Philosophiae Doctor - PhD / The use of ontologies in the mapping of gene expression events provides an effective and comparable method to determine the expression profile of an entire genome across a large collection of experiments derived from different expression sources. In this dissertation I describe the development of the developmental human and mouse eVOC ontologies and demonstrate the ontologies by identifying genes showing a bias for developmental brain expression in human and mouse, identifying transcription factor complexes, and exploring the mouse orthologs of human cancer/testis genes.Model organisms represent an important resource for understanding the fundamental aspects of mammalian biology. Mapping of biological phenomena between model organisms is complex and if it is to be meaningful, a simplified representation can be a powerful means for comparison. The implementation of the ontologies has been illustrated here in two ways.Firstly, the ontologies have been used to illustrate methods to determine clusters of genes showing tissue-restricted expression in humans. The identification of tissue restricted genes within an organism serves as an indication of the finetuning in the regulation of gene expression in a given tissue. Secondly, due to the differences in human and mouse gene expression on a temporal and spatial level, the ontologies were used to identify mouse orthologs of human cancer/testis genes showing cancer/testis characteristics. With the use of model systems such as mouse in the development of gene-targeted drugs in the treatment of disease, it is important to establish that the expression characteristics and profiles of a drug target in the model system is representative of the characteristics of the target in the system for which it is intended.

The present thesis discusses how gene regulation by transforming growth factor β (TGFβ) family cytokines is affected by post-translational modifications of different transcription factors. The thesis also focuses on gene regulation by transcription factors involved in TGFβ signaling. The importance of the poly ADP-ribose polymerase (PARP) family in controlling gene expression in response to TGFβ and bone morphogenetic protein (BMP) is analyzed first. PARP2, along with PARP1, ADP-ribosylates Smad2 and Smad3, the signaling mediators of TGFβ. On the other hand, poly ADP-ribose glycohydrolase (PARG) removes the ADP-ribose from Smad2/3 and antagonizes PARP1 and PARP2. ADP-ribosylation of Smads in turn affects their DNA binding capacity. We then illustrate how PARP1 and PARG can regulate gene expression in response to BMP that signals via Smad1, 5. Over-expression of PARP1 suppressed the transcriptional activity of Smad1/5. Knockdown of PARP1 or over-expression of PARG enhanced the transcriptional activity of BMP-Smads on target genes. Hence our data suggest that ADP-ribosylation of Smad proteins controls both TGFβ and BMP signaling.  I then focus on elucidating novel genes that are regulated by ZEB1 and Snail1, two key transcriptional factors in TGFβ signaling, known for their ability to induce EMT and cancer metastasis. Chromatin immunoprecipitation-sequencing (ChIP-seq) and targeted whole genome transcriptomics in triple negative breast cancer cells were used, to find binding regions and the functional impact of ZEB1 and Snail1 throughout the genome. ZEB1 binds to the regulatory sequences of a wide range of genes, not only related to cell invasion, pointing to new functions of ZEB1. On the other hand, Snail1 regulated only a few genes, especially related to signal transduction and cellular movement. Further functional analysis revealed that ZEB1 could regulate the anchorage-independent growth of the triple negative breast cancer cells, whereas Snail1 could regulate the expression of BMP6 in these cells. We have therefore elucidated novel functional roles of the two transcription factors, Snail1 and ZEB1 in triple negative breast cancer cells.

In Saccharomyces cerevisiae, telomeric and HM silencing requires the histone deacetylase Sir2 and the chromatin binding proteins Sir3 and Sir4, which interact to form the SIR complex. Silent chromatin formation begins with a nucleation step, followed by spreading of Sir proteins along chromatin. Overexpression of Sir3 extends silent chromatin domains, however the role of Sir protein interactions within silent chromatin extensions remains unknown. Here, we generated the Sir3 mutant, Sir3-4A, which cannot interact with Sir4 but is capable of forming silent chromatin extensions when overexpressed. Within extended silent domains, Sir2 and Sir4 enrichments are similar whether Sir3 or Sir3-4A is overexpressed, suggesting that silent chromatin extensions require Sir4 but not the interaction between Sir3 and Sir4. Tethering Sir3-4A at an HMR silencer cannot nucleate silencing in the absence of Sir3, suggesting that in addition to Sir3 recruitment, the Sir3-Sir4 interaction has at least one other function during silent chromatin nucleation.

Visando a construção de três biossensores bacterianos para os metais mercúrio, arsênio e chumbo, Cupriavidus metallidurans CH34 foi escolhida para obtenção dos fragmentos de DNA (por PCR) correspondentes aos operons mer, ars e pbr. Os fragmentos foram inseridos à montante do gene EGFP, no plasmídeo pBB-EGFP, obtendo três novos plasmídeos: pGHg, pGAs e pGPb. Estes foram clonados em C. metallidurans CH34 e Escherichia coli DH5<font face=\"Symbol\">&#945;. As linhagens recombinantes foram submetidas a várias situações de cultivo e diferentes intensidades de fluorescência, detectadas em microscopia e quantificadas por citometria de fluxo. As linhagens recombinantes C. metallidurans CH34 / pGHg, E. coli DH5<font face=\"Symbol\">&#945; / pGHg, C. metallidurans CH34 / pGAs e C. metallidurans CH34 / pGPb mostraram-se eficazes e, consequentemente, poderão ser utilizadas em rápidos diagnósticos de amostras contaminadas por mercúrio, arsênio e chumbo. / Aiming at the construction of three bacterial biosensors for metals mercury, arsenic and lead, Cupriavidus metallidurans CH34 was chosen to obtain the DNA fragments (by PCR) corresponding to operons mer, ars and pbr. The fragments were inserted upstream of the EGFP gene, in plasmid pBB-EGFP, getting three new plasmids: pGHg, pGAs and pGPb. They were cloned in C. metallidurans CH34 and Escherichia coli DH5<font face=\"Symbol\">&#945;. The recombinant strains were subjected to various conditions of cultivation. Different intensities of fluorescence were detected microscopy and quantified by flow cytometry. The recombinant strains C. metallidurans CH34 / pGHg, E. coli DH5<font face=\"Symbol\">&#945; / pGHg, C. metallidurans CH34 / pGAs and C. metallidurans CH34 / pGPb were effective and therefore may be used in rapid diagnosis of samples contaminated by mercury, arsenic and lead.

CspD é uma das quatro proteínas de choque frio de Caulobacter crescentus, sendo maior que as outras CSPs por possuir dois domínios de choque frio, e tem seu papel na célula ainda desconhecido. O objetivo deste trabalho foi identificar e caracterizar os fatores in cis e in trans envolvidos na regulação da expressão do gene cspD em C. crescentus. Neste trabalho foi visto que a expressão de cspD é induzida pela carência de glicose no meio, mas não pela carência de nitrogênio. Esta indução é dependente do sinalizador ppGpp, indicando que ppGpp está envolvido na sinalização de carência de carbono. Um regulador de resposta de um sistema de dois componentes (SpdR) foi identificado como responsável pela indução em fase estacionária, sendo fosforilado pela histidina quinase cognata SpdS. Através de ensaios de EMSA e DNAseI footprinting pudemos verificar que a proteína SpdR se liga em uma sequência repetida invertida no promotor de cspD, e que essa ligação é dependente da fosforilação no resíduo de aspartato na posição 64 da proteína. Foi construído um mutante nulo do gene SpdR e este foi testado quanto à resistência a estresse oxidativo causado por H2O2 e viabilidade em fase estacionária; o mutante não apresentou diferença na resposta em relação à linhagem selvagem. / CspD is one of the four cold shock proteins of Caulobacter crescentus being larger than the other ones by possessing two cold shock domains, and its cellular role is still unknown. The aim of this work was to identify and characterize factors in cis and in trans involved in regulation of cspD expression in C. crescentus. In this work we determined that cspD expression is induced by glucose starvation but not by nitrogen starvation. This induction is dependent on the signalling molecule ppGpp, indicating that it is involved in carbon starvation signaling. A response regulator of a two-component system (SpdR) was identified as responsible for stationary phase induction, being phosphorylated by the cognate histidine kinase SpdS. EMSA and DNAseI footprinting assays showed that the SpdR protein binds to an inverted repeat at the cspD promoter, and that this binding is dependent on phosphorylation at the aspartate residue at position 64. A null spdR mutant strain was constructed and its resistance to H2O2 and stationary phase viability were determined, however the mutant showed no difference in response when compared to the wild type.

Submitted by Michele Mologni (mologni@unoeste.br) on 2017-06-13T13:30:56Z No. of bitstreams: 1 Viviane Cacefo.pdf: 1556310 bytes, checksum: 3f9d8dc2ff4a08b8acc2ec7e1a731117 (MD5) / Made available in DSpace on 2017-06-13T13:30:56Z (GMT). No. of bitstreams: 1 Viviane Cacefo.pdf: 1556310 bytes, checksum: 3f9d8dc2ff4a08b8acc2ec7e1a731117 (MD5) Previous issue date: 2017-02-17 / C4 plants have been classified into three groups based on the enzymes used to decarboxylate C4 acids in the bundle sheath: NADP-ME, NAD-ME and PEPCK subtypes. Numerous molecular, biochemical and physiological evidences indicate that C4 plants could exhibit a certain degree of flexibility in the use of the three established decarboxylation mechanisms, depending on environmental factors. In this context, the objective of this work was to evaluate the modulation of the pathways of decarboxylation of the C4 photosynthesis in sugarcane (NADP-ME specie) under water deficit. The experiment was realized with two genotypes - RB92579 (tolerant to water deficit) and SP80-3280 (susceptible to water deficit) submitted to four treatments: control (normal conditions of water supply), moderate stress (-1,5 to -1,8 MPa), severe stress (below -2,0 MPa) and recovery (48 hours after rehydration). Leaf water potential, leaf gas exchange and biomass production were measured for the physiological characterization of the plants. Changes in the transcriptional responses of genes encoding C4-cycle enzymes (NADP-ME, NAD-ME, PEPCK, AspAT, AlaAT, PEPC, NADP-MDH and PPDK) and the activities of the enzymes NADP-ME, NAD-ME, PEPCK, AspAT and AlaAT were analyzed by RT-qPCR and spectrophotometry, respectively. Under water deficit conditions the genotypes SP80-3280 was more sensitive to drought stress in terms of increased loss of above ground biomass and lower leaf water potential. It also showed less capacity of photosynthetic recovery following rehydration compared to the tolerant genotypes RB92579. The analysis of transcriptionals and of the activities of enzymes involved in the C4 photosynthetic pathway showed that sugarcane uses the PEPCK pathway as a decarboxylation mechanism in addition to the NADP-ME, which was more evident under water deficit conditions for both the drought tolerant and susceptible genotypes. Finally, the results here obtained, together with the existing information, do not support the established classification of sugarcane (Saccharum spp.) as a classical NADP-ME C4 subtype, but instead should be considered as a NADP-ME + PEPCK species. / As plantas C4 são classificadas em três grupos de acordo com as enzimas utilizadas para descarboxilação de ácidos C4 na bainha dos feixes vasculares: subtipos NADP-ME, NAD-ME e PEPCK. Inúmeras evidências moleculares, bioquímicas e fisiológicas indicam que plantas C4 podem exibir certo nível de flexibilidade na utilização dos três mecanismos de descarboxilação, dependendo de fatores ambientais. Neste contexto, o objetivo deste trabalho foi avaliar a modulação das vias de descarboxilação da fotossíntese C4 em cana-de-açúcar (espécie NADP-ME) sob déficit hídrico. O experimento foi realizado com dois genótipos - RB92579 (tolerante ao déficit hídrico) e SP80-3280 (suscetível ao déficit hídrico) submetidos a quatro tratamentos: controle (condições normais de suprimento de água), estresse moderado (-1,5 a -1,8 MPa), estresse severo (abaixo de -2,0 MPa) e recuperação (48 hrs após a reidratação). Foram realizadas análises de potencial de água foliar, trocas gasosas foliares e biomassa para caracterização fisiológica das plantas. As alterações nas respostas transcricionais dos genes codificadores de enzimas do ciclo C4 (NADP-ME, NAD-ME, PEPCK, AspAT, AlaAT, PEPC, NADP-MDH e PPDK) e atividades das enzimas NADP-ME, NAD-ME, PEPCK, AspAT e AlaAT, foram analisadas por RT-qPCR e espectrofotometria, respectivamente. Em condições de déficit hídrico, o genótipo SP80-3280 foi mais sensível ao estresse por seca em termos de aumento da perda de biomassa e menor potencial de água foliar. Também mostrou menor capacidade de recuperação fotossintética após reidratação do que o genótipo tolerante RB92579. As análises transcricionais e das atividades das enzimas envolvidas na via fotossintética C4 mostraram que a cana-de-açúcar utiliza a via PEPCK como mecanismo de descarboxilação, além da NADP-ME, mais evidente em condições de déficit hídrico tanto para genótipos tolerantes à seca como para suscetíveis. Finalmente, os resultados aqui obtidos, juntamente com a informação existente, não suportam a classificação estabelecida da cana-de-açúcar (Saccharum spp.) como um subtipo C4 NADP-ME clássico, mas considerá-la como uma espécie NADP-ME + PEPCK.

No description available.

QepA is a plasmid-mediated efflux pump found in some strains of Escherichia coli, in which it significantly elevates the resistance against quinolones. The protein has similarities with 14-TMS major facilitator superfamily transporters and is situated in the inner membrane of the bacteria. It was acquired by horizontal gene transfer and integrated into a now inactivated class 1 integron, also harbouring several other antibiotic resistance genes such as rmtB and blaTEM-1. QepA alone is not sufficient to raise the resistance level over the clinical breakpoint and is in clinical isolates therefore associated with other quinolone antibiotic resistance genes or quinolone target point mutations. The mechanisms regulating qepA expression are not yet understood. Therefore, in this study the qepA gene was amplified from an E. coli clinical isolate and, together with its upstream promotor sequence, was inserted into the E. coli chromosome. It was shown that qepA gene expression can be induced by exposure to 0.5-fold MIC concentrations of ciprofloxacin, trimethoprim and other DNA damaging antimicrobials. The deletion of a LexA binding site situated after a PcW promotor, which was predicted to drive qepA expression, did not alter this induction behaviour. Nested deletions of up to 200 nts downstream sequence of the PcW promotor, led to the identification of a sequence region required for expression induction. This study showed that qepA expression is induced by environmental factors leading to DNA damage and further identified a previously unknown DNA sequence required for expression regulation.

Corticotropin releasing factor (CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter (NET) in vitro. SK-N-BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [3H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status.

Glucocorticoids affect cellular and molecular events in brains by modulating the expression of many genes during stress. In the present study, we examined the regulatory effect of corticosterone on the expression and function of the norepinephrine transporter (NET) in vitro. The results show that exposure of SK-N-BE(2)C cells to corticosterone for 14 days significantly increased mRNA (up to 43%) and protein (up to 71%) levels of NET in the concentration- dependent manner. Longer exposure (21 days) resulted in greater increases in the levels of mRNAs (up to about 160%) and proteins (up to about 250%) of the NET. The up-regulatory effect of corticosterone on NET expression lasted a persistent period after cessation of exposure. Associated with the corticosterone-induced enhancement in NET expression, there was a parallel increase in the uptake of [3H]norepinephrine by SK-N-BE(2)C cells. Increased NET expression and function were abolished after exposure of cells to corticosterone in combination with mifepristone or spironolactone, two specific antagonists of corticosteroid receptors. This is consistent with the hypothesis that corticosterone-induced NET up-regulation is mediated by corticosteroid receptors. Nevertheless, there was no synergistic effect for a combination of both corticosteroid receptor antagonists. A similar up-regulation of NET protein levels was also observed after exposing PC12 cells to corticosterone. The present findings demonstrate that corticosterone up-regulates the expression and function of NET in vitro, indicating the action of corticosterone on the noradrenergic phenotype may play an important role in the correlation between stress and the development of depression.