Thesis (M.S.)--State University of New York at Binghamton, Department of Physics, Applied Physics and Astronomy, 2009. / Includes bibliographical references.

Thesis (M.A.)--Carleton University, 1993. / Also available in electronic format on the Internet.

Malignant brain tumors are diseases with poor prognosis and/or severe long-term side effects of treatment. This thesis aimed to discover novel regulators in brain tumor development, based on studying neural stem cell and progenitor cell (NSPC) differentiation and using animal models to introduce new insights to mechanisms of human brain tumors. The enzyme heparanase (HPSE) that degrades heparan sulfate (HS) is active in cell signaling and ECM remodeling. In paper I, we found an enhanced differentiation to oligodendrocytes in ES cell-derived NSPCs overexpressing HPSE. Further analysis suggested that this enhanced formation of oligodendrocytes was associated with alterations in receptor tyrosine kinase signaling, and that HPSE might also exert anti-apoptotic functions. Subsequently, in paper II we studied the involvement of HPSE in glioma development. We observed that high HPSE levels associated with poor survival in glioma patients. In experimental models, we found that HPSE promoted glioma growth, and that an inhibitor of HPSE reduced glioma progression both in vitro and in vivo. We hypothesize that regulators in NSPC differentiation could have a potential role in brain tumor development. In paper III, we explored the function of NRBP2, a pseudokinase that is up-regulated during NSPC differentiation. We found low expression of NRBP2 in brain tumors, in comparison to normal brain. In medulloblastoma, in particular, low NRBP2 expression is linked to poor prognosis. Overexpression of NRBP2 in medulloblastoma cells led to impaired cell growth and migration, concomitant with an increased cell death. In paper IV, we searched for novel glioma susceptibility genes by sequencing dog breeds from the same ancestor but with different glioma incidence. In this way we identified three new glioma-associated genes. Two of these are significantly regulated in human glioma and one of those might have a role in glioblastoma stem cell differentiation.

Increased HIV susceptibility has been associated with the progesterone-dominant luteal phase of the menstrual cycle and the use of progesterone-only contraceptives, yet the mechanism is poorly understood. Here, we performed mass spectrometry-based analyses of cervicovaginal fluids collected from women with differing ovarian hormone levels as demonstrated by menstrual cycle phase or exogenous progesterone-only contraceptive use. We found that proteins associated with maintaining the integrity of epithelial barrier were enriched during times of high estradiol, whereas during times of high progesterone, there was a loss of barrier integrity proteins and an enrichment of proteins with known roles in inflammatory processes including leukocyte infiltration. Progesterone-based proteomic profiles were also strongly associated with neutrophil signatures with some evidence of CD4+ T cell signatures. This study generates new hypotheses about the potential mechanisms of hormone-associated HIV susceptibility including a weakened epithelial barrier and increased HIV target cell recruitment during times of increased progesterone. / February 2016

Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-14Bitstream added on 2014-06-13T20:04:32Z : No. of bitstreams: 1 matsumoto_mt_dr_arafcf.pdf: 7554122 bytes, checksum: 0910afc354605fb9e82889bf3660ea86 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / Cryptococcus neoformans é patógeno importante, principalmente em pacientes imunocomprometidos. A principal porta de entrada deste patógeno é pela via respiratória, disseminando-se posteriormente e atingindo principalmente o sistema nervoso central, provocando a meningite criptocóccica. A primeira parte deste estudo teve como objetivos determinar, nos 106 isolados clínicos de C. neoformans obtidos de dois Estados (São Paulo e Rio de Janeiro), (1) as variedades e (2) os mating-types por PCR (Polymerase Chain Reaction), (3) analisar a diversidade genética por PCR-fingerprinting com a seqüência iniciadora específica para regiões microssatélite (GACA)4, (4) por RAPD (Random Amplification of Polymorphic DNA) com o iniciador 6 e (5) por PCR-RFLP (Restriction Fragment Length Polymorphism) do gene da fosfolipase B (PLB1) digerido com a enzima de restrição AvaI e (6) determinar a sensibilidade a quatro antifúngicos (fluconazol, itraconazol, 5-fluorocitosina e anfotericina B) seguindo o método de referência (documento M27-A2) do CLSI (Clinical and Laboratory Standards Institute). A segunda parte teve como objetivo, analisar transcritos diferencialmente expressos durante a infecção pulmonar de C. neoformans, pela técnica de RDA (Representational Difference Analysis). Entre os 106 isolados, 104 foram identificados como C. neoformans e apenas dois foram C. gattii (=C. neoformans var. gattii), todos MATa. O tipo molecular VNI (C. neoformans var. grubii, sorotipo A) foi o mais prevalente entre os isolados (97/106), seguido do tipo VNII (C. neoformans var. grubii, sorotipo A) (7/106) e VGII (C. gattii, sorotipos B ou C) (2/106) quando analisados por PCR-fingerprinting e PCR-RFLP. Homogeneidade alta foi obtida com o iniciador (GACA)4, com a maioria dos isolados apresentando correlação em torno de 0,9. Os resultados do RAPD, por sua vez, revelaram maior heterogeneidade com número maior de perfis moleculares. / Cryptococcus neoformans is an important pathogen, mainly in immunocompromised patients. The pathogen penetrates mainly by respiratory way, disseminate afterward and reach specially the central nervous system causing the cryptococcal meningitis. The first part of this study had the objective to determine, in the 106 clinical isolates of C. neoformans obtained from São Paulo and Rio de Janeiro State, (1) the varieties and (2) mating-types by PCR (Polymerase Chain Reaction), (3) analyze the genetic diversity by PCR-fingerprinting with specific primer to microsatellite regions (GACA)4, (4) by RAPD (Random Amplification of Polymorphic DNA) with primer 6 and (5) by PCR-RFLP (Restriction Fragment Length Polymorphism) of the phospholipase B gene (PLB1) digested with restriction enzyme AvaI and (6) to determine the susceptibility to four antifungal (fluconazole, itraconazole, 5-flucytosine and amphotericin B) following the reference method (document M27-A2) from CLSI (Clinical and Laboratory Standard Institute). The second part had the goal to analyze differentially expressed transcription during pulmonary infection of C. neoformans, by RDA (Representational Difference Analysis) technique. Among 106 isolates, 104 were identified as C. neoformans and only two were C. gattii (=C. neoformans var. gattii), all MATa. The molecular type VNI (C. neoformans var. grubii, serotype A) was the most prevalent among the isolates (97/106), followed by VNII (C. neoformans var. grubii, serotype A) (7/106) and VGII (C. gattii, serotypes B ou C) (2/106) when analyzed by PCR-fingerprinting and PCR-RFLP. High homogeneity was obtained with the primer (GACA)4, with most of the isolates showing correlation around 0.9. By contrast, the RAPD analysis revealed more heterogeneous with more numbers of molecular profiles.

Orientador: George de Paula Bernardes / Banca: Eduardo Dell'Avanzi / Banca: Heraldo Luiz Giacheti / Resumo: A região das nascentes do ribeirão Guaratinguetá, localizada no contraforte da Serra da Mantiqueira, Vale do Paraíba, tem sido intensamente modificada por ações antrópicas criando condições que favorecem os movimentos de massa, princiapalmente devido à supressão da mata nativa. Estas ações, somada ao aumento a intensidade de chuvas sobre uma complexa geologia da região, têm potencializado os escorregamentos translacionais, de modo que foram registradas mais de 40 ocorrências, nas chuvas do final de 2008 e início de 2009. Neste contexto, as proposta desta dissertação é de identificar os mecanismos que geram essas instabilidades através da avaliação das características geotécnicas de dois escorregamentos característicos na bacia do ribeirão Guaratinguetá. Para isso, as atividades envolveram ensaios de laboratório e de campo. No laboratório foram realizados ensaios para a caracterização completa das amostras, ensaios de sucção, para determinação das curvas características, e ensaios de cisalhamento direto, para determinação dos parâmetros de resistência. No campo, foram determinadas as propriedades hidráulicas através do permeâmetro de Guelph e a resistência à penetração dos horizontes utilizando o Penetrômetro Dinâmico de Cone. O estudo dos possíveis processos de identificação de instabilidade foi realizado a partir de retro-análises dos dois escorregamentos estudados, através do software SLOPE/W, da GeoSlope, e de simulações de cenários, através do modelo matemático Shalstab. Nestas análises, foram consideradas diferentes hipóteses relativas à presença de água no subsolo, onde foram verificados os respectivos fatores de segurança. Os resultados das retro-análises indicam coerência entre as superfícies previstas e observadas no campo, para a condição de solo saturado acima da superfície de ruptura e solo na umidade residual... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The land degradation process as consequences of deforestation and farming activities at the northem part of the Guaratinguetá creek watershed, Paraíba Valley, is compromising the environmental balance of the region traduced by increase in the numbers of shallow landslides occurring during rain seasons. In summer 2009, more than 40 shallow landslides occurred during an intense rainfall with serious consequence for the local population. In this context, the purpose of this dissertation is to understand the mechanism that triggered the soil movement based on geotechnical investigation of two landslides in the Guaratinguetá creek watershed. The geotechnical profiles of two landslides and the hydraulic parameters were determined by field tests using Dynamic Cone Penetrometer and Guelph Permeameter. In laboratory, physical and mechanical soil properties were determined. The shear strength parameters were obtained by direct shear tests on undisturbed speciments for three initial gravimetric water contents (residual, natural and saturated). The instability process was inferrred by back-analysing these two landslides using the SLOPE/W, GeoSlope, and simulating scenarios using SHALSTAB model. These analyses took into account the physical properties and the shear strength parameters of the solis for different water contents. The predicted slip surfaces were in agreement with the observed ones when the analyses considered the soil parameters above the slip surface as saturated and the soil parameters below the slip surface as in residual water content condition. The susceptibility map using SHALSTAB were then carried out for the soil parameters for satured condition. The influence of the vegetation (root strength) was inserted in the analyses by a scale factor based onn Algebra Map Technique. The validation of the applied methodology was verified by crossing the predicted unstable... (Complete abstract click electronic access below) / Mestre

Archean greenstone belts typically form narrow sheared basins separating bulbous tonalo-trondjhemo-granodioritic (TTG) batholiths. The role played by gravity in the development of such dome-and-keel structures is a key question in Archean tectonics. The Pukaskwa batholith - Hemlo shear zone (HSZ) is a representative example of the dome-and-keel structures that are common in Archean terrains. This region has received considerable attention because the HSZ hosts several major gold deposits that are currently being mined. Late dextral strike-slip kinematics of the HSZ are well recorded by abundant strain markers in greenstone rocks, whereas the quartzofeldspathic coarse-grained rocks of the Pukaskwa batholith bear no macroscopically visible fabric. The goal of this study is to understand the structural history of this greenstone belt-batholith system. The Pukaskwa batholith is a heterogeneous assemblage of TTG gneisses bounded by the Hemlo greenstone belt to the north. The density of the Pukaskwa batholith rocks (density = 2700 kg/m3) is on average less than that of the Hemlo greenstone rocks (density = 3000 kg/m3). Since Archean geotherms were considered higher than modern equivalents, the effective viscosity of the TTG rocks might have been sufficiently low to allow their diapiric ascent through denser greenstone rocks. Alternatively, the emplacement of the TTG batholith might have been driven primarily by transpressive tectonics. The anisotropy of magnetic susceptibility (AMS) provides valuable information on the internal fabric of the Pukaskwa batholith. This study provides the kinematic information needed to support either the diapiric or the transpressive tectonic model. AMS recorded east-west trending prolate and plano-linear fabrics across the northern section along the contact, suggesting that transpressional forces from the Hemlo shear zone affected the emplacement of the Pukaskwa batholith. Away from the contact, fabrics are generally flattened, indicative of doming through diapiric processes. Also, in order to fully evaluate the diapiric hypothesis, it is necessary to obtain reliable data on rock densities across the Pukaskwa batholith. The density of about 360 representative specimens from the Pukaskwa batholith has been measured and will constitute a valuable database for future gravimetric investigations by mining companies. The significant degree of correlation between high-field magnetic susceptibility and density in the Pukaskwa batholith should be taken into account in geophysical exploration in Archean terrains, only as a proxy for iron content.

O Brasil é líder mundial em exportação de carne bovina. A qualidade e a segurança dos produtos cárneos são essenciais para permanecer como exportador no competitivo mercado internacional. Atualmente, critérios microbiológicos são usados para avaliar produtos cárneos, e Salmonella tem sido usada como um importante indicador da segurança dos alimentos por diversos países. No presente estudo, um total de 120 carcaças foram analisadas em três diferentes pontos do processo de abate em um abatedouro-frigorífico exportador localizado no sul do Brasil. Os resultados demonstraram que quatro carcaças (3,3%) foram positivas para Salmonella. Todas as amostras positivas foram coletadas do couro depois da sangria (Primeiro ponto de coleta – P1), e Salmonella não foi encontrada nas amostras coletadas após a retirada do couro (Segundo ponto de coleta – P2) e antes do último toalete (Terceiro ponto de coleta – P3). Seis isolados foram obtidos e classificados em três sorovares diferentes; S. Newport (n=3), S. Saintpaul (n=2) e S. Anatum (n=1). As cepas de Salmonella também foram analisadas quanto à susceptibilidade antimicrobiana, usando 15 antimicrobianos recomendados pelo CLSI. Nenhuma cepa apresentou resistência a qualquer um dos antimicrobianos testados. Além da investigação de Salmonella, a contagem de Escherichia coli foi realizada em todos os três pontos do processo. O ponto mais contaminado foi o primeiro (couro) que apresentou contagens variando de 0,31 a 5,07 log UFC/100 cm². O segundo e o terceiro ponto apresentaram contagens variando de N. D (não detectado) a 2,42 log UFC/100 cm² e N. D a 2,10 log UFC/100 cm², respectivamente. Uma redução significativa da contagem de E. coli foi observada entre o primeiro e os outros demais pontos. Os resultados indicaram que o risco de contaminação das carcaças ainda existe principalmente do couro. A implementação de Boas Práticas de Fabricação e dos princípios do APPCC são importantes ferramentas para prevenir a contaminação microbiana em carcaças. / Brazil is the leading exporter of beef worldwide. The quality and safety of the meat products are essential to remain as an exporter in the very competitive international market. Currently, microbiological criteria are used to evaluate meat products, and Salmonella has been used as an important food safety indicator in many countries. In the present study, a total of 120 carcasses were analyzed in three different steps of the slaughter process in an exporter slaughterhouse located in southern Brazil. The results demonstrated that four carcasses (3.3%) were positive for Salmonella. All the positive samples were collected on the hides after the bleeding (First collection step – P1), and Salmonella was not found in the samples collected after the removal of the hides (Second collection step – P2) and before the last toilet (Third collection step – P3). Six isolates were obtained and were classified in three different serovars, i. e. S. Newport (n=3), S. Saintpaul (n=2) and S. Anatum (n=1). The Salmonella strains were also examined for antimicrobial susceptibility using 15 drugs recommended for CLSI. None strains exhibited resistance to any of the antimicrobials tested. In addition to the Salmonella investigation, Escherichia coli counts were carried out in the three processing points. The most contaminated point was the first (hide) that showed counts ranged from 0.31 to 5.07 log CFU/100 cm². The second and the third points demonstrated counts ranged from N.D (not detected) to 2.42 log CFU/100 cm² and N.D to 2.10 log CFU/100 cm², respectively. A significant reduction of the E. coli counts was observed among the first and the other points. Results indicated that the risk of contamination of the carcasses still exists, mainly from the hides. The implementation of Good Manufacturing Practices and HACCP principles are important to avoid microbial contamination.

Considerando a importância do felino doméstico nos relatos zoonóticos da esporotricose, assim como os problemas relacionados a terapêutica da micose nessa espécie, o estudo objetiva avaliar a atividade in vitro da terbinafina e itraconazol frente ao S. schenckii; estudar a ação desses fármacos no tratamento da esporotricose sistêmica experimental; analisar as enzimas hepáticas e hemograma dos animais submetidos ao tratamento antifúngico; estudar as possíveis alterações macroscópicas nos animais inoculados com S. schenckii assim como realizar o retroisolamento do agente. Para o teste in vitro, foram utilizados 10 isolados de S. schenckii (seis felinos, três humanos e um de cão), os quais foram avaliados quanto a suscetibilidade frente aos fármacos através da técnica de microdiluição em caldo de acordo com o CLSI para as fases leveduriforme e filamentosa do agente. A leitura da CIM foi visual, correspondendo à menor concentração do antifúngico em que não houve crescimento visível do S. schenckii quando comparado ao controle positivo. O teste in vivo, foi realizado através da inoculação pelas vias intraperitonial e veia lateral da cauda de células fúngicas (2x103 cél/mL) de dois isolados de S. schenckii (felino e canino) em 120 ratos norvergicus, divididos em quatro grupos: tratado terbinafina (250mg/kg) e itraconazol (100mg/kg); diluente terbinafina (250mg/kg de solução de água destilada com 5% de DMSO e 1% de Tween 20) e itraconazol (100mg/kg de água destilada estéril), sendo administrados através de sondagem oral uma vez ao dia, durante 30 dias. Paralelamente foram realizados hemogramas e avaliação das enzimas hepáticas (ALT e FA), assim como o acompanhamento da evolução clínica e análise histopatológica de todos animais experimentais. No final do período experimental os animais foram necropsiados e realizado análise macroscópica com a coleta dos órgãos para a obtenção do retroisolamento do S. schenckii. A CIM para a terbinafina obtida entre todos os isolados na fase leveduriforme de esporotricose felina variaram de 0,055μg/mL a 0,109μg/mL, enquanto que para os isolados humanos e de cão foi de 0,055μg/ml. Para o itraconazol, a CIM frente aos isolados de esporotricose felina foi de 0,875μg/mL a 1,75μg/mL, já para os casos humanos foi de 0,219μg/mL e para o isolado de cão de 1,75μg/mL. Para a fase filamentosa do agente foi observada uma CIM para a terbinafina entre os isolados de felinos de 0,875 μg/mL, enquanto que os isolados humanos e de cão a CIM foi de 0,219μg/mL. Para o itraconazol os isolados felinos resultaram numa CIM de 3,5μg/mL, já nos isolados de esporotricose humana e em cão a CIM foi de 1,75μg/mL. Quanto ao estudo in vivo, os animais tratados com terbinafina e itraconazol resultaram em menor freqüência de alterações macroscópicas assim como na obtenção do retroisolamento do agente em relação aos grupos controle. Não foi detectada alterações nas enzimas hepáticas, hemograma e nas avaliações macro e histopatológicas dos animais submetidos ao tratamento antifúngico. Com esses resultados conclui-se que a terbinafina e itraconazol possuem atividade in vitro frente ao S. schenckii e que as doses estudadas dos fármacos são eficazes no tratamento da esporotricose experimental sistêmica, assim como não produzem alterações das enzimas hepáticas avaliadas e do hemograma. / Considering the importance of the domestic feline in the zoonotic reports of the esporotrichosis, as well as the related problems the therapeutics of the mycosis in that species, the study objective was evaluate the activity in vitro of terbinafine and itraconazole against to S. schenckii; study the activity of those drugs in the treatment of the experimental systemic esporotrichosis; analyze the hepatic enzymes and blood count of the animals submitted to the antifungal treatment; study the possible macroscopic alterations in the animals inoculated with S. schenckii as well as realized the agent retroisolament. For the test in vitro, 10 isolated of S. schenckii were used (six felines, three humans and one canine), which were performed as the susceptibility against to the drugs through the microdiluição technique in broth in accordance with the CLSI for the phases yeast and mycelial of the agent. The reading of CIM was visual, corresponding to smallest concentration of the antifungal in that there was not visible growth of S. schenckii when compared to the positive control. The test in vivo, was performed through the of the inoculation intraperitonial and lateral vein of the tail of fungal cells (2x103 cél/mL) of two isolated of S. schenckii (feline and canine) in 120 mice norvergicus, divided in four groups: treaty terbinafine (250mg/kg) and itraconazole (100mg/kg); diluent terbinafine (250mg/kg of solution of water distilled with 5% of DMSO and 1% of Tween 20) and itraconazol (100mg/kg of water distilled sterile), being administered by gavage once a day, for 30 days. Parallel blood counts and evaluation of the hepatic enzymes were accomplished (ALT and F.A), as well as the accompaniment of the clinical evolution and analysis histopathologic of all animal experimental. In the end of the experimental period the animals were autopsied and performed macroscopic analysis with the collection of the organs for the obtaining of the detected S. schenckii growth. CIM for the terbinafine obtained among all the isolated in the phase yeast of feline esporotrichosis oscillated between 0,055μg/mL the 0,109μg/mL, while for the isolated humans and canine was of 0,055μg/ml. For the itraconazole, the CIM against of the isolated of feline esporotrichosis was from 0,875μg/mL to 1,75μg/mL, for the human cases it went of 0,219μg/mL and for the isolated canine of 1,75μg/mL. For the mycelial phase a CIM was observed for the terbinafine among the isolated of felines of 0,875 μg/mL, while the isolated humans and canine CIM was of 0,219μg/mL. For the itraconazole the isolated felines resulted in a CIM 3,5μg/mL, in the isolated of human esporotrichosis and canine CIM was of 1,75μg/mL. As for the study in vivo, the animals treaties with terbinafine and itraconazole resulted in smaller frequency of macroscopic alterations as well as in the obtaining of the detected agent growth in relation to the groups control. It was not detected alterations in the hepatic enzymes, blood count and in the evaluations macro and histopathologic of the animals submitted to the antifungal treatment. With those results we are to report that the terbinafine and itraconazol apresent activity in vitro against to S. schenckii and that the studied doses of the drugs are effective in the treatment of the systemic experimental esporotrichosis, as well as they don't produce alterations of the appraised hepatic enzymes and blood count.