Orientador: Renata de Oliveira Mattos-Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia / Made available in DSpace on 2018-08-18T21:04:54Z (GMT). No. of bitstreams: 1 Vizoto_NataliaLeal_M.pdf: 1501643 bytes, checksum: 855281de051e36237ad28b6d3f428f1b (MD5) Previous issue date: 2011 / Resumo: Streptococcus mutans é o principal patógeno da cárie dental em humanos, por ser capaz de se acumular no biofilme dentário principalmente na presença de sacarose e produzir e tolerar grandes concentrações de ácidos, os quais causam a desmineralização dos dentes. Além disto, S. mutans pode estar associado à etiologia da endocardite bacteriana, doença cuja evolução envolve a formação de biofilmes em válvulas cardíacas lesadas. S. mutans expressa diversas proteínas de superfície envolvidas na adesão e acúmulo bacteriano em biofilmes. Estas incluem as glucosiltransferases (codificadas por gtfB, gtfC e gtfD), as quais sintetizam glucanos extracelulares a partir da sacarose, e as proteínas ligantes de glucano (codificadas por gbpA, gbpB, gbpC e gbpD). Os genes gtfB/C/D, gbpB/C são controlados diretamente pela proteína reguladora CovR. Em diversas espécies patogênicas de Streptococcus, como Streptococcus pyogenes e Streptococcus agalactiae, CovR compõe o sistema regulador de transcrição de dois componentes CovRS (Cov, de control of virulence), o qual regula diversos genes de virulência. Em S. mutans, CovR foi identificado como um regulador órfão pois o gene que codifica o receptor de membrana cognato não foi identificado no mesmo locus gênico de covR. Em S. mutans, CovR regula diversos genes envolvidos na formação de biofilmes (gtfB/C, gbpC e gbpB), na biossíntese do envelope celular e na resposta a estresses. Os estímulos que ativam CovR em S. mutans ainda não são conhecidos. O presente projeto visa caracterizar a participação de CovR na resposta de S. mutans ao contato com a saliva e sangue humano. Para isto, uma cepa knock-out de covR- foi comparada com a respectiva cepa selvagem quanto ao crescimento e/ou sobrevida em saliva e sangue de voluntários saudáveis. O efeito da exposição à saliva na expressão dos genes regulados diretamente por CovR também foi avaliado / Abstract: Streptococcus mutans is the main pathogen of dental caries in humans, because it can accumulate in the biofilm in the presence of sucrose and produce and tolerate high concentrations of acids, which cause demineralization of teeth. Moreover, S. mutans is involved in the etiology of bacterial endocarditis, a disease whose evolution involves the formation of biofilms on damaged heart valves. S. mutans expresses several surface proteins involved in bacterial adhesion and accumulation in biofilms. These include the glucosiltransferases (gtfB, gtfC and gtfD), which synthesize extracellular glucans from sucrose, and glucan binding proteins (encoded by gbpA/B/C/D). The genes gtfB/C/D, and gbpB/C are directly controlled by the regulatory protein CovR. In several pathogenic species of Streptococcus, such as S. pyogenes and S. agalactiae, CovR is the transcriptional regulator that compose the two-component system CovRS (Cov for control of virulence), which regulates several virulence genes. In S. mutans, CovR was identified as an orphan regulator, because the gene encoding its cognate membrane receptor was not identified at the same locus of covR. In S. mutans, CovR regulates several genes involved in biofilm formation (gtfB/C, and gbpB/C), in biosynthesis of the cell envelope and in stress responses. The stimuli which activate CovR in S. mutans are not yet known. This project aims to characterize the participation of CovR in S. mutans response to contact with saliva and blood. To this purpose, a knock-out mutant of covR was compared with parent strain regarding growth and/or survival in human saliva and blood. The effect of exposure to saliva in the expression of genes directly by covR was also analyzed / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (CuZn SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. In order to determine whether additional SOD activity affects longevity and oxygen metabolism in Drosophila, our approach was to clone the Sod gene and introduce additional copies of the gene back into the genome via P element mediated transformation. The effects of increased SOD activity on Drosophila life span and oxygen free radical metabolism were investigated. The CuZn SOD cDNA and gene were cloned from Drosophila melanogaster. The sequence of the Sod cDNA and gene revealed an additional C-terminal triplet coding for valine not found in the mature SOD protein. The nucleotide sequence of the coding region has 56% and 57% identity when compared to the corresponding human and rat Sod genes, respectively. A probe of the cloned gene hybridizes to position 68A4-9 on Drosophila polytene chromosomes. In wild-type Drosophila the Sod cDNA hybridizes to a 0.7-0.8 kb transcript which is greatly diminished in a SOD 'null' mutant that produces only 3.5% of the SOD protein. A 1.8 kb EcoRI gene fragment containing the Sod gene was cloned into the P vector pUChsneo and microinjected into Drosophila embryos. Five transformed lines, each of which contain an additional copy of the Sod gene at different chromosomal sites were constructed. The chromosomal positions of the transposed Sod sequence were determined by in situ hybridization of the Sod gene to salivary gland polytene chromosomes. Analysis of RNA from the transformed flies revealed that the transposed Sod gene was expressed. The range of SOD activity for the five transformed lines was 131% to 170% of the value of wild-type. There was good correlation between the amount of Sod mRNA and the level of SOD activity in the transformed lines. Increased SOD levels in the transformed lines did not confer greater resistance to paraquat-generated superoxide radicals, nor increase their lifespan. The SOD 'null' mutant with 3.5% of the wild-type SOD activity was hypersensitive to paraquat when compared to wild-type, whereas the heterozygous SOD deficiency Df(3L)1xd⁹/TM3SbSer with 50% of the wild-type SOD activity was not. Mutants lacking SOD are dramatically impaired in oxygen metabolism and a few percent of wild-type activity appears to provide significant protection against superoxide, while 50% of the wild-type levels confers essentially the same resistance as wild-type. Despite the observation that the SOD activities found in a wide range of animals correlates directly with their longevity, Drosophila melanogaster appears to be well protected against the toxic effects of oxygen by its native levels of SOD. / Arts, Faculty of / Philosophy, Department of / Graduate

Orientadores: Marcelo Menossi Teixeira, Ricardo Aparicio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T14:13:51Z (GMT). No. of bitstreams: 1 Soares_JoseSergiodeMacedo_D.pdf: 4995080 bytes, checksum: ad3458f447f7044749e0ee6cb95f4315 (MD5) Previous issue date: 2013 / Resumo: O agronegócio da cana de açúcar movimenta cerca de R$ 40 bilhões por ano no Brasil. A cadeia produtiva da cana de açúcar como atividade na economia é responsável por 1,5% do produto interno bruto (PIB) nacional e um dos principais componentes econômicos é a quantidade de sacarose acumulada nos colmos. No entanto, a síntese de sacarose e sua acumulação em plantas superiores é o resultado do produto de uma extensa rede de interações. Quando descarregada nas células do parênquima de armazenamento, a sacarose é metabolizada por diferentes enzimas, sendo a UDP-glucose pirofosforilase (UGPase) uma das enzimas responsáveis pela síntese de sacarose em cana de açúcar. O objetivo deste trabalho foi avaliar o padrão de expressão do gene ScUGPase-1 e os mecanismos regulatórios que controlam a atividade da proteína UGPase de cana de açúcar. Análises por RT-qPCR revelaram que a expressão do gene ScUGPase-1 diminui ao longo da maturação dos colmos e o gene é mais expresso nos entrenós em comparação com o tecido de folha. Porém, nenhuma diferença de expressão significativa foi observada entre dois cultivares contrastantes em teor de sacarose. In vivo, a localização subcelular da proteína ScUGPase-1 indicou uma associação à membrana nos tecidos de folha e colmo. Utilizando anticorpo primário fosfo-específico, observamos a fosforilação da proteína ScUGPase-1 apenas na fração solúvel e microssomal do tecido de folha. In vitro, a proteína ScUGPase-1 formou um complexo com a proteína recombinante caseína quinase 1 (CK1) e sua atividade foi afetada por agentes óxido-redutores. Para complementar os dados de óxido-redução, análises de espalhamento de luz a baixo ângulo (SAXS) forneceram o primeiro modelo estrutural do dímero da proteína ScUGPase-1 em solução, destacando que a interface de dimerização está localizada na região C-terminal. Os dados indicam que a fosforilação, interação protéica e oligomerização podem exercem um papel importante na regulação da proteína ScUGPase-1 durante a síntese de sacarose em cana de açúcar. / Abstract:The sugarcane agribusiness generates around R$ 40 billion per year in Brazil, while the entire supply chain of sugarcane is responsible for 1.5% of the gross domestic product (GDP). Sugarcane productivity is mainly determined by the accumulation of sucrose in the culms. However, the synthesis and accumulation of sucrose in plants is the result of an extensive network. When sucrose is unloaded in the storage parenchyma cells, it is metabolized by different enzymes, and UDP-glucose pyrophosphorylase (UGPase) is one of the enzymes responsible for the synthesis of sucrose in sugarcane. The objective of this work was to gain insights on the ScUGPase-1 expression pattern and the regulatory mechanisms that control protein activity. ScUGPase-1 transcript levels were negatively correlated with sucrose content in the internodes and only a slight difference in the expression pattern was observed between two cultivars that differ in their sucrose content. The intracellular localization of ScUGPase-1 indicated association with membranes in both leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo in the soluble and membrane fractions from leaves, but not from internodes. In vitro, the purified recombinant enzyme interacted with recombinant protein casein kinase 1 and its activity was affected by redox modification. To complement the redox data, Small-Angle X-ray Scattering provided the first structural model of the dimer of sugarcane UGPase in solution, highlighting that the dimer interface is located at the C-terminal. The data indicated that phosphorylation, protein interaction and oligomerization may play an important role in the regulation of ScUGPase-1 activity / Doutorado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

The overall objective of this study has been to address some of the longstanding questions concerning functional organization of the interphase nucleus. This was achieved by using recently developed high-resolution fluorescence in situ hybridization techniques for a precise localization of specific DNA and RNA sequences in conjunction with immunocytochemistry and biochemical fractionation. This study is based on the philosophy that new insights may be gained by an approach that attempts to interrelate genomic organization, spatial arrangement of RNA metabolism, and nuclear substructure within the mammalian cell nucleus. The nuclear distribution of an exogenous, viral RNA (Epstein-Barr Virus, EBV) within nuclear matrix preparations was studied by developing an approach which couples in situhybridization with biochemical fractionation procedures. EBV RNA molecules accumulate in highly localized foci or elongated tracks within the nucleus of lymphoma cells. These RNA tracks were retained with spatial and quantitative fidelity in nuclear matrix preparations even after biochemical fractionation which removes 95% of cellular protein, DNA, and phospholipid. This provided direct evidence that the primary transcripts are localized via their binding to, or comprising part of, a non-chromatin nuclear substructure. Then the nuclear distribution of RNA from an endogenous gene, fibronectin, was investigated using fluorescence techniques modified for more sensitive detection of endogenous RNAs within nuclear morphology. A series of in situhybridization experiments were performed using different combinations of intron, cDNA, and genomic probes for RNA/RNA or RNA/DNA analysis in intact cells. Fibronectin RNAs were highly localized in the nucleus, forming foci or tracks. Both intron and exon sequences were highly concentrated at the same site within the nucleus, indicating the presence of primary unspliced transcripts. Double-color hybridization using a nontranscribed 5' flanking sequence probe and a genomic DNA probe showed that the gene and RNA track for fibronectin were spatially overlapped, with the gene consistently towards one end of the track. These results provided evidence that the accumulation of RNA molecules occurs directly at or near the site of transcription, and further indicated a structural polarity to the RNA track formation with the gene towards one end. It was further discovered that within a single cell, cDNA probes produced longer tracks than those formed with intron probes, i.e. intron signals were generally confined to a smaller part of the track than the exon signals, indicating that splicing occurs within the RNA track. Additional experiments using poly(A) RNA hybridization or anti-SC-35 antibody staining combined with fibronectin RNA hybridization have shown that the fibronectin tracks were associated with recently discovered transcript domains enriched in poly(A) RNA and splicing factors. To further determine whether other specific genes and RNAs are functionally organized within the nucleus, the nuclear distribution of several active or inactive genes was analyzed in terms of their spatial relationship to transcript domains. The results indicated that in addition to fibronectin, the genes or their primary transcripts from two other active genes, collagen and actin, were also closely associated with the domains. For both of these, over 90% of the gene/RNA sites were either overlapping or directly contacting the domains. In contrast. for two inactive genes, cardiac myosin heavy chain and neurotensin, it was found that both genes were separated from the domains in the majority of nuclei. Histone genes, which have several unique features, showed a relatively complex result with about half of the gene signals extremely close to the domains. Therefore, three actively expressed genes were demonstrated to be tightly associated with the domains and, moreover, their RNAs showed distinct and characteristic spatial relationships with the domains. In contrast, two inactive genes were not associated with the domains. One potential implication of these finding is that active genes may be preferentially localized in and around these transcript domains. The nuclear localization of another RNA, XIST, standing for X-inactivation specific transcript, was studied because of its potentially unique biological role. XIST is the only gene which is known to be expressed from the inactive human X chromosome but not from the active X chromosome, and was believed to be important in X inactivation. Using fluorescence in situhybridization, it was found that XIST RNA was highly localized within the nucleus and always completely overlapped the Barr body which is the condensed, inactive X chromosome. The different fine distribution pattern of XIST RNA within the nucleus as compared to other protein coding RNAs suggested a unique function for this RNA, possibly involving a structural role in inactivating the X chromosome. The final area of my thesis research was to study and acquire expertise in the applications of fluorescence in situ hybridization in gene mapping and cancer genetics. A retinoblastoma (RB)-related putative tumor suppressor gene, p107, was mapped to human chromosome 20 in band q11.2. Localization of p107 to 20q11.2 was of particular interest because of the correlation of breakpoints in this area with specific myeloid disorders such as acute nonlymphocytic leukemia and myelodysplastic syndrome. Other applications of in situ hybridization including the search for unknown genes at a known chromosomal breakpoint, detection of deletions, translocations or other chromosomal rearrangements associated with specific tumors were also explored and reviewed.

RNA interference (RNAi) is an important pathway that regulates gene expression in several organisms. The role of RNAi in post-transcriptional gene silencing in the cytoplasm is well characterized. In contrast, the role of RNAi components in the nucleus remains to be elucidated. Previous reports have indicated that RNAi components (Dicer and Argonaute proteins) and small RNAs act in the nucleus to regulate various pathways including heterochromatin formation, transposable elements repression, RNA Pol II processivity and alternative splicing. Nuclear Ago1 and Dicer have also been found to associate with active promoters and enhancers in mammalian cells, however their functional roles and mechanisms remain elusive. In this work, I investigated the functional role of nuclear RNAi components in gene expression regulation during skeletal muscle differentiation. To address this question, I undertook genomic and biochemical approaches applied to myogenic cells (C2C12) as a model system. I found that Ago1 and Dicer are present in the nucleus of C2C12 cells and expressed during differentiation. Chromatin Immunoprecipitation (ChIP) coupled with high throughput sequencing and quantitative real-time PCR indicate that Ago1 and Dicer are enriched at promoters and enhancer regions of myogenic genes. Interestingly, I found that depletion of Ago1 and Dicer reduces enhancer RNAs (eRNAs) levels at enhancer regions and expression of MyoD during differentiation. I observed that loss of Ago1 impacts differentiation, whereas, loss of Dicer leads to cell death and has severe effects on C2C12 cells. Moreover, using Chromosome Conformation Capture (3C), I revealed that Ago1 is involved in enhancer-promoter interaction at MyoD locus. The knockdown of Ago1 destabilizes these interactions and decreases the expression of MyoD. Finally, I demonstrated that Ago1 binds to eRNAs and interacts with CBP Acetyl-transferase in the nucleus of myotube cells. Ago1 depletion leads to loss of eRNA-CBP interaction and consequent impairment of CBP acetyltransferase activity and failure of MyoD mediated activation of the myogenic program. Taken together, these finding indicate that nuclear Ago1 together with eRNAs and CBP regulates MyoD expression by stimulating histone acetylation during differentiation. This study uncovered a novel function of chromatin associated Ago1 in gene expression regulation during mammalian skeletal muscle differentiation.

The pathological conse-quences of ischemic heart disease involve signaling through the autonomic nervous system. Although early activation may serve to maintain hemodynamic stability, persistent aberrant sympathoexcitation contributes to the development of lethal arrhythmias and heart failure. We hypothesized that as the myocardium reacts and remodels to ischemic injury over time, there is an analogous sequence of gene expression changes in the thoracic spinal cord dorsal horn, the processing center for incoming afferent fibers from the heart to the central nervous system. Acute and chronic myocardial ischemia (MI) was induced in a large animal model of Yorkshire pigs, and the thoracic dorsal horn of treated pigs, along with control nonischemic pigs, was harvested for transcriptome analysis. We identified 32 differentially expressed genes between healthy and acute ischemia cohorts and 46 differentially expressed genes between healthy and chronic ischemia cohorts. The canonical immediate-early gene c-fos was upregulated after acute MI, along with fosB, dual specificity phosphatase 1 and 2 (dusp1 and dusp2), and early growth response 2 (egr2). After chronic MI, there was a persistent yet unique activation of immediate-early genes, including fosB, nuclear receptor subfamily 4 group A members 1±3 (nr4a1, nr4a2, and nr4a3), egr3, and TNF-β-induced protein 3 (tnfaip3). In addition, differentially expressed genes from the chronic MI signature were enriched in pathways linked to apoptosis, immune regulation, and the stress response. These findings support a dynamic progression of gene expression changes in the dorsal horn with maturation of myocardial injury, and they may explain how early adaptive autonomic nervous system responses can maintain hemodynamic stability, whereas prolonged maladaptive signals can predispose patients to arrhythmias and heart failure. NEW & NOTEWORTHY Activation of the autonomic nervous system after myocardial injury can provide early cardiovascular support or prolonged aberrant sympathoexcitation. The later response can lead to lethal arrhythmias and heart failure. This study provides evidence of ongoing changes in the gene expression signature of the spinal cord dorsal horn as myocardial injury progresses over time. These changes could help explain how an adaptive nervous system response can become maladaptive over time.

Induction of germline (GL) ε transcripts, an essential step preceding immunoglobulin (Ig) isotype switching to IgE, requires activation of transcription factors by IL-4 and a B cell activator, e.g. CD40 ligand (CD40L). AP-1 (Fos and Jun), induced transiently by CD40L, binds a DNA element in the mouse GL ε promoter. AP-1 synergizes with Stat6 to activate both the intact GL ε promoter and a minimal heterologous promoter driven by the AP-1 and Stat6 sites of the mouse GL ε promoter. By contrast, C/EBPβ, which transactivates the human GL ε promoter, inhibits IL-4 induction of the mouse promoter, probably by attenuating the synergistic interaction between AP-1 and Stat6. Furthermore, AP-1 does not transactivate the human GL ε promoter. Thus, due to selective binding of either AP-1 or C/EBP proteins, induction of GL ε transcripts in mouse and human may be regulated differently. In addition to AP-1, NF-кB activity is also induced by CD40L stimulation in normal B cells. Using GST pulldown assays and coimmunoprecipitation techniques I show that NF-кB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. When Stat6 and NF-кB proteins are co-expressed in human embryonic kidney 293 (HEK 293) cells, an IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4. Furthermore, the same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-кB proteins in IL-4-stimulated B lymphoma cells I.29μ. Consistently, by using nuclear extracts from transfected HEK 293 cells and from I.29μ B cells in electrophoretic mobility shift assays (EMSAs), I show that Stat6 and NF-кB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-кB. I conclude that the direct interaction between Stat6 and NF-кB may provide the basis for synergistic activation of the GL ε promoter. Finally, although mouse GL ε transcripts have a half-life of approximately 100 min, the RNA level continues to increase for up to 24 h and the promoter appears to be active for at least 2 days after B cell activation. These data suggest that induction of AP-1 and NF-кB activities by CD40L, although transient, is required for activation of the mouse GL ε promoter by IL-4-induced Stat6.

Maternally inherited poly(A)+ RNAs are important for directing early development in many animal species. This thesis investigates the regulation of maternal mRNA in the South African clawed frog, Xenopus laevis. The first portion of this thesis examines an unusual class of maternal RNA, interspersed poly(A)+ RNA, which is composed of co-linear repeat and single copy sequences. A cDNA clone, called pXR, contains the repeat portion of an interspersed RNA that hybridizes to several different oocyte transcripts of diverse size that persist until the neurula stage. DNA sequence analysis of the cDNA and hybrid selection of the oocyte transcripts followed by in vitro translation show that molecules of this repeat family are not translatable. This data, combined with the developmental profile of XR containing RNAs, indicate that members of this repeat family are not likely to be maternal messenger RNAs. The second part of this thesis investigates the expression of a class of maternal mRNAs that are regulated by cytoplasmic polyadenylation during progesterone induced oocyte maturation. One particular mRNA G10, is stored as a polyadenylated RNA in the cytoplasm of stage VI oocytes until maturation when the process of poly(A) elongation stimulates its translation. Injection of mutant and wild-type mRNAs, synthesized in vitro, revealed that two sequence elements, UUUUUUAUAAAG and AAUAAA, were both necessary and sufficient for polyadenylation and polysomal recruitment of G10. Maturation promoting factor and cyclin as well as progesterone can induce polyadenylation but in each case protein synthesis is required. Extracts from oocytes and unfertilized eggs were employed to identify factors that may be responsible for maturation-specific polyadenylation. An 82 kd protein that binds to the UUUUUUAUAAAG in egg, but not oocyte extracts, was identified by UV crosslinking. This data suggests that p82 is a good candidate for a developmentally regulated protein that controls the expression of maternal messenger RNAs in early Xenopus development.

Insertion of transposable elements within an operon has been known not only to abolish expression of the gene interrupted by the insertion, but also to exert a strong polar effect on the expression of downstream genes in the same operon. In this dissertation, I have shown that insertions of the transposable ampicillin-resistance element Tn1, either in the polar or nonpolar orientation, in the 3' untranslated region of the bacteriophage P22 antirepressor (ant) operon reduce the rate of upstream ant gene expression; insertions of Tn1 in the nonpolar orientation reduce the rate of ant gene expression more significantly than those in the polar orientation. This effect appears to be due to reduced stability of ant mRNA. Tn1 deletion mutants of one of the nonpolar Tn1 insertion mutations have been isolated. Two classes of Tn1 deletions are obtained. Class I retains a 68 bp Tn1 sequence that shows a potential 14 bp stem and 37 bp loop conformation, while class II retains 147 bp Tn1 sequence that shows a potential 69 bp stem and 6 bp loop conformation. These two classes of Tn1 deletions do not delete any P22 sequences. Class I but not class II Tn1 deletion mutants restore the rate of ant gene expression and ant mRNA stability. Six different Ant+ revertants of the class II Tn1 deletion mutant simultaneously restore the rate of ant gene expression and ant mRNA stability. They all have deletions that remove all or part of the class II Tn1 sequence. In one case, the Tn1 sequence retained shows a potential 15 bp stem and 8 bp loop conformation, in the other cases, no secondary structure is predicted to form. The results of the Tn1 deletion mutants suggest that the stem-and-loop structures and the length of stems potentially formed by the Tn1 sequences in mRNA may affect its stability.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21332号 / 理博第4428号 / 新制||理||1636(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 秋山 芳展, 准教授 竹田 一旗 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM