Aromatase is an enzyme that converts androgens to estrogens. In teleosts, brain aromatase, also known as aromatase B (cy19a1b), is only expressed in radial glial cells (RGCs). This is in contrast to aromatase A, which is expressed in gonads. Estrogens such as estradiol (E2) modulate neurogenesis in the adult teleost brain. Recent studies show that E2 also differentially regulates aromatase B expression in goldfish RGCs. As a result, teleost RGCs are suggested to be involved in regulating neurogenesis. In addition, aromatase B expression in goldfish RGC is under the control of dopamine suggesting that neurons and neurotransmitters can regulate RGC function. Interestingly, goldfish RGC transcriptome data shows the expression of one group of metabotropic glutamate receptors (mGluRs), group III mGluRs, which suggests that glutamate may affect RGC function. In this thesis, I present my findings regarding potential glutamatergic regulation of RGCs. Firstly, I investigated the distribution of glutamatergic synaptic vesicles and RGCs in the female goldfish forebrain. Double-staining immunohistochemistry shows that vesicular glutamate transporter (vGLUT) 1/2-labelled glutamatergic synaptic vesicles are in close anatomical proximity to aromatase B-labelled RGCs, which suggests potential regulation of RGCs by glutamate. Glutamatergic regulation of cyp19a1b, cyclin D1 (ccnd1), cyclin A2 (ccna2), mGluR6b (grm6b), mGluR7 (grm7), and mGluR8b (grm8b) expression in cultured adult female goldfish RGCs was also examined. Results from pharmacological manipulations and qPCR data analysis show that selective activation of group III mGluRs decreased cyp19a1b, ccnd1, and ccna2 mRNA via inhibition of cAMP/PKA signalling. Furthermore, grm7 mRNA is positively regulated by cAMP-dependent signalling. The glutamate analog L-glutamic acid decreased cyp19a1b mRNA and increased ccnd1 and grm6b mRNA in a dose-dependent manner. This suggests that ccnd1 and grm6b expression may be regulated by glutamate receptors other than group III mGluRs, for example, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are expressed in cultured goldfish RGCs. It was found that E2 upregulated cyp19a1b, ccnd1 and grm7 mRNA. However, selective activation of group III mGluRs decreases the stimulatory effect of E2 on ccnd1 expression. My findings show that glutamate finely regulates RGC neurogenic and steroidogenic genes, which may implicate glutamate in the regulation of RGC differentiation, RGC proliferation, and neurogenesis in surrounding cells.

The common human GNB3 825C > T variant, which is present in 50% of the world’s chromosomes, has previously been shown to predispose individuals to hypertension, cardiac and neural disorders. This variant causes the production of a stable and gain of function protein Gβ_3S- This thesis describes the discovery of a novel D153del mutation that produces an unstable, loss of function, protein Gβ_3D in the recessively inherited, retinopathy globe enlarged (rge) chickens. This thesis also demonstrates that the normal Gβ₃ downstream phosphorylation signalling pathways are significantly altered in a tissue specific manner in rge chicken organs and in a human GNB3 825TT lymphoblast cell line. In rge tissues expressing Gβ_3D protein, the cAMP induced GRK2 phosphorylation activity is significantly altered. Moreover MAPK1 (ERK2) phosphorylation is significantly decreased compared to normal tissues. In contrast human 825TT cell lines expressing the Gβ_3S protein, showed enhanced cAMP induced GRK2 and MAPK (ERK1 and ERK2) phosphorylation activity. These results confirm previous findings of 825C > T Gβ₃ studies, that Gβ_3S is indeed a hyper-activating structural variant, in contrast to the D153del Gp3D is a classical recessively inherited non-functional mutation.

No description available.

The Tec family tyrosine kinases Itk, Tec and Rlk are expressed in T cells. Previous studies have established that these kinases are critical for TCR signaling, leading to the activation of PLCγ1. To further understand the functions of Tec kinases in T cell activation, we took three different approaches. First, we performed a thorough analysis of CD28-mediated signaling events and functional responses with purified naïve T cells from Itk-/- mice and a highly controlled stimulation system. Data from this set of studies definitively demonstrate that CD28 costimulation functions efficiently in naïve CD4+ T cells in the absence of Itk. Second, in order to further study the functions of Tec kinases in vivo, we generated transgenic mouse lines expressing a kinase-dead (KD) mutant of Tec on the Itk-/-Rlk-/- background, hoping to study mice that are functionally deficient for all three Tec kinases. The results hint the importance of the Tec kinases in T cell development and/or survival. Finally, in order to identify potential transcriptional targets of Itk, we used microarray technology to compare global gene expression profiles of naïve and stimulated Itk-/- versus Itk+/- CD4+ T cells. This analysis provided a short list of differentially expressed genes in Itk-/- versus Itk+/- CD4 T cells, providing a starting point for further studies of Itk in T cell activation. Collectively, these studies clarified the role of Itk in CD28 signaling, revealed some unexpected aspects of Tec family kinases in T cells, and indicated potential targets of Itk-dependent signaling pathways in T cells.

Endocytosis is important for the regulation of signal transduction and for the movement of essential cellular components from outside the cell to their appropriate intracellular compartment(s). Two established mechanisms of endocytosis are clathrinmediated (CME) and clathrin-independent endocytosis, and they are responsible for internalization of different ligands. In this study, the newly established technique of total internal reflection fluorescent microscopy (TIRF-M) was used, along with standard biochemical and molecular biological tools, to systematically study the sorting and early trafficking of two established ligands of endocytosis, transferrin (Tf) and epidermal growth factor (EGF). TIRF-M studies revealed that Tf binds its receptor that is located in large clathrin arrays positioned just below the surface of the cell and that these large clathrin platforms serves as the major site of CME at the plasma membrane. EGF endocytosis is very different and occurs as follows 1) the liganded EGFR recruits Rab5 to the plasma membrane, 2) Rab5 concentrates around vesicles containing liganded EGFR and 3) these vesicles co-localize with EEA1 enriched endosomes. EEA1 was shown to play a pivotal role in EGF endocytosis, establishing a new role for EEA1 in vesicle trafficking in addition to its role in tethering and fusion. Finally, WDFY2, a new FYVE domain protein was shown to decorate a specific subset of vesicles, upstream of the EEA1 vesicle pool that appear to participate in Tf endocytosis. These studies establish new functions and components of endocytosis that enhances our understanding of this complex process.

According to the cancer stem cells (CSCs) hypothesis, CSCs are responsible for the treatment failures. CSCs are a subset of cells possessing stemness properties within the heterogeneous tumor mass. Therapeutic intervention on Wnt signaling is of our great interest because an aberrant Wnt signaling is an important driver to maintain the potency of CSCs. In nasopharyngeal carcinoma (NPC), deregulated expression of the Wnt signaling components is frequently observed. ICG-001 is a selective Wnt modulator (CBP antagonist) that specifically interrupts the interaction between β-catenin and CBP, thereby encourages the interaction between β-catenin and p300 and the subsequent differentiation and reduction of the CSCs subset. For this reason, the present study aimed to evaluate the therapeutic potential of ICG-001 in NPC. Results showed that ICG-001 inhibited both the migration of the NPC cells and the formation of tumor spheres. In the first part of the mechanistic studies (Chapter 3), ICG-001 was found to restore the expression of miR-150 in NPC cells. MiR-150 was further found to directly reduce CD44 expression and inhibit NPC cell migration. In the second part of the mechanistic studies (Chapter 4), ICG-001 was found to reduce the expression of Evi1 in NPC cells. The effect was accompanied with the inhibition of both the NPC cells migration and the tumor spheres formation. Two molecular axes, namely miR-96/Evi1/miR-449a and survivin/Evi1/miR-449a, were found to be involved in the inhibition of the tumor cell migration and spheroids formation. The therapeutic potential of using this CBP antagonist (ICG-001) in NPC, namely the in vitro and in vivo efficacy of ICG-001 combined with cisplatin, was examined (Chapter 5). Concurrent treatment of ICG-001 and cisplatin exhibited a synergistic inhibition on the in vitro growth and the tumor sphere forming capacity of NPC cells as well as the growth of NPC xenografts. Taken together, results presented in this thesis suggested that ICG-001 (PRI-724 is the analog of ICG-001 currently used in clinical trials) has a therapeutic potential in NPC.

Orientadores: Carmen Verissima Ferreira, Maikel Petrus Peppelenbosch / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T22:42:24Z (GMT). No. of bitstreams: 1 Queiroz_KarlaCristianedeSouza_D.pdf: 5700883 bytes, checksum: 11ab46407bbf600912fa23adf313688d (MD5) Previous issue date: 2007 / Resumo: A eficiência do tratamento do câncer sob vários aspectos, mesmo com os avanços ¿farmacotecnológicos¿, ainda permanece como desafio para a medicina. Diante desse fato, novos agentes que atuem de forma alvo-específico, apresentem poucos efeitos colaterais e possam impedir o ¿escape¿ das células tumorais à indução de morte, são extremamente desejáveis. No presente trabalho, foram abordados 3 aspectos da atividade antitumoral da riboflavina: indução da apoptose das células de câncer prostático, indução da diferenciação de células leucêmicas e aumento da biodisponibilidade intracelular do quimioterápico mitoxantrona. Sob esses 3 aspectos, através do estudo de vias de sinalização, identificamos mediadores moleculares responsáveis pela ação da riboflavina como antitumoral. A atuação da riboflavina irradiada em células de câncer de próstata foi dependente da inibição da PI3K. De forma interessante, observamos uma potencial ação inibitória da metástase, evidenciada pela inibição das metaloproteinases 2 e 9 e da angiogênese pela diminuição da expressão do VEGF. Em relação à diferenciação das células leucêmicas evidenciamos o envolvimento do receptor TNFR1, bem como das proteínas ciclina D, JNK, Src quinase e das proteínas tirosinas fosfatases SHP2 e PTP!. Portanto, proteínas com diferentes localizações celulares foram afetadas, culminando com a diminuição da proliferação, manutenção da sobrevivência celular, interrupção da progressão do ciclo celular e reorganização do citoesqueleto, efeitos metabólicos essenciais para a ocorrência da diferenciação. Esse trabalho de tese também demonstrou a aplicabilidade da técnica do Pepchip para a identificação de diferenças metabólicas entre duas linhagens das células da leucemia eritroblástica, K562 e Lucena. Outra abordagem interessante nesse trabalho foi o uso da riboflavina com a finalidade de aumentar a biodisponibilidade celular de quimioterápicos tradicionais e a utilização de inibidores de proteínas fosfatases como estratégia para reverter a resistência de células tumorais. De acordo com os resultados, esse trabalho aponta de forma inédita uma nova função da vitamina B2 como antitumoral / Abstract: Cancer therapy efficiency, under several aspects, even with the progress of ¿pharmacotechnology¿, remains as a challenge for the medicine. According to this factor, new agents that act on specific target, present low side-effects and prevent cancer cells ¿escaping¿ from death induction, are extremely desirable. In this work, 3 aspects of antitumoral property of riboflavin were evaluated: apoptosis induction of prostate cancer cells, leukaemic cells differentiation and increase of intracellular bioavailability of chemotherapic (mitoxantrone). Under these 3 aspects, and by signal transduction approach, we identified molecular mediators responsible for antitumoral activity of riboflavin. The action of irradiated riboflavin on prostate cancer cells was dependent on PI3K inhibition. Interestingly, we also observed a potential inhibitory action of metastasis, as demonstrated by the inhibition of metalloproteinases 2 and 9 and decreasing of angiogenesis by downregulation of VEGF. In relation to leukaemic cells differentiation we demonstrated the involvement of TNFR1, as well as cyclin D, JNK, Src kinase and protein tyrosine phosphatases SHP2 and PTP!. Therefore, proteins with different cellular localizations were affected culminating in decreasing of cell proliferation, maintaining cell survival, cell cycle arrest and cytoskeleton rearrangement, crucial metabolic effects for the occurrence of differentiation process. This work also demonstrated the applicability of Pepchip technique for identifying the differences between 2 erytroblastic leukaemia cell lines, K562 and Lucena. Other interesting approach, in this work, was the use of riboflavin for improving chemotherapic cellular bioavailability and the strategical use of protein phosphatase inhibitors for reverting tumor cells resistance. According to our findings, this work spotlights the novel function of the vitamin B2 as an antitumoral agent / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Orientador: Otávio Rizzi Coelho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T21:29:46Z (GMT). No. of bitstreams: 1 Tornatore_ThaisFranchini_D.pdf: 5344920 bytes, checksum: 13bb7f4ae7264a0aca117131579c9b8e (MD5) Previous issue date: 2011 / Resumo: Estudamos o gene da FAK (focal adhesion kinase) na mitocondriogênese cardíaca induzida por estresse mecânico. O estresse prolongado (2-12 hs) de miócitos do ventrículo esquerdo de ratos neonatos(NRVM) aumentou a expressão do regulador transcricional mitocondrial PGC-1? (peroxisome proliferator activated receptor coactivator-1), NRF-1 (nuclear respiratory factor) e o Tfam (mitochondrial transcription factor A). A ativação da cascata transcricional mitocondrial em cardiomiócitos estirados foi acompanhada pelo aumento da mitocondriogênese (aumento da densidade mitocondrial e número de cópias do DNA - mtDNA) e hipertrofia(tamanho da célula e transcrição do ANP). Estresse mecânico também aumentou a fosforilação da FAK, a localização nuclear e associação com PGC1-?. FAK C-terminal Recombinante, mas não a N-terminal ou domínio kinase, precipitaram PGC-1? em extratos nucleares de NRVMs. Além disso, a inibição da expressão da FAK por RNA de interferência suprimiu a hiper regulação do PGC-1? e NRF-1, e atenuou o aumento da mitocondriogênese e hipertrofia em cardiomiócitos. Ao mesmo tempo, a inibição da FAK reduziu os níveis de ATP em cardiomiócitos estirados. Estudos complementares em ventrículos esquerdo de camundongos adultos revelaram aumento da expressão de PGC-1?, NRF-1 e mtDNA em resposta à sobrecarga pressórica. In vivo o silenciamento da FAK atenuou a hiper regulação do PGC-1?, NRF-1, mtDNA e a hipertrofia do ventrículo esquerdo induzida por sobrecarga pressórica. A ativação da sinalização da FAK parece ter importante relação no aumento da mitocondriogênese paralelamente à resposta do aumento hipertrófico relacionado ao estresse mecânico dos cardiomiócitos / Abstract: We studied the implication of FAK (focal adhesion kinase) in cardiac mitochondriogenesis induced by mechanical stress. Prolonged stretching (2-12 hs) of neonatal rat ventricular myocytes (NRVM) enhanced the expression of mitochondria transcriptional regulator PGC-1? (peroxisome proliferator activated receptor coactivator-1), NRF-1 (nuclear respiratory factor) and transcripts of Tfam (mitochondrial transcription factor A). The activation of the mitochondria transcriptional cascade in stretched NRVM was accompanied by enhanced mitochondriogenesis (increases in mitochondrial density and DNA copy number - mtDNA) and hypertrophy (cell size and ANP transcripts). Cell stretching also enhanced FAK phosphorylation, nuclear localization and association with PGC1?. Recombinant FAK C-terminal, but not the N-terminal or kinase domain, precipitated PGC-1? from NRVM nuclear extracts. Moreover, depletion of FAK by RNA interference suppressed the upregulation of PGC-1? and NRF-1, and markedly attenuated the enhanced mitochondriogenesis and hypertrophy of stretched NRVMs. Concomitantly, FAK depletion induced a marked reduction of ATP levels of stretched NRVM. Complementary studies in adult mice left ventricle revealed enhanced expression of PGC-1?, NRF-1 and mtDNA in response to pressure overload. In vivo FAK silencing attenuated the upregulation of PGC-1?, NRF-1, mtDNA and left ventricular hypertrophy induced by pressure overload. Activation of FAK signaling seems to be important for conferring enhanced mitochondriogenesis coupled to the hypertrophying growth response to mechanical stress in cardiomyocytes / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Orientadores: Marcelo Menossi Teixeira, Eugenio Cesar Ulian. / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T07:07:14Z (GMT). No. of bitstreams: 1 Felix_JulianadeMaria_D.pdf: 2208995 bytes, checksum: 381046fcda413b39719db5fa437f223e (MD5) Previous issue date: 2006 / Resumo: Os processos associados ao acúmulo de sacarose e o metabolismo envolvido durante o crescimento e a maturação da cana-de-açúcar tem sido objeto de estudo intensivo. Uma estratégia para associar expressão gênica com uma característica desejada é comparar o perfil de expressão entre indivíduos pertencentes a uma população segregante para aquela determinada característica. Essa estratégia, denominada 'genetical genomics¿ foi utilizada nesse presente trabalho ao avaliar o perfil de expressão gênica de uma população de cana-de-açúcar que segrega para teor de sacarose. Para tal nós utilizamos cDNA microarrays que contém 1,280 elementos envolvidos em transdução de sinal, transcrição, desenvolvimento, ciclo celular, resposta à estresse e interação com patógenos, designado chip SUCAST (Sugarcane Signal Transduction). Com o objetivo de desvendar o envolvimento desses elementos no metabolismo de sacarose, foram comparados os perfis de expressão entre amostras de indivíduos de cana-de-açúcar de uma população segregante para teor de sacarose. Foram analisadas amostras de folha madura, sendo que 24 genes foram identificados como diferencialmente expressos. Também foram analisadas amostras de entrenós em desenvolvimento, sendo que 88 genes foram identificados como diferencialmente expressos em pelo menos uma amostra de entrenó analisada. Dada a complexidade genética da cana-de-açúcar, esta estratégia nos permitiu identificar genes candidatos que podem controlar vias metabólicas sinalizadas por açúcar envolvida na síntese e acúmulo de sacarose em cana-de-açúcar. E finalmente, no trabalho descrito no capítulo 4 nós procuramos por genes codificantes para proteínas tranportadoras de açúcar no banco de dados do SUCEST. A análise de seus diferentes padrões de expressão observado nesse trabalho sugerem seu possível envolvimento tanto em tecidos fonte quanto dreno, e também em diferentes estágios de desenvolvimento / Abstract: Due to the capacity of sugarcane for storing high concentrations of sucrose in the culm, the processes associated with sucrose accumulation and metabolism during sugarcane growth and maturation have been the subject of intensive study. A strategy for associating gene expression with a trait is described in which individuals exhibiting particular traits are selected from segregating populations of sugarcane and their gene expression profiles compared. In this current work we used the 'genetical genomics¿ strategy on a sugarcane population segregating for sugar content. For this purpose we used cDNA microarrays that contains 1,280 components involved in several aspects of signal transduction, transcription, development, cell cycle, stress responses and pathogen interaction, that was designed Sugarcane Signal Transduction (SUCAST) chip. In order to assess the involvement of these elements in sucrose metabolism, the expression profiles of individuals from a sugarcane population segregated for sugar content were compared. In the work described at chapter 2 we analyzed mature leaf samples, and twenty-four genes were identified as differentially expressed. We also analyzed maturing internodes samples, and 88 genes were identified as differentially expressed at least in one internode sample. Given the complex genetics of sugarcane, this strategy could identify candidate genes that may control sugar-signaling pathways involved in sucrose synthesis and accumulation in sugarcane. And finally, in the last chapter we searched for genes encoding sugar transport proteins in the SUCEST database. The differential expression patterns of these putative transporter genes in sugarcane plants observed in this work suggest that they have many roles in both source and sink tissues, and at different developmental stages / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Orientadores: Carmen Verissima Ferreira, Silvia Mika Shishido / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T19:46:07Z (GMT). No. of bitstreams: 1 Machado_Daisy_M.pdf: 1450123 bytes, checksum: e8dc961f855a7ea88daa10bb8ee00685 (MD5) Previous issue date: 2009 / Resumo: A riboflavina (RF) é uma vitamina hidrossolúvel pertencente ao complexo vitamínico B2 e precursora das coenzimas FMN e FAD. Além da função biológica como componente de coenzimas, a RF apresenta atividade antitumoral e fotossensibilizante. A proposta principal desse estudo se baseou na avaliação da fototoxicidade da RF, bem como na determinação de marcadores moleculares da inflamação, senescência e morte celular, ações deletérias normalmente desencadeadas pela radiação UVA. Os resultados obtidos indicam que a RF tem potencial aplicação na terapia fotodinâmica, levando se em conta os modelos celulares utilizados, fibroblastos (BALB/c 3T3) e queratinócitos humanos (HaCaT). As células BALB/c 3T3 tratadas com RF 6,0 mM e dose de 5 J/cm² de radiação UVA, apresentaram indução de apoptose principalmente pela via intrínseca. A indução de senescência foi evidenciada pelo aumento da p-caveolina e aumento da atividade da MMP-2 (principal protease responsável pela degradação de colágeno). De acordo com os níveis de NFkB e p- IKKa/b, a RF não alterou significativamente o processo de inflamação desencadeado pela radiação UVA. Nas células HaCaT, o tratamento com 5,0 mM de RF e dose de 5 J/cm² de radiação UVA levou a ativação da apoptose induzida tanto pela via extrínseca como pela intrínseca. O aumento nos níveis de p-caveolina, p21 e da atividade das MMPs-2 e -9 sugerem à indução de processo de senescência precoce também nos queratinócitos. Além disto, a diminuição da expressão do NF?B indica que o mesmo esteja se translocando para o núcleo e, portanto, regulando a resposta inflamatória. Outro aspecto avaliado foi a influência do copolímero F-127 na fototoxicidade da RF. A irradiação do hidrogel contendo F-127 e riboflavina manteve a propriedade fototóxica da mesma. Nossos dados sugerem que a RF apresenta potencial para uso em terapia fotodinâmica, uma vez que a mesma se mostrou fototóxica quando irradiada com doses subtóxicas de radiação UVA. / Abstract: Riboflavin (RF) is a water soluble vitamin which belongs to vitamin B2 complex, an essential precursor of FMN and FAD coenzymes. Besides being a component of coenzymes, RF displays antitumoral and photossensitizing activities. The main proposal of this study was to evaluate the RF phototoxicity, as well as to determine molecular markers of inflammation, senescence and cellular death, deleterious actions normally triggered by the UVA radiation. Taking into consideration both cell lines used as models, fibroblasts (BALB/c 3T3) and human keratinocytes (HaCaT), the results obtained indicate that RF has potential application in photodynamic therapy. BALB/c 3T3 cells treated with 6.0 µM RF associate with UVA radiation (5 J/cm²) showed apoptosis induction mainly via intrinsic pathway. An increase of p-caveolin and MMP-2 activity (major protease responsible for degradating collagen) evidenced senescence induction. According to NF?B and p-IKKa/ß levels, RF did not significantly change the process of inflammation triggered by UVA radiation, in fibroblast cells. In HaCaT cells 5.0 µM RF associated with UVA radiation (5 J/cm ²) was observed apoptosis induction thorough extrinsic and intrinsic pathways. Senescence process was also observed in keratinocytes as indicated by an increase of pcaveolin, p21 and MMPs-2 and -9 activities. Besides, the decrease of NF?B expression indicates that this transcription factor translocates into the nucleus and in turn, regulates inflammatory response. Other aspect evaluated in this work was the influence of the F-127 in the RF phototoxicity. The irradiation of hydrogel containing F-127 and RF remained RF phototoxicity property. Our findings suggest that RF displays potential for use in photodynamic therapy, once it was phototoxic when irradiated with subtoxic UVA dose / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular