Orientador: Licio Augusto Velloso / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T01:56:53Z (GMT). No. of bitstreams: 1 Morari_Joseane_M.pdf: 1671782 bytes, checksum: ebca1c2f8d19c88433316cd9134da3a3 (MD5) Previous issue date: 2009 / Resumo: Insuficiência hepática causada por cirrose decorrente da progressão da esteato-hepatite não alcoólica é hoje uma das principais indicações para transplante hepático no mundo. A ingestão de dietas ricas em lípides é uma das principais causas de esteato-hepatite não alcoólica, e, uma vez estabelecida, poderá ou não evoluir para cirrose, dependendo da ativação de uma resposta inflamatória local. Nem todos os indivíduos que desenvolvem esteatose progridem para a esteato-hepatite. Acredita-se que, dentre os fatores que desempenham papel protetor na progressão para a esteato-hepatite e, por conseguinte, para a cirrose, encontra-se o equilíbrio entre a expressão de citocinas pró- e anti-inflamatórias no fígado. Níveis locais elevados da citocina anti-inflamatória IL-10 reduzem a resistência hepática à insulina e diminuem o risco de desenvolvimento de esteato-hepatite. Em estudos recentes observamos que a inibição da atividade da IL-10 em um modelo animal de esteato-hepatite induzida por dieta contribui para a piora da disfunção hepática. Por outro lado, a redução da expressão do co-ativador de transcrição gênica PGC-1a reverte a esteatose hepática induzida por dieta. Tal reversão é acompanhada pela modulação da expressão de IL-10 e de seu receptor, IL-10R. No presente estudo avaliamos se o PGC-1a?interage com dois fatores de transcrição que participam do controle da expressão de IL-10, c-Maf e NF?Bp50. Para tal utilizamos as técnicas de imunoblot, imunoprecipitação, imunocitoquímica, histologia convencional e imunoprecipitação de cromatina. Os nossos resultados revelam que o PGC-1a?se associa a ambos, c-Maf e p50, após estímulo por ácidos graxos saturados e insaturados. Tal associação ocorre em paralelo ao aumento da expressão de IL-10 e se acompanha da migração de todas as três proteínas de uma localização preferencialmente citosólica para o interior do núcleo de hepatócitos. Além disso, sob estímulo por ácidos graxos, as três proteínas se ligam ao DNA do promotor do gene da IL-10, numa região localizada entre as bases -493 e -254, antes do códon iniciador do gene. Pelo menos a associação de PGC-1a?com p-50 e a indução da expressão de IL-10 por ácidos graxos pode ser inibida pela pré-exposição de hepatócitos ao ácido acetil salicílico, um inibidor da enzima ativadora do NF?B. Portanto, PGC-1a?interage com dois fatores de transcrição e com o DNA da região promotora do gene da IL-10 e emerge como potencial regulador da expressão de IL-10 no fígado. / Abstract: Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis. Reducing IL-10 expression increases pro-inflammatory activity in the steatotic liver and worsens insulin resistance. Because in diet-induced steatosis the transcriptional co-activator PGC-1a plays a central role in the dysfunctional hepatocytic activity, we hypothesized that at least part of PGC-1a activity could be mediated by its effect on the transcriptional control of IL-10 expression. Here, we used immunoblot, real-time PCR, immunocytochemistry and ChIP assay to investigate the role of PGC-1a in the control of IL-10 expression in hepatic cells. First, we show that in the intact steatotic liver, the expressions of IL-10 and PGC-1a are increased. Inhibiting PGC-1a expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes the treatment with saturated and unsaturated fatty acids, increase IL-10 expression. This is accompanied by increased association of PGC-1a with c-Maf and p50-NF?B, two transcription factors known to modulate IL-10 expression. In addition, following fatty acid treatment PGC-1a, c-Maf and p50- NF?B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region. Inhibiting NF?B activation with salicylate reduces IL-10 expression and PGC-1a association with p50-NF?B. Thus, PGC-1a emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver. / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Abstract Cardiac hypertrophy and remodelling are mechanisms of adaptation to increased workload and acute injuries of the heart. In the long-term, these initially beneficial mechanisms become detrimental and ultimately lead to the development of heart failure. The molecular determinant of myocardial remodelling and heart failure is altered intracellular signal transduction and a modified gene expression pattern in the individual cardiomyocyte. This study was aimed at characterising the changes in mitogen-activated protein kinases (MAPKs) and their nuclear effector, GATA-4, and their functional significance and interaction in experimental models of increased cardiac workload and remodelling. To study the effects of increased cardiac workload on MAPKs and GATA-4, isolated perfused rat hearts were subjected to increased left ventricular wall stress and their activities were determined using western blot and gel mobility shift assays. Left ventricular wall stress rapidly activated the DNA binding of GATA-4, and this activation was abolished in the presence of endothelin-1 (ET-1) and angiotensin II receptor antagonists. Furthermore, the activation of GATA-4 DNA binding was significantly attenuated by p38 MAPK and extracellular signal regulated kinase (ERK) inhibition. To gain further insights into the role of p38 MAPK as a regulator of cardiac transcription factors, gene expression and remodelling, a gene transfer protocol of increased p38 MAPK activity was established. Direct adenovirus-mediated gene transfer of wild-type p38α and constitutively active upstream kinase mitogen-activated kinase kinase 3b (MKK3b) selectively increased p38 MAPK activity in the left ventricle, which was followed by up-regulation of cardiac gene expression, myocardial inflammation and fibrosis. Using a DNA microarray approach, the cardiac target genes of p38 MAPK were identified, including several cell division, inflammation and signal transduction-associated genes. Furthermore, p38 MAPK over-expression was found to increase the DNA binding activities of several transcription factors, including GATA-4. Finally, the functional role of p38 MAPK was determined using adenovirus-mediated gene transfer in an experimental model of myocardial infarction. Post-infarction remodelling was characterised by a sustained down-regulation of p38 MAPK, while rescue of p38 MAPK activity attenuated post-infarction remodelling through anti-apoptotic and angiogenic mechanisms. These results indicate that p38 MAPK is a key regulator of GATA-4 transcription factor and cardiac gene expression during left ventricular wall stress and remodelling. They demonstrate that p38 MAPK, being cardioprotective in the infarcted heart but promoting inflammation and fibrosis in the normal heart, has a unique dual role in the myocardium.

Abstract Cardiac pathologies, including ventricular hypertrophy, are the primary cause of death in industrialized countries. Cardiac hypertrophy is often the consequence of work overload on the heart and characterizes several cardiovascular diseases, including atherosclerosis and hypertension. Cardiac hypertrophy is accompanied by genetic reprogramming characterized by the reexpression of several embryonic and growth response genes. Two of these genes encode A- and B-type natriuretic peptides (ANP and BNP), two cardiac-specific hormones secreted by myocytes, which play an important role in blood pressure regulation. The aim of the present study was to study the effect of acute pressure overload on BNP gene expression in the hearts of normal and hypertensive rats and then to examine the role of a passerine factor, angiotensin II (Ang II), on volume and pressure overload -induced ANP and BNP secretion and synthesis. Further, the aim was to characterize elements on the BNP promoter mediating hemodynamic stress in vivo. BNP gene expression was studied in conscious spontaneously hypertensive (SHR) rats and together with ANP in two hypertensive, ream Transgenic rat models. The increased workload of the heart was produced by the infusion of vasopressin (AVP), phenylephrine (PHE) or bolus saline infusion. The increased workload caused rapid increases in cardiac BNP mRNA levels. Daring both AVP and PHE infusions, substantial increases in ventricular BNP mRNA levels were already evident after I h, and peak levels of BNP mRNA were reached at 4 h. Transgenic rats carrying one extra mouse renin gene showed impaired secretion and synthesis of ANP and BNP, while double transgenic rats carrying both human angiotensinogen and human renin genes showed augmentation of left atrial, but not ventricular BNP gene expression in response ta acute pressure overload. To characterize the elements mediating hemodynamic stress, bi-lateral nephrectomy was performed. GATA motif transduced the hemodynamic stress stimulus 26–28 hrs postnephrectomy in BNP gene expression.In conclusion, these results show that pressure overload abruptly stimulates the cardiac expression of a noncontractile protein gene BNP, suggesting that it may be used as a myocyte-specific marker of mechanical loading. BNP gene expression was augmented in atria hut nut in ventricles in response to pressure overload in an experimental model of hypertension, suggesting that high local levels of Ang II may differentially regulate cardiac gene expression in atrial and ventricular myocytes in double transgenic rats. At the transcriptional level, acute hemodynamic stress produced by nephrectomy increases BNP reporter expression through a GATA-dependent pathway.

A transcription factor (TF) is a protein that binds to specific DNA sequences as part of the initiation stage of transcription. Various methods of finding these transcription factor binding sites (TFBS) have been developed. In vivo technologies analyze DNA binding regions known to have bound to a TF in a living cell. Most widely used in vivo methods at the moment are chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and DNase I hypersensitive sites sequencing. In vitro methods derive TFBS based on experiments with TFs and DNA usually in artificial settings or computationally. An example is the Protein Binding Microarray which uses artificially constructed DNA sequences to determine the short sequences that are most likely to bind to a TF. The major drawback of this approach is that binding of TFs in vivo is also dependent on other factors such as chromatin accessibility and the presence of cofactors. Therefore TFBS derived from the PBM technique might not resemble the true DNA binding sequences. In this work, we use PBM data from the UniPROBE motif database, ChIP-seq data and DNase I hypersensitive sites data. Using the Spearman’s rank correlation and area under receiver operating characteristic curve, we compare the enrichment scores which the PBM approach assigns to its identified sequences and the frequency of these sequences in likely binding regions and the human genome as a whole. We also use central motif enrichment analysis (CentriMo) to compare the enrichment of UniPROBE motifs with in vivo derived motifs (from the JASPAR CORE database) in their respective TF ChIP-seq peak region. CentriMo is applied to 14 TF ChIP-seq peak regions from different cell lines. We aim to establish if there is a relationship between the occurrences of UniPROBE 8-mer patterns in likely binding regions and their enrichment score and how well the in vitro derived motifs match in vivo binding specificity. We did not come out with a particular trend showing failure of the PBM approach to predict in vivo binding specificity. Our results show Ets1, Hnf4a and Tcf3 show prediction failure by the PBM technique in terms of our Spearman’s rank correlation for ChIP-seq data and central motif enrichment analysis. However, the PBM technique also matched the in vivo binding specificities of FoxA2, Pou2f2 and Mafk. Failure of the PBM approach was found to be a result of variability in the TF’s binding specificity, the presence of cofactors, narrow binding specificity and the presence ubiquitous binding patterns.

Characterising transcription factor binding sites (TFBS) is an important problem in bioinformatics, since predicting binding sites has many applications such as predicting gene regulation. ChIP-seq is a powerful in vivo method for generating genome-wide putative binding regions for transcription factors (TFs). CentriMo is an algorithm that measures central enrichment of a motif and has previously been used as motif enrichment analysis (MEA) tool. CentriMo uses the fact that ChIP-seq peak calling methods are likely to be biased towards the centre of the putative binding region, at least in cases where there is direct binding. CentriMo calculates a binomial p-value representing central enrichment, based on the central bias of the binding site with the highest likelihood ratio. In cases where binding is indirect or involves cofactors, a more complex distribution of preferred binding sites may occur but, in many cases, a low CentriMo p-value and low width of maximum enrichment (about 100bp) are strong evidence that the motif in question is the true binding motif. Several other MEA tools have been developed, but they do not consider motif central enrichment. The study investigates the claim made by Zhao and Stormo (2011) that they have identified a simpler method than that used to derive the UniPROBE motif database for creating motifs from protein binding microarray (PBM) data, which they call BEEML-PBM (Binding Energy Estimation by Maximum Likelihood-PBM). To accomplish this, CentriMo is employed on 13 motifs from both motif databases. The results indicate that there is no conclusive difference in the quality of motifs from the original PBM and BEEML-PBM approaches. CentriMo provides an understanding of the mechanisms by which TFs bind to DNA. Out of 13 TFs for which ChIP-seq data is used, BEEML-PBM reports five better motifs and twice it has not had any central enrichment when the best PBM motif does. PBM approach finds seven motifs with better central enrichment. On the other hand, across all variations, the number of examples where PBM is better is not high enough to conclude that it is overall the better approach. Some TFs bind directly to DNA, some indirect or in combination with other TFs. Some of the predicted mechanisms are supported by literature evidence. This study further revealed that the binding specificity of a TF is different in different cell types and development stages. A TF is up-regulated in a cell line where it performs its biological function. The discovery of cell line differences, which has not been done before in any CentriMo study, is interesting and provides reasons to study this further.

Transcription factors (TFs) are key regulators of gene expression whose failure has been implicated in many diseases, including cancer. They bind at various sites at different specificity depending on the prevailing cellular conditions, disease, development stage or environmental conditions of the cell. TF binding specificity is how well a TF distinguishes functional sites from potential non-functional sites to form a useful regulatory network. Owing to its role in diseases, various techniques have been used to determine TF binding specificity in vitro and in vivo, including chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq). ChIP-seq is an in vivo technique that considers how the chromatin landscape affects TF binding. Motif enrichment analysis (MEA) tools are used to identify motifs that are over-represented in ChIP-seq peak regions. One such tool, CentriMo, finds over-represented motifs at the center since peak calling software are biased to declaring binding regions centered at the TF binding site. In this study, we investigate the use of CentriMo and other MEA tools to determine the difference in motif enrichment attributed presence of Chronic Myeloid leukemia (CML)), treatment with Interferon (IFN) and Dexamethasone (DEX) compared to control based on Fisher’s exact test; using uniform peaks ChIP-seq data generated by the ENCODE consortium. CentriMo proved to be capable. We observed differential motif enrichment of TFs with tumor promoter activity: YY1, CEBPA, Egr1, Cmyc family, Gata1 and JunD in K562 while Stat1, Irf1, and Runx1 in Gm12878. Enrichment of CTCF in Gm12878 with YY1 as the immuno-precipitated (ChIP-ed) factor and the presence of significant spacing (SpaMo analysis) of CTCF and YY1 in Gm12878 but not in K562 could show that CTCF, as a repressor, helps in maintaining the required YY1 level in a normal cell line. IFN might reduce Cmyc and the Jun family of TFs binding via the repressive action of CTCF and E2f2. We also show that the concentration of DEX treatment affects motif enrichment with 50nm being an optimum concentration for Gr binding by maintaining open chromatin via AP1 TF. This study has demonstrated the usefulness of CentriMo for TF binding specificity analysis.

The idea of morphogen gradients encoding positional information for a developing organism has long been discussed in the field of developmental biology, but only recently have quantitative models been proposed that relate measured transcription factor concentrations to enhancer activity. However, successful models are typically computationally time-consuming, thus limiting full exploration and interpretation of the data. This thesis addresses these problems using standard statistical techniques applied to a comprehensive data set with the even skipped (eve) locus as a test case. The first part of the thesis introduces the data set. This is the precellular Virtual Embryo from the Berkeley Drosophila Transcription Network project. It comprises expression measurements of almost 100 genes in more than 6,000 individual nuclei at six time points. Different modelling approaches are evaluated in the context of this data set leading to a justification of logistic regression and the methods used to prepare the data set for further analysis. The second part applies logistic regression to describe the response of the eve enhancers to known regulating transcription factors such as Hunchback. Predictions of behaviour under regulator perturbation are consistent with experimental results and the functional form is shown not to be arbitrarily flexible, both in terms of the regulators and regions of the embryo included. The third part uses the framework developed above to find minimal explanatory models in the context of statistical model selection. It is found that the best scoring models depend on well-known regulators. The model selection techniques are then extended by directing the process using previous biological observations to analyse the eve 2 and eve 3+7 enhancers. The results are consistent with published research, but suggest specific additional hypotheses for the enhancers' regulation. Finally, the thesis concludes by proposing a general model of positional information and discussing the biological implications of the results. Overall, the results show how transcriptional control can be allocated to discrete enhancers and that characterising their activity in relatively simple terms is sufficient to explain their precise spatially-defined response to transcription factor concentrations.

GLK1 and GLK2 transcription factors have been suggested to be involved in the regulation of chloroplast development, organic nitrogen signaling, disease resistance and circadian rhythmicity (Waters et al. 2009; Gutiérrez et al. 2008; Savitch et al. 2007; Sprott et al. 2010). This implies that multiple factors may play roles in regulation of GLK genes. In the present study, transcriptional regulation of GLK1 and GLK2 in Arabidopsis by various endogenous and environmental stimuli was investigated with the objective of elucidating the primary signalling pathway affecting expression of these two genes. Collectively, results of GLK1 and GLK2 expression in response to the experimental treatments of Arabidopsis point to the regulation of the two genes by changes in photosynthetic metabolism and reactive oxygen species (ROS) levels, and by organic nitrogen signalling. Changes in ROS levels and organic nitrogen signalling may also affect the two genes indirectly by interfering with or altering photosynthetic metabolism.

Autophagy is the major cellular, conservative, lysosomal catabolic process to eliminate and recycle intracellular waste and organelles through autophagosomes. Enhancing autophagy to promote the clearance of toxic proteins is developing as a promising approach to treat proteinopathy disorders like Alzheimer's disease (AD). AD is the most common aging-associated neurodegenerative disease. It is characterized by the aggregation of aberrantly hyperphosphorylated tau (p-Tau) and excessively produced Amyloid-beta (Aβ) into neurofibrillary tangles (NFTs), and amyloid plaques (AP) respectively. Reprogramming autophagy lysosomal pathway (ALP) through autophagy master controller, transcription factor EB (TFEB), is developing as an attractive strategy to treat AD. It is already proven that TFEB overexpression can promote Aβ and p-Tau lysosomal clearance, attenuate NFT and AP deposition and restore the behavioural deficits in AD mice models. Previously Song et al., 2016 have identified a small molecule curcumin derivative C1. They reported that C1 could bind to recombinant TFEB and enhance ALP both in vitro and in vivo conditions independent of mTOR inhibition. In the current study, C1 is systematically evaluated for its bioavailability, anti-AD efficacy in vitro, and in vivo AD experimental models. To validate TFEB mediated anti-AD efficacy of C1 in vitro, we tested the C1 effect on amyloid precursor protein (APP) and p-Tau degradation in vitro neuronal AD cell culture models. In N2a cells overexpressed with APP (695) and EGFP-P301L tau plasmids, C1 induced APP, CTFβ, and Tau lysosomal degradation. To demonstrate the TFEB dependent autophagic clearance effects of C1, TFEB is silenced in N2a cells with lentiviral shRNA particles. Under TFEB silenced condition, C1 induced reduction of FL-APP, CTFβ, and Tau was compromised. Overall In vitro experiments show that C1 induced lysosomal digestion of FL-APP, CTFβ, and p-Tau in a TFEB dependent manner. To further demonstrate C1 brain bioavailability, C1 and curcumin comparative pharmacokinetic studies (Pk study) in both mice (time course Pk study) and rat (single time point analysis) are conducted. In Pk studies, both C1 and curcumin are dosed at 10 mg/kg to determine their concentration in the whole brain (mice), separate brain regions (rat), CSF (rat), and plasma. The WinNonlin analysis of C1 and curcumin mice Pk study data revealed that C1 is significantly more bioavailable than curcumin in both brain and plasma, which is also corroborated by the single time point analysis in rats. To illustrate C1 anti-AD activity in vivo, C1 is screened in homozygous P301S (Tau), heterozygous 5xFAD (Aβ), and homozygous 3xTg (both Aβ and Tau) AD transgenic mice models. These mice were started to treat with C1 before the onset of AD pathology until the AD pathological phenotype is expressed to cause impairment in mice behaviour. In mice behavioural examination, C1 treatment has significantly improved mice motor function (Rotarod-P301S), restored cognitive impairment related to the cortex (contextual fear conditioning-5xFAD), hippocampus (Morris water maze-3xTg) and improved cholinergic activation (open field-3xTg). In the brain biochemical examination, C1 activated the TFEB mediated ALP pathway to degrade FL-APP, CTFα/β, Aβ, and p-Tau and reduced the amyloid plaque load, extra neuronal-NFT positive cells. Notably, C1 treatment in 5xFAD mice has significantly restored hippocampal synaptic function. In summary, the current study validates C1 as an orally bio-available potent small molecule TFEB activator which restores mice cognitive impairment, altered behaviour, and synaptic plasticity by reducing Aβ and tau levels in AD experimental models. Overall, the TFEB activator C1 can be a promising drug to treat AD.

Cyanobateria are promising cell factories due to their minimal nutrient requirements and utilization of asmospheric carbon di-oxide as its sole carbon source. In particular, polyhydroxybutyrate (PHB) is an industrially useful bio plastic that is produced naturally by some cyanobacteria. Furthermore, PHB biosynthetic pathway is a starting point for production of the biofuel, 1-butanol. There has been much genetic engineering effort toward increasing the production of PHB from cyanobacteria. These have been focused on increasing the pool of acetyl-CoA precursor, or increasing the amount of the reductant NADPH. The upstream process for increasing these reactants is complex and involves many genes. In this contect, cyanobacteria libraries will contribute to reveal genes or gene fragments that are responsible for production of PHB, alkanes and other high value compounds. In pursuit of finding these novel genes or genefragments, a transcription factor library is created in this study with 50 transcription factors. Furthermore, the process is optimized towards the creation of genomic fragment library and metagenomic fragment library with 26 diverse strains. Membersof the transcription factor library are over-expressed by a PHB - producing host Synechocystis PCC 6803 and the process towards creation of genomic and metagenomic libraries is optimized. The members of the metagenomic library can be screened for increased PHB, alkanes, lactate and other high value products and the potential members can be isolated and characterized.