Abstract not available

Orientador: Maria Cristina Cintra Gomes Marcondes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T11:32:15Z (GMT). No. of bitstreams: 1 Pertile_Tatiane_M.pdf: 1613871 bytes, checksum: 2d715c00e32e2a411050801a7a1ac95f (MD5) Previous issue date: 2011 / Resumo: O câncer pode promover a morte do hospedeiro, pois durante sua evolução há modificações da homeostasia dos processos metabólicos, promovendo profundas alterações caracterizadas como caquexia, que por sua vez relaciona-se à diminuição da qualidade e do tempo de vida do hospedeiro. Assim, no presente estudo, analisamos os efeitos da evolução do crescimento de neoplasia - carcinossarcoma de Walker 256 - em ratos jovens e adultos e os efeitos modulatórios do tratamento desses animais com EPA (ácido eicosapentaenóico) sobre o processo de caquexia e a concentração de citocinas anti e proinflamatórias no músculo gastrocnêmio, pois o tecido muscular é o tecido mais afetado no processo de caquexia. Foram utilizados 108 ratos Wistar machos, com idade de 30 dias (jovens) e 100 dias (adultos), os quais foram distribuídos de acordo com o local de implante tumoral, intraperitônio e subcutâneo, e tratamento ou não com ácido eicosapentaenóico, 100?g/Kg de peso corpóreo. Os animais receberam gavagem diária do EPA (animais tratados) ou de óleo mineral (grupos sem tratamento) até a fase pré-agônica. A partir dos órgãos coletados, foram calculados o ganho de peso corpóreo, os pesos relativos de cada órgão, do tumor e da carcaça. Com o objetivo de identificarmos a via de degradação protéica predominante nos grupos experimentais, foram avaliados, no tecido muscular, o teor de proteína muscular total e as atividades das seguintes enzimas: chymotrypsin-like, catepsinas B e H, calpaína e fosfatase alcalina,. A partir do sangue desses animais foram feitas análises fluorimétricas do fator de crescimento semelhante à insulina (IGF) e das citocinas - interleucinas 4 (IL-4), 6 (IL-6) e 10 (IL-10), interferon gama (INF-?) e leptina, utilizando-se kits específicos para citômetro de fluxo de fluorescência (Luminex). Também foi analisada a expressão gênica, no tecido muscular, por reação em cadeia da polimerase em tempo real (PCR-RT), para a via proteossômica e também para os fatores eucarióticos de inicialização. Os dados indicam efeitos modulatórios do EPA sobre o tecido muscular (manutenção da proteína e do peso), principalmente para os grupos jovens, e também no processo inflamatório crônico (aumento de citocinas pró e antiinflamatórias). Entretanto, efeitos mais expressivos do EPA não foram verificados na prevenção da espoliação de gordura (gordura perirrenal e leptina), no processo de síntese protéica (manutenção da expressão gênica de fatores eucarióticos de inicialização) ou também sobre a via proteossômica / Abstract: Cancer can promotes the host death, because during its evolution there are modifications in metabolic processes of homeostasis, promoting deep changes characterized as cachexia, which in turn relates with reduction in quality and lifetime of the host. Thus, in this study, we analyze the effects of development in Walker 256 carcinoma evolution - in young and adults rats and the modulatory effect of treatment with EPA (eicosapentaenoic acid) in gastrocnemius muscle as this tissue is the most affected in the cachexia process. Wistar males rats were used (n=108 animals), 30 days-old (young) and 100 days-old (adults), which were distributed according to the tumour implant, intraperitoneally and subcutaneously and treatment or not with eicosapentaenoic acid, 100 ?g/Kg of body weight. The animals receive daily EPA by gavage (treated animals) or nujol (sham groups) and were cared up to pre-agonic state. The bodies weight were measured and the body weight gain was calculated, as well the relative weights of tissues, tumour, and carcass. In order to identify the predominant pathway of protein degradation the total muscle protein content and proteolytic enzymes activities (chymotrypsin-like, cathepsin B and H, calpain and alkaline phosphatase) were measured in gastrocnemius muscle. The blood samples were assessed to measure the insulin-like growth factor 1 (IGF-1), leptin and the cytokines - interleukins (IL-4), 6 (IL-6) and 10 (IL-10), gamma interferon (INF-?), using specific kits for cytometer fluorescence (Luminex). It was also examined gene expression, in the muscle tissue, by real-time polymerase chain reaction (RT-PCR), assessing keys of ubiquitin-proteasome pathway and also on the eukaryotic initiation factors. The data indicated some modulatory effects of EPA on the muscle tissue (maintenance of protein and weight), mainly for the young rats, and also the chronic inflammatory process. However, more expressive effects of EPA have not been verified as preventing fat wasting (perirenal fat and leptin), nor in the process of protein synthesis (maintenance of eukaryotic initiating factors gene expression) or also in the ubiquitin-proteasome via / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Calcium (Ca2+) plays an important role as a second messenger, transmitting the message of arrival of stimuli such as hormones and neurotransmitters to the intracellular system that carries out the cellular response to the stimulus. The universality of Ca2+ as an intracellular messenger depends on its enormous versatility. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of Ca2+ being highly toxic. Ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs) are Ca2+ -release channels located on intracellular membranes of the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) that perform essential functions as key targets of hormone/neurotransmitter action to initiate intracellular Ca2+ signals. The purpose of this project was to study the role of RyR2 in Ca2+ signaling in the NG115-401L neuronal cell line. siRNA transfection methods were employed to knockdown RyR2 expression levels in NG115-401L cells. We used reverse transcription and real-time PCR to evaluate RyR2 gene expression in transfected/untransfected cells. We also evaluated cytosolic Ca2+ changes induced by RyR activators or regulators, using fura-2 AM as the Ca2+ indicator. Successful RyR2 gene knockdown allowed us to carry out some initial experiments to characterize the specific roles played by the RyR2 receptor isoform. We examined cell responses to FK-506 under the condition of RyR2 knockdown, finding that RyR2 appears to confer selectivity to this response. Finally, the effects of siRNA transfection and FK-506 treatment on NG115-401L cell growth were evaluated. These experimental results may contribute to future studies of RyR2, and help develop novel treatments for RyR2-base d dysfunctional diseases.

eDNA microarray technology has generated a tremendous amount of interest among biologists because of its promise to monitor the entire genome on a single chip, thus enabling researchers to have a better picture of the interaction among thousands of genes simultaneously. In the current study, this technology was used to print over 3,000 unknown genes from various silk glands of the black widow spider to profile their expression patterns and to identify novel candidates. Spiders are remarkable creatures because of their ability to make different silks, each with a specific function. Some of these silks have amazing mechanical properties, comparable to those of the finest synthetic materials. Several silk genes have been cloned from various spiders over the last few years, and the contribution of each of those genes in silk production has been identified. However, the majority of cellular and biochemical processes involved in silk manufacture remain a mystery. In our research, we attempt to identify genes that might be involved in silk assembly, on a global scale and investigate more about those genes and their interplay with other key biological molecules involved in silk manufacture. Our study showed that silking spiders for a certain period of time resulted in down-regulation of two important silk genes, ECP-1 and ECP-2. Both these genes are key molecules implicated for their role in maintaining the egg case architecture in the black widow spider.,-and we believe that these genes are also directly or indirectly involved in the manufacture of dragline silk. Microarray analyses also enable the discovery of several other interesting molecules, two of which could be accessory proteins involved in silk formation. Furthermore, in a separate study we also characterized a novel silk glue protein with unique ensemble repeats. In conclusion, we believe that the findings of this study will indeed be significant to silk researchers and material scientists alike and it will enhance our knowledge in understanding the mystery behind silk production.

Indiana University-Purdue University Indianapolis (IUPUI) / The root system architecture of higher plants plays an essential role in the uptake of water and nutrients as well as the production of hormones. These root systems are highly branched with the formation of post-embryonic organs such as lateral roots. The initiation and development of lateral roots has been well defined. WAG1 and WAG2 are protein-serine/threonine kinases from Arabidopsis that are closely related to PINOID and suppress root waving. The wag1;wag2 double mutants exhibit a strong root waving phenotype on vertical hard agar plates only seen in wild-type roots when the seedlings are grown on inclined plates. Here an additional root phenotype in the wag1;wag2 mutant is reported. The wag1;wag2 double mutant displays both an increased total number and density of emerged lateral roots (approximately 1.5-fold). An increased LRP density of 1.5-fold over wild-type is observed. To ascertain the role of WAG1 and WAG2 in lateral root development we examined promoter activity in the WAG1::GUS and WAG2::GUS lines. The WAG1 promoter showed no detectable activity at any stage of development. The WAG2 promoter was active in stage IV onward, however there was no detectable activity in the cell types associated with initiation events. The lateral root density and spatial patterning in wild-type, when grown on inclined hard agar plates, was similar to wag1;wag2 on vertical plates. Seedlings of both genotypes were treated with hormones such as auxin and MeJA, and inhibitors. Auxin response in wag1;wag2 was normal with a similar number of LR as the wild-type after treatment. Treatment with MeJA resulted in a similar induction of LRP in both genotypes, however the percent lateral root emergence in wag1;wag2 was reduced while Col-0 was increased compared to controls. Treatment with the calcium blocker tetracaine resulted in wag1;wag2 displaying a wild-type level of LR but had no significant effect on wild-type. Genetic analysis of the wag1;wag2 LR pathway revealed that WAG1 and WAG2 are acting in the same pathway as AUX1, AXR1and PGM1. pgm1-1 was not previously reported to have a LR defect but showed decreased LR formation here, while pgm1;wag1;wag2 had a similar LR density to wag1;wag2. TIR7 and ARG1 were both deduced to operate in separate pathways from WAG1 and WAG2. The data presented here shows that the wag1;wag2 double mutant has an increased number of LR compared to Col-0. This defect appears to be caused by increased pre-initiation events and seems to be tied to the root waving phenotype. However, the treatment with MeJA revealed a possible role for WAG1 or WAG2 in LRP development, potentially under stress conditions. Calcium also seems to play a significant role in the wag1;wag2 LR phenotype, possibly independent of the root waving phenotype.

Polycyclic aromatic hydrocarbons are environmental chemicals that are produced during incomplete combustion of coal, oil, gas, and garbage. Toxic effects of these compounds are mediated via the ligand activated Aryl Hydrocarbon Receptor (AHR) signaling pathway. To enable the study of the AHR signaling mechanism, our lab has generated many human proteins using recombinant DNA technology. This thesis documents the design and synthesis of a number of proteins of the AHR deletion construct CΔ553. The bacterial expressed and purified fusion proteins could be utilized as antigen to generate antibodies and be used for cellular delivery. The purified protein was immunogenic in rabbits and produced significant amount of polyclonal antibodies. In western blot analysis, the antibodies were able to the detect baculovirus expressed AHR and different recombinant proteins of the AHR. The polyclonal antibodies were also used in the gel-shift assay to show the AHR dependent gel shift. Cellular delivery CΔ553 was achieved using the protein transduction domain from the HIV-1 virus transactivating protein (TAT). In order to deliver the CΔ553 into mammalian cells, an expression vector was constructed to generate the TAT-CΔ553 fusion protein. The TAT-CΔ553 fusion protein was successfully transduced into two mammalian cells-HeLa and HepG2. The in vivo function of TAT-CΔ553 was determined using the luciferase reporter plasmid assay. The transduced protein was functional; it competed with the AHR and heterodimerize with ARNT in both HeLa and HepG2 cells at a concentration of 3.8x103 nM and 18 nM respectively. Since there an apparent similarity between the basic region of TAT-PTD and CΔ553, we examined the transduction potential of CΔ553. Western blot analysis indicated that the extent of denatured protein transduction was comparable for CΔ553 and TAT-CΔ553 in HepG2 cells. Thus CΔ553 might have intrinsic transduction capability.

The effects of light, light "mimicking" chemicals, and protein synthesis inhibitors on the photo-induced carotenogenesis of Corynebacterium 7EIC were studied. Changes in the dosage of fluorescent light applied to dark grown cells showed a dose related carotenogenic response. Maintaining the same dosage but varying the wavelength of monochromatic light revealed that light with a wavelength of 280 to 450nm was responsible for photo-induction. It further showed a peak of photo-induction between the wavelengths of 370 and 430nm. The light "mimicking" chemicals antimycin A and p-Chloromercurybenzoate were shown to have no light "mimicking" effects. The transcriptional inhibitor of protein synthesis actinomycin D partially inhibited, and chloramphenicol a translational inhibitor, completely inhibited photo-induced carotenogenesis.

Orientador: Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T15:29:14Z (GMT). No. of bitstreams: 1 Coltri_PatriciaPereira_D.pdf: 4564852 bytes, checksum: f11b831da981a8969c20f8f03ae8c617 (MD5) Previous issue date: 2007 / Resumo: A síntese de ribossomos é um processo conservado em eucariotos e se inicia com a transcrição dos rRNAs no nucléolo. Mais de 170 fatores atuam de forma transitória no processamento dos precursores para gerar os rRNAs maduros que formarão as subunidades ribossomais no citoplasma. Entre as proteínas envolvidas na síntese de ribossomos está a Nip7p, uma proteína nucleolar de 21 kDa associada ao complexo pré-60S em Saccharomyces cerevisiae. Nip7p é conservada e possui ortólogas em eucariotos e em Archaea. A análise da seqüência primária revela a presença de um domínio conservado na região C-terminal, denominado PUA, encontrado em diversas proteínas associadas a modificações no RNA. Neste trabalho, foram realizadas análises estruturais e funcionais com o objetivo de investigar a função molecular da proteína Nip7 no processamento e modificação do rRNA. A estrutura tri-dimensional de PaNip7, ortóloga de Nip7p em Pyrococcus abyssi foi resolvida por difração de raios-X até 1,8Å de resolução, utilizando o método SIRAS. Comparação estrutural seguida por ensaios in vitro confirmaram o envolvimento do domínio PUA na interação com RNA. Além disso, tanto Nip7p como suas ortólogas PaNip7 e HsNip7 interagem com seqüências ricas em uridina, indicando que atuam de forma semelhante no processamento do rRNA. Essa preferência por uridina pode ainda explicar a afinidade da proteína Nip7p de S. cerevisiae pelo RNA da região ITS2, conforme observado em ensaios de interação utilizando UV-crosslinking. De fato, uma análise funcional realizada por primer extension comprovou que ocorre um bloqueio no processamento da região espaçadora ITS2 na ausência de Nip7p. Nip7p interage com várias proteínas do complexo pré-60S, entre as quais Nop8p e Nop53p, ambas associadas ao processamento do pré-27S. Embora os ensaios de co-purificação tenham confirmado a interação com as proteínas do complexo H/ACA box, deficiência em Nip7p não afeta a pseudo-uridinilação do rRNA. O duplo-híbrido realizado com a ortóloga humana de Nip7p, HsNip7, revelou interações com FTSJ3 e com a proteína SUMO-2. A interação direta de HsNip7 com estas proteínas foi confirmada por ensaios in vitro. HsNip7 e FTSJ3 colocalizaram na região nucleolar de células HEK293. FTSJ3 é uma proteína não caracterizada que possui o domínio FtsJ, descrito inicialmente para rRNA metiltransferases de procariotos. Além disso, FTSJ3 apresenta similaridade de sequência à proteína Spb1p de levedura, cuja função na metilação do rRNA 25S na posição Gm2922 já foi estabelecida. Embora a Nip7p não interaja com a Spb1p, estes dados indicam que FTSJ3 deve ser a ortóloga humana da Spb1p. As proteínas SUMO estão envolvidas na modificação pós-traducional (sumoylation) que regula a localização subcelular de proteínas. Em levedura, a provável ortóloga de SUMO, Smt3p, foi descrita na partícula pré-60S, portanto a interação HsNip7-SUMO-2 pode ser específica. Estes dados sugerem que as proteínas atuem no mesmo complexo da formação da subunidade 60S também em células humanas / Abstract: Ribosome biogenesis is conserved throughout eukaryotes and takes place in the nucleolus, a specialized nuclear compartment where the rRNA precursors are transcribed. More than 170 trans-acting factors coordinately interact to generate the mature rRNAs. Among the proteins identified in the pre-60S particle in Saccharomyces cerevisiae is Nip7p. Highly conserved Nip7p orthologues are found in all eukaryotes and Archaea. The analysis of Nip7p sequence reveals a conserved C-terminal domain named PUA, also found in a number of RNA-interacting proteins. In this work, we performed structural and functional analysis to investigate Nip7p molecular role on rRNA processing and modification. The structure of Pyrococcus abyssi Nip7p ortholog, PaNip7, was solved using X-ray diffraction data to 1,8Å resolution. Structural analysis followed by in vitro assays confirmed the involvement of PUA domain in RNA interaction. S. cerevisiae Nip7p and its archaeal and human counterparts show preference for binding uridine-rich sequences, indicating conserved functional features among the orthologues. The preference for uridine can explain the higher affinity of S. cerevisiae Nip7p for ITS2 sequence, as observed by UV-crosslinking assays. Consistently, functional analysis revealed pre-rRNA processing in the ITS2 region is seriously impaired. Yeast two-hybrid analysis confirmed by pull down assays revealed Nip7p interacts with Nop8p and Nop53p, two nucleolar proteins involved in pre-27S processing and components of pre-60S particle. Although yeast two-hybrid and pull down assays indicated that Nip7p interacts with H/ACA box core proteins, pseudouridylation is not affected under conditions of Nip7p depletion. In addition, yeast two-hybrid analysis confirmed by GST-pull down revealed HsNip7 interaction with FTSJ3 and SUMO-2. Both HsNip7 and FTSJ3 showed nucleolar subcellular localization in HEK293 cells. FTSJ3 is an uncharacterized protein containing the FtsJ domain, initially described in prokaryotic rRNA methyl-transferases. FTSJ3 shows sequence similarity to yeast Spb1p, an rRNA methyl-transferase involved in methylation of Gm2922, indicating that FTSJ3 may be the human orthologue of Spb1p. Sumoylation is a post-transcriptional covalent modification involved in regulation of protein subcellular localization. Putative yeast orthologues of SUMO, such as Smt3p, have been described in the pre-60S ribosomal particle, suggesting that SUMO-2 might play a specific role in 60S subunit biogenesis / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Indiana University-Purdue University Indianapolis (IUPUI) / Eukaryotic cells rapidly modulate protein synthesis in response to environmental cues through the reversible phosphorylation of eukaryotic initiation factor 2 (eIF2α~P) by a family of eIF2α kinases. The eIF2 delivers initiator Met-tRNAiMet to the translational apparatus, and eIF2α~P transforms its function from a translation initiation factor into a competitive inhibitor of the guanine nucleotide exchange factor (GEF) eIF2B, which is responsible for the recycling of eIF2-GDP to the translationally-competent eIF2-GTP state. Reduced eIF2-GTP levels lower general protein synthesis, which allows for the conservation of energy and nutrients, and a restructuring of gene expression. Coincident with global translational control, eIF2α~P directs the preferential translation of mRNA encoding ATF4, a transcriptional activator of genes important for stress remediation. The term Integrated Stress Response (ISR) describes this pathway in which multiple stresses converge to phosphorylate eIF2α and enhance synthesis of ATF4 and its downstream effectors. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2α~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKα as being subject to both translational and transcriptional induction during eIF2α~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKα mRNA involves the stress-induced relief of two inhibitory uORFs in the 5’-leader of the transcript. Also identified as being subject to preferential translation is mRNA encoding the bifunctional aminoacyl tRNA synthetase EPRS. During eIF2α~P, translational regulation of EPRS is suggested to occur through the bypass of a non-canonical upstream ORF encoded by a CUG start codon, highlighting the diversity by which upstream translation initiation events can regulate expression of a downstream coding sequence. This body of work provides for a better understanding of how translational control during stress is modulated genome-wide and for the processes by which this mode of gene regulation in the ISR contributes to cell fate.

Indiana University-Purdue University Indianapolis (IUPUI) / Giant axonal neuropathy (GAN) is a rare genetic disease that causes progressive damage to the nervous system. Neurons in GAN patients develop an abnormal organization of cytoskeletal proteins called intermediate filaments (IFs), which normally provide strength and support for the overall cell structure. The irregular IF structure in GAN patient neurons leads to a progressive loss of motor skills in children and subsequent death in adolescence. GAN is caused by reduced levels of the gigaxonin (Giga) protein. Giga functions to control the degradation of other cellular proteins, and the loss of Giga in GAN cells results in significantly elevated levels of the galectin-1 (Gal-1) protein. Gal-1 stabilizes the active form of the Ras signaling protein, which functions as a molecular switch to regulate the phosphorylation and subsequent organization of IFs. The connection between these pathways led us to propose that Giga regulates IF phosphorylation and structure by modulating Ras signaling through the degradation of Gal-1. Using GAN patient cells, we demonstrated that restoring Giga reduced Gal-1 protein levels, decreased IF phosphorylation, and reestablished normal IF organization. Similar effects of reduced IF phosphorylation and improved IF structure were also obtained in GAN cells by directly decreasing the protein levels of either Gal-1, or downstream Ras signaling proteins. Taken together, these results demonstrate that the loss of Giga induces Gal-1 mediated activation of Ras signaling, thereby leading to the increased IF phosphorylation and abnormal IF structure observed in GAN cells. Identification of aberrant Ras signaling is significant because it is the first to specify a mechanism by which the loss of Giga leads to the development of GAN and provides targets for novel drug therapies for the treatment of this currently immedicable genetic disease.