Astrocytes, the most abundant glial cell type in the brain, undergo a number of roles in brain physiology; among them, the energetic support of neurons is the best characterized. Contained within astrocytes is the brain’s obligate energy store, glycogen. Through glycogenolysis, glycogen, a storage form of glucose, is converted to pyruvate that is further reduced to lactate and transferred to neurons as an energy source via MCTs. Glycogen is a multi-branched polysaccharide synthesized from the glucose uptaken in astrocytes. It has been shown that glycogen accumulates with age and contributes to the physiological ageing process in the brain. In this study, we compared glycogen distribution between young adults and geriatric mice to understand the energy consumption of synaptic terminals during ageing using computational tools. We segmented and densely reconstructed neuropil and glycogen granules within six (three 4 month old old and three 24 month old) volumes of Layer 1 somatosensory cortex mice brains from FIB-SEM stacks, using a combination of semi-automated and manual tools, ilastik and TrakEM2. Finally, the 3D visualization software, Blender, was used to analyze the dataset using the DBSCAN and KDTree Nearest neighbor algorithms to study the distribution of glycogen granules compared to synapses, using a plugin that was developed for this purpose. The Nearest Neighbors and clustering results of 6 datasets show that glycogen clusters around excitatory synapses more than inhibitory synapses and that, in general, glycogen is found around axonal boutons more than dendritic spines. There was no significant accumulation of glycogen with ageing within our admittedly small dataset. However, there was a homogenization of glycogen distribution with age and that is consistent with published literature. We conclude that glycogen distribution in the brain is not a random process but follows a function distribution.

To prevent damaging ATP depletion during periods of intense activity‚ intrinsic mechanisms within skeletal muscle are activated and lead to myoprotection; a process known as muscle fatigue. It has been proposed that the primary mechanism of fatigue is a submaximal sarcoplasmic reticulum Ca2+ release and decreased force generation‚ however‚ what triggers this mechanism remains controversial. It is possible that glycogen may act as a trigger as studies have repeatedly shown a direct correlation between glycogen content at the beginning of activity and time to fatigue. In previous studies‚ a fatigue bout and/or period of fasting to deplete glycogen was used. However, this leaves investigators to differentiate between the effects of glycogen depletion methodology causing a metabolic stress and effects of glycogen itself. One objective of this M.Sc. project was to produce a low glycogen model without a prior metabolic stress that could forgo these limitations. It was extended to differentiate between the role of glycogen and extracellular glucose during fatigue as well as fatigue pre-conditioning (FPC)‚ a phenomenon in which fatigue resistance increases for about 2 hours after a first fatigue bout. During a single‚ first fatigue bout (one contraction every sec for 3 min) a mean decrease in glycogen from 95 to 20 μmol/g dry wt. had no effect on the decrease in tetanic [Ca2+]i‚ i.e. the [Ca2+]i during tetanic contractions‚ whereas removing glucose from the physiological solution led to a 46% greater decrease in tetanic [Ca2+]i than when glucose was present. During a subsequent fatigue bout (i.e. FPC) a greater amount of glycogen was used as glycogen content was 27% greater than prior to the first fatigue bout. When glycogen and/or glucose was limited‚ FPC was abolished. It is concluded that extracellular glucose is critical to prevent fatigue. Additionally, whereas glycogen is important for FPC‚ it appears to be much less important during a first fatigue bout initiated in absence of any prior metabolic stress.

Orientador: Maria Célia Bertolini / Banca: Iran Malavazi / Banca: Sergio Akira Uyemura / Resumo: O fungo filamentoso Neurospora crassa é um organismo modelo utilizado na compreensão de diversos aspectos da biologia dos eucariotos, e tem sido usado, em nosso laboratório, para estudos celulares básicos, como os mecanismos bioquímicos e moleculares envolvidos na regulação do metabolismo de glicogênio. Uma análise sistemática realizada com uma coleção de linhagens mutantes em genes codificadores de fatores de transcrição permitiu identificar várias proteínas potencialmente envolvidas na regulação do metabolismo do glicogênio neste organismo. Algumas linhagens mutantes apresentaram alterações no perfil de acúmulo de glicogênio e na expressão dos genes que codificam as enzimas glicogênio sintase (gsn) e glicogênio fosforilase (gpn) quando comparadas à linhagem selvagem. Dentre estas, duas linhagens mutantes em genes que codificam para a proteína RCO-1 (regulator of conidiation-1) e para uma proteína hipotética foram selecionadas para o presente estudo, levando em consideração que ambas linhagens também apresentaram variações na progressão do ciclo celular quando analisadas por citometria de fluxo. Como a proteína RCO-1 é uma provável parceira da proteína RCM-1 (regulator of conidiation and morphology-1), então a linhagem mutante no gene codificador de RCM-1 foi incluída neste trabalho. Portanto, foi feita a caracterização de um fator de transcrição anotado como proteína hipotética e de dois cofatores transcricionais RCO-1 e RCM-1, ortólogos ao complexo corepressor Tup1-Ssn6 de Saccharomyces cerevisiae. As proteínas RCO-1, RCM-1 e a codificada pela ORF NCU09739 estão envolvidas na regulação do metabolismo do glicogênio, atuando na regulação da expressão dos genes gsn e/ou gpn. Estas mesmas proteínas também são necessárias para o crescimento e desenvolvimento normal do... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The filamentous fungus Neurospora crassa is a model organism used to understand various aspects of eukaryotic biology. It has been used, in our laboratory, in basic cellular studies, such as the biochemical and molecular mechanisms involved in the regulation of glycogen metabolism. A systematic analysis performed with a collection of mutant strains in genes encoding transcription factors led to the identification of proteins likely involved in the regulation of glycogen metabolism in this organism. Some mutant strains showed changes in the glycogen accumulation profile and in the expression of the genes encoding the enzymes glycogen synthase (gsn) and glycogen phosphorylase (gpn) when compared to the wild-type strain. Among these, two mutant strains in the genes encoding RCO-1 (regulator of conidiation-1) and a hypothetical proteins were selected for the present study. Both strains presented variations in cell cycle progression when analyzed by flow cytometry. RCO-1 protein is likely a partner of RCM-1 (regulator of conidiation and morphology-1) protein, thus the mutant strain in the gene encoding RCM-1 was included in this work. Therefore, we performed the characterization of a transcription factor annotated as a hypothetical protein and the two transcriptional cofactors RCO-1 and RCM-1, orthologs of the Saccharomyces cerevisiae corepressor complex Tup1-Ssn6. RCO-1, RCM-1 and the product of the ORF NCU09739 are involved in the regulation of glycogen metabolism, acting in the regulation of gsn and/or gpn gene expression. The same proteins are necessary for growth and normal development of the fungus, since the mutant strains showed changes in hyphae length, pigmentation and conidiation. Gene expression analysis showed that the NCU09739 gene was highly expressed at the beginning of the conidia germination, showing the importance... (Complete abstract click electronic access below) / Mestre

Orientador: Maria Célia Bertolini / Banca: Paulo Sergio Rodrigues Coelho / Banca: Otavio Henrique Thiemann / Banca: Gustavo Henrique Goldman / Banca: Aline Maria da Silva / Resumo: Resultados prévios, em nosso laboratório, permitiram identificar fatores de transcrição provavelmente atuando como reguladores do metabolismo de glicogênio no fungo Neurospora crassa utilizando uma coleção de linhagens mutantes do fungo contendo ORFS codificadoras de fatores de transcrição individualmente nocauteadas. O presente trabalho teve como objetivo realizar análises funcionais de alguns dos fatores de transcrição anteriormente identificados, tais como o regulador transcricional XlnR, descrito em outros fungos filamentosos como regulador de genes codificadores de enzimas xilanolíticas e/ou celulolíticas e o produto da ORF NCU04390, uma proteína sem função conhecida. Inicialmente foram realizadas análises de acúmulo de glicogênio e da expressão dos genes gsn (codificador da enzima glicogênio sintase) e gpn (codificador da enzima glicogênio fosforilase) durante o crescimento vegetativo (30ºC) e sob condição de estresse térmico (45ºC) nas linhagens mutantes e comparadas à linhagem selvagem. Ambas as linhagens mutantes apresentaram alterações tanto no conteúdo de glicogênio como na expressão dos genes citados anteriormente, o que indicou o possível envolvimento dos fatores de transcrição na regulação do metabolismo do carboidrato. Foram realizados experimentos com o objetivo de investigar a função do fator de transcrição XlnR na regulação do metabolismo de glicogênio. Foi verificado que esta proteína regula os níveis do carboidrato, bem como a expressão do gene gsn quando o fungo foi crescido em fontes alternativas de carbono. Entretanto, o fator de transcrição XLR-1 de N. crassa produzido na forma recombinante em E. coli (inteiro e truncado) não foi capaz de ligar ao motif presente no promotor gsn. Genes codificadores de duas endoxilanases foram identificados no genoma de N. crassa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Previous results from our laboratory using a collection of Neurospora crassa strains mutated in genes encoding transcription factors allowed us to identify transcription factors likely involved in glycogen metabolism regulation. The present work aimed to perform a functional analysis of some transcription factors previously identified, such as the transcriptional regulator XlnR, described in many filamentous fungi as a transcriptional regulator of xylanolitic and/or cellulolitic genes and a protein with unknown function, the ORF NCU04390 product. Initially, analysis of glycogen accumulation and gsn (coding glycogen synthase) and gpn (coding glycogen phosphorylase) gene expression were performed under vegetative growth (30 °C) and under heat shock cond ition (45 ºC) in the two mutant strains and compared to the wild-type strain. Both mutant strains showed changes in the glycogen levels as well as in the gsn and gpn expression, suggesting an involvement of these transcription factors in the regulation of glycogen metabolism. Additional analysis was performed in order to investigate the function of the XlnR transcription factor. It was observed that this transcription factor regulates the glycogen levels as well as the gsn expression under alternative carbon sources. However, the recombinant XLR-1 transcription factor produced in E. coli (entire and truncated proteins) was not able to bind to the XLR-1 motif present in the gsn promoter. Two endoxylanases coding genes were identified in the N. crassa genome database and analysis of their gene expression showed the upregulation of both genes during growth in xylan instead of xylose as carbon sources. This result suggested that the XLR-1 transcription factor is not the only protein that regulates the endoxylanases genes. The transcription factor encoded by NCU04390 ORF was... (Complete abstract click electronic access below) / Doutor

Glioblastoma is the most malignant form of brain cancer. Due to its aggressive nature, extensive research has been performed, but little progress has been made in identifying effective treatment options. Glycogen synthase kinase-3 (GSK-3) is a ubiquitous, multifaceted protein kinase. Previous studies have shown that small molecule inhibitors of GSK-3 block the migration of glioblastoma cells and may prevent spread of tumor in the brain. However, these studies were performed using non-selective GSK-3 inhibitors (LiCl and an indirubin derivative, BIO); thus, it was unclear whether GSK-3 was the most important target. In this study, we used recently generated highly selective GSK-3 inhibitors (CHIR99021, AZD1080, and AZD2858, as well as BIO) to investigate these questions. These were applied to four glioblastoma cell lines: G30, G9, U251, and U1242, in three migration assays: transwell, spheroid, and wound healing (scratch) assay to further assess the suitability of GSK-3 as a target in glioblastoma. We also utilized the ATP Luciferase reporter assay for cell viability to assess the influence of our panel of drugs on cell migration versus viability. In addition, the TOPFlash Luciferase reporter assay was performed as an indicator of the level of GSK-3 inhibition. The TOPFlash assay showed that all GSK-3 inhibitors were able to increase luciferase levels. This indicates that GSK-3 was inhibited in our cells after drug treatment. The transwell assays showed us that the GSK-3 inhibitors were able to block migration significantly in all cell lines tested in a dose-dependent manner. The effectiveness of GSK-3 inhibition in the three-dimensional collagen spheroid assays was cell line-dependent, with the non-selective GSK-3 inhibitor BIO showing the most potent effects. Cell migration was not blocked by any of the three selective GSK-3 inhibitors in the wound healing scratch assay. Thus we have found that the three distinct highly selective inhibitors of GSK-3 block glioblastoma cell migration, but only work consistently in the transwell assay. Therefore, we conclude that GSK-3 might be important in the contraction and morphological changes necessary for glioblastoma cells to migrate through the 8 micron pores in the transwell. Further investigation into this observation is necessary. Though results were variable between assays, we conclude that the inhibition of GSK-3 is a promising potential therapeutic strategy for glioblastoma treatment.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-07-11Bitstream added on 2014-06-13T20:41:02Z : No. of bitstreams: 1 goncalves_rd_dr_araiq_parcial.pdf: 284569 bytes, checksum: f7e48a4c9ff942697ece9e42f05b6fea (MD5) Bitstreams deleted on 2015-06-25T13:01:23Z: goncalves_rd_dr_araiq_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:34Z : No. of bitstreams: 1 000713132_20161231.pdf: 275112 bytes, checksum: 63f307a03521b081835abe229f60fbf4 (MD5) Bitstreams deleted on 2017-01-02T15:03:46Z: 000713132_20161231.pdf,. Added 1 bitstream(s) on 2017-01-02T15:05:06Z : No. of bitstreams: 1 000713132.pdf: 8332325 bytes, checksum: 76068ad08200d9297dc8e7da373fb7ed (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Resultados prévios, em nosso laboratório, permitiram identificar fatores de transcrição provavelmente atuando como reguladores do metabolismo de glicogênio no fungo Neurospora crassa utilizando uma coleção de linhagens mutantes do fungo contendo ORFS codificadoras de fatores de transcrição individualmente nocauteadas. O presente trabalho teve como objetivo realizar análises funcionais de alguns dos fatores de transcrição anteriormente identificados, tais como o regulador transcricional XlnR, descrito em outros fungos filamentosos como regulador de genes codificadores de enzimas xilanolíticas e/ou celulolíticas e o produto da ORF NCU04390, uma proteína sem função conhecida. Inicialmente foram realizadas análises de acúmulo de glicogênio e da expressão dos genes gsn (codificador da enzima glicogênio sintase) e gpn (codificador da enzima glicogênio fosforilase) durante o crescimento vegetativo (30ºC) e sob condição de estresse térmico (45ºC) nas linhagens mutantes e comparadas à linhagem selvagem. Ambas as linhagens mutantes apresentaram alterações tanto no conteúdo de glicogênio como na expressão dos genes citados anteriormente, o que indicou o possível envolvimento dos fatores de transcrição na regulação do metabolismo do carboidrato. Foram realizados experimentos com o objetivo de investigar a função do fator de transcrição XlnR na regulação do metabolismo de glicogênio. Foi verificado que esta proteína regula os níveis do carboidrato, bem como a expressão do gene gsn quando o fungo foi crescido em fontes alternativas de carbono. Entretanto, o fator de transcrição XLR-1 de N. crassa produzido na forma recombinante em E. coli (inteiro e truncado) não foi capaz de ligar ao motif presente no promotor gsn. Genes codificadores de duas endoxilanases foram identificados no genoma de N. crassa... / Previous results from our laboratory using a collection of Neurospora crassa strains mutated in genes encoding transcription factors allowed us to identify transcription factors likely involved in glycogen metabolism regulation. The present work aimed to perform a functional analysis of some transcription factors previously identified, such as the transcriptional regulator XlnR, described in many filamentous fungi as a transcriptional regulator of xylanolitic and/or cellulolitic genes and a protein with unknown function, the ORF NCU04390 product. Initially, analysis of glycogen accumulation and gsn (coding glycogen synthase) and gpn (coding glycogen phosphorylase) gene expression were performed under vegetative growth (30 °C) and under heat shock cond ition (45 ºC) in the two mutant strains and compared to the wild-type strain. Both mutant strains showed changes in the glycogen levels as well as in the gsn and gpn expression, suggesting an involvement of these transcription factors in the regulation of glycogen metabolism. Additional analysis was performed in order to investigate the function of the XlnR transcription factor. It was observed that this transcription factor regulates the glycogen levels as well as the gsn expression under alternative carbon sources. However, the recombinant XLR-1 transcription factor produced in E. coli (entire and truncated proteins) was not able to bind to the XLR-1 motif present in the gsn promoter. Two endoxylanases coding genes were identified in the N. crassa genome database and analysis of their gene expression showed the upregulation of both genes during growth in xylan instead of xylose as carbon sources. This result suggested that the XLR-1 transcription factor is not the only protein that regulates the endoxylanases genes. The transcription factor encoded by NCU04390 ORF was... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-09-03Bitstream added on 2014-06-13T19:19:50Z : No. of bitstreams: 1 paula_rm_dr_araiq.pdf: 2077296 bytes, checksum: aa9a17baf3828015b873a455a57c08c8 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Glicogênio representa um dos principais carboidratos de reserva em muitos organismos e seu metabolismo está sob o controle de um complexo mecanismo envolvendo o balanço de nutrientes e sinais ambientais. A proteína glicogenina constitui a molécula iniciadora do processo de síntese de glicogênio, sendo as etapas seguintes efetuadas pelas enzimas glicogênio sintase e enzima ramificadora. Glicogênio sintase é a enzima limitante no processo e é regulada alosterismo e fosforilação reversível. Neste trabalho foi realizada uma caracterização do metabolismo de glicogênio no fungo N. crassa enfocando as enzimas glicogenina, glicogênio sintase e glicogênio sintase quinase-3. A proteína glicogenina (GNN) foi caracterizada do ponto de vista molecular, bioquímico e funcional. O cDNA codificando para esta proteína foi isolado e a seqüência polipeptídica deduzida mostrou uma proteína de 664 aminoácidos, uma das maiores proteínas glicogenina já isoladas. A inativação do gene gnn resultou em uma linhagem mutante incapaz de acumular glicogênio. A produção da proteína GNN em E. coli resultou em um polipeptídeo altamente susceptível à proteólise e formas truncadas da proteína mostraram ser mais estáveis e igualmente ativas nos processos de auto- e trans-glicosilação, além de servirem de substrato para ação da glicogênio sintase. Tais formas também foram capazes de complementar funcionalmente mutantes de S. cerevisiae. Além disso, a expressão do gene gnn foi mostrado ser regulado durante crescimento vegetativo e deprivação de carbono. Os resíduos Tyr196 e Tyr198 foram identificados como os sítios de glicosilação, os quais contribuem diferencialmente para este processo. Análise das interações entre GNN e a proteína glicogênio sintase de N. crassa (GSN) demonstrou que a região C-terminal da GNN é a mais importante para a interação. Entretanto, o... / Glycogen represents one of the main reserve carbohydrates in many organisms and its metabolism is under control of a complex mechanism involving the balance of nutrients and environmental signals. The protein glycogenin is the initiator molecule in glycogen biogenesis and the subsequent steps are carried out by the enzymes glycogen synthase and branching enzyme. Glycogen synthase is the rate-limiting enzyme in the process and is regulated both by alosterism and reversible phosphorylation. In this work we performed a characterization of the glycogen metabolism in the filamentous fungus Neurospora crassa, focusing on the enzymes glycogenin, glycogen synthase and glycogen synthase kinase-3. The protein glycogenin (GNN) was characterized under the molecular, biochemical and functional aspects. The cDNA encoding for this protein was isolated and the deduced polypeptide sequence showed a protein with 664 residues, one of the largest glycogenins isolated so far. The inactivation of the gnn gene rendered a mutant strain that was no longer able to accumulate glycogen. The production of GNN protein in E. coli cells resulted in a polypeptide highly susceptible towards proteolysis and truncated forms were more stable and equally active, judged by their abilities to self- and trans-glucosylate, and to serve as substrate for glycogen synthase elongation. These proteins were also able to recover the glycogen deficiency phenotype in a S. cerevisae mutant strain. Moreover, the gnn gene expression was shown to be ...(Complete abstract, click electronic access below)

The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.

The intracellular delivery of molecules to modulate signaling pathways and gene expression is a powerful approach to control stem cell fate decision. For applications in gene therapy and regenerative medicine, the use of genetic material and viral vectors raise concerns because stem cells persist throughout life, and long-term effects of uncontrolled genetic modifications could affect the cellular progeny. An alternative is to deliver directly peptides or proteins using cell-permeable peptides (CPPs) which have the ability of crossing the plasma membrane and carrying cargos into cells. CPPs can therefore be used to deliver factors to direct stem cells proliferation, survival and differentiation. This thesis describes an approach to control stem cell fate based on the delivery of a CPP-conjugated bioactive peptide. A first significant contribution from this work is the development of a flow cytometric assay to accurately quantify the uptake of a panel of CPPs. This study revealed that HIV-transactivator of transcription (TAT) and Antennapedia (Antp) offered the highest level of translocation in different cell types. The uptake was improved by treating the cells with a single, low-voltage electrical pulse that selectively enhances the amount of TAT-conjugated peptides and proteins delivered by at least an order of magnitude, without causing cellular toxicity or apoptosis. Subsequently, flow cytometry, confocal microscopy, capillary electrophoresis and mass spectrometry were used to examine the intracellular fate of TAT-conjugated peptides in order to define the parameters that limit their bioactivity and point to specific sequence modifications that can improve their efficacy. The advances described in this thesis were applied to the development of TAT-eIF2B, a peptide-inhibitor of glycogen synthase kinase-3 (GSK-3). TAT-eIF2B was found to be specific for GSK-3 and had a significant positive effect on the formation of neurospheres in embryonic stem cell cultures and on the survival of myeloid progenitors in cytokine-starved fetal liver cell cultures. On the other hand, GSK-3 inhibition reduced the number of neurospheres generated by human olfactory neuroepithelium cells due to lower proliferation and increased neuronal differentiation. In summary, this work describes the development of a peptide-based technology to deliver bioactive cargoes in cells, and it demonstrates its utility for modulating the activity of a master regulator of stem cell fate decision.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2002. / Bibliography: leaves 131-146.