resultados indicaram que a RNAP pode ser vista como um motor molecular e sua motilidade possui características do modelo de catraca browniana. Entretanto, esses modelos consideram a presença de apenas uma polimerase realizando a transcrição. Experimentos recentes mostraram que a ocorrência de colisões entre essas enzimas durante a transcrição múltipla de um mesmo gene altera seu comportamento. Baseados nesses resultados, propomos a generalização de um dos modelos estocásticos que consideram a sequência molde para o estudo desse fenômeno. Em nossa aproximação, colisões entre as moléculas RNAP modificam a taxa de ocorrência da transcrição. A implementação do modelo foi realizada em ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The transcription of the information encoded within the DNA to the RNA molecule is exquisitely controlled during the development of the organisms and to its vital functions and has as the protagonist the RNA polymerase enzyme (RNAP). The development of single-molecule techniques, such as the magnetic and optical tweezers, atomic-force microscopy and single-molecule uorescence, increased our understanding of the process, complementing traditional biochemical studies. The non-homogeneity of the RNAP movement due to the occurrence of \pauses" at speci c sites during elongation was revealed using electrophoresis gels. It is believed that these pauses ensure concurrency between transcription and translation in bacteria, allow the correct folding of RNA secondary and tertiary structures, facilitate the binding of regulating factors during elongation and preceding the transcriptional termination step. Theoretical models have been proposed to explain and predict the RNAP kinetics during the polymerization. Models based on the thermodynamic stability of the transcription elongation complex recover much of the kinetics and indicate that its movement has a Brownian ratchet mechanism. However, experiments showed that if more than one RNAP molecule initiate from the same promoter, their behavior changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAP molecules and predicts their cooperative behavior during multi-round transcription. The model generalizes a stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica and compared the results of the single and the multiple-molecule transcription with experimental... (Complete abstract click electronic access below) / Mestre

Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.

The cellular stress response (CSR) is one of the most highly conserved mechanisms among all organisms. Cellular stress can be defined as damage or the threat of damage to proteins, macromolecules and/or DNA. The response to damage can involve cell cycle regulation, protein chaperoning, DNA repair or, if macromolecular damage is too severe, apoptotic mechanisms can be initiated. This thesis details experiments that were designed to examine the cellular response to non-lethal environmental stressors at the protein level, using two fish species as study models. Two proteins that can cause cell cycle arrest and apoptosis mechanisms were examined. Expression of the CCAAT enhancer binding protein-delta (C/EBP-[delta]) was examined in the zebrafish, Danio rerio, exposed to acute, non-lethal hypoxic conditions. While C/EBP-[delta] was expressed constitutively in control individuals during all time points, exposure to hypoxic conditions did not have a consistent significant effect on C/EBP-[delta] expression (two-way ANOVA, P>0.05) in zebrafish white muscle tissue. In a second study, the expression of the growth arrest and DNA damage 45-alpha protein (gadd45-[alpha], a mediator of cell cycle arrest and perhaps apoptosis was examined in heat-stressed liver tissue of an extremely cold-adapted Antarctic fish, Trematomus bernacchii. Gadd45-[alpha] levels were higher in fish exposure to 2°C across all time points (one-way ANOVA; P

ABF -1 is a human class II bHLH transcription factor that is expressed predominantly in activated B cells and EBV immortalized cell lines. A portion of this study sought to characterize the homolog of ABF- l in Caenorhabditis e/egans. The nematode gene product, ceABF -1, is capable of forming heterodimers with E2A gene products and binding E box binding sites. HeLa cells transfected with ceABF-1 reveal that it is capable of blocking E2A mediated gene transcription. In order to maintain full repression capabilities, two conserved amino acid residues within helix I ofthe HLH domain are required. These results show a conserved mechanism of gene repression between invertebrates and vertebrates. This study also sought to analyze ABF-1 mediated regulation of both ld3 and cellular growth. Using a human ABF-1 stably transfected cell line, ID3 protein levels and transcript levels were shown to increase in response to overexpression of ABF-1 via western and northern blot, respectively. Flow cytometry analysis and Real-time PCR revealed that ABF-1 programs a slow down in the cell cycle, however this growth arrest is not mediated by ID3.

The vertebrate eye lens functions to focus light onto the retina to produce vision. The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a core of organelle free fiber cells. The lens develops and grows throughout life by the successive layering of lens fiber cells via their differentiation from lens epithelial cells. Lens developmental defect and damage to the lens are associated with cataract formation, an opacity of the lens that is a leading cause of visual impairment worldwide. The only treatment to date for cataract is by surgery. Elucidating those molecules and mechanisms that regulate the development and lifelong protection of the lens is critical toward the development of future therapies to prevent or treat cataract. To determine those molecules and mechanisms that may be important for these lens requirements we employed high-throughput RNA sequencing of microdissected differentiation statespecific lens cells to identify an extensive range of transcripts encoding proteins expressed by these functionally distinct cell types. Using this data, we identified differentiation state-specific molecules that regulate mitochondrial populations between lens epithelial cells that require the maintenance of a functional population of mitochondria and lens fiber cells that must eliminate their mitochondria for their maturation. In addition, we discovered a novel mechanism for how lens epithelial cells clear apoptotic cell debris that could arise from damage to the lens and found that UVlight likely compromises this system. Moreover, the data herein provide a framework to determine novel lens cell differentiation state-specific mechanisms. Future studies are required to determine the requirements of the identified molecules and mechanisms during lens development, lens defense against damage, and cataract formation. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

The assembly and maintenance of central synapses is a complex process, requiring myriad genes and their products. Highwire is a large gene containing a RING domain, characteristic of ubiquitin E3 ligases. Highwire has been shown to restrain axon growth and control synaptogenesis at a peripheral synapse. Here I examine the roles of Highwire at a central synapse in the adult Drosophila Giant Fiber System (GFS). Highwire is indeed necessary for proper axonal growth as well as synaptic transmission in the GFS. Differences arise between the central synapse and the neuromuscular junction (NMJ), where highwire was initially characterized : expresion from the postsynaptic cell can rescue highwire synaptic defects, which is not seen at the NMJ. In addition, a MAP kinase signaling pathway regulated by highwire at the NMJ has differing roles at a central synapse. Wallenda MAPK can rescue not only the highwire anatomical phenotype but also the defects seen in transmission. Another distinction is seen here : loss of function basket and Dfos enhance the highwire anatomical phenotype while expression of dominant negative basket and Dfos suppress the highwire phenotype. As a result we have compared the signaling pathway in flies and worms and found that the NMJ in the two organisms use a parallel pathway while the central synapse uses a distinct pathway. / by Kimberly Diane Rowland. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Transcription and translation of proteins are required for the consolidation of episodic memory. Arc, an effector immediate early gene, has been linked to synaptic plasticity following learning and memory. It is well established that the rodent hippocampus is essential for processing spatial memory, but its role in processing object memory is a point of contention. Using immunohistochemical techniques, hippocampal sections were stained for arc proteins in the CA1 region of the dorsal hippocampus in mice following two variations of the novel object recognition (NOR) task. Results suggest mice that acquired strong object memory showed significant hippocampal activation. In mice that acquired weak object memory, hippocampal activation was not significantly different from controls. Arc expression was also examined in other hippocampal sub-regions, as well as in the perirhinal cortex. These results suggest that the mice must acquire a threshold amount of object information before the hippocampal CA1 region is engaged. / Includes bibliography. / Thesis (M.A.)--Florida Atlantic University, 2015 / FAU Electronic Theses and Dissertations Collection

Thesis (Ph.D.)--University of Melbourne, The Walter and Eliza Hall Institute of Medical Research, The Division of Immunology and the Division of Molecular Medicine, Dept. of Medical Biology, Faculty of Medicine and Dentistry, 2009. / Typescript. Includes bibliographical references (p. 133-165)

Calcium is a crucial intracellular messenger controlling a plethora of important intracellular events. Elevated [Ca2+]i levels regulate numerous processes due to the versatility of the Ca2+ signaling in terms of speed, amplitude and spatia-temporal patterning. Depletion of intracellular calcium stores functions as a primary trigger for a message that is translated to the plasma membrane, resulting in the slow activation of plasma membrane Ca2+ influx channels, which allow entry of external calcium. Since these channels depend upon the state of filling of the intracellular Ca2+stores, these influx channels are called store-operated channels (SOCs). It is unclear how empty intracellular stores signal activation of plasma membrane capacitative calcium entry (CCE). In order to bridge the gaps in our understanding of calcium's role in T cell regulation, this project was designed to look at the effects of various pharmacological regulators of T cell calcium stores. At the start, the effort was directed at the regulation of the microsomal calcium A TPases, considering these are perhaps the most essential mediators of intracellular calcium storage. Thapsigargin (TG) and cyclopiazonic acid (CPA) were shown to be the most potent inhibitors of the Ca2+ -ATPase, also a new Ca2+ regulator aaptamine was shown to exert more modest inhibition of the Ca2+ pump. We also characterized a novel compound, gingerol, findings its actions are to stimulate Ca2+ pumps. Cell growth assays revealed an important role for ryanodine receptors (RyRs) in regulating T cell growth. RyR activators CMC and PCB95 dramatically altered T cell growth patterns leading to significantly reduced cell viability. In contrast RyR antagonist dantrolene appeared to induce growth arrest in that cell proliferation was curtailed, yet cells remained viable. Cell viability studies revealed that the Ca2+ pump regulators TG and aaptamine were also observed to reduce cell growth rates, presumably as a result of their ability to deplete Ca2+ stores (100 nM TG was able to decrease cell viability by 90% within 24 hrs of exposure). PCB95 was able to decrease cell viability by 50% within 24 of hrs exposure and CMC decreased cell viability by 75% within 24 hrs and further over a 48 hr period. 2-APB and aaptamine were cytotoxic at higher doses. The inositol 1 ,4,5 -trisphosphate receptor (IP3R) pathway was also found to be critically linked to T cell growth control. We observed that the IP3R modulator, 2- aminoethoxydiphenyl borate (2-APB) induced antigen-like Ca2+ spike that correlates with suppression of T cell growth rates. In this study we have identified two novel T cell Ca2+ store regulators, aaptamine and gingerol. We also find that Ca2+ stores are indeed sensitively linked to T cell growth regulation. Depletion of Ca2+ stores with SERCA inhibitors as well as both RyR and IP3R activators profoundly suppress T cell proliferation most likely via activation of apoptosis.