This Thesis presents the synthesis, characterization and mechanistic explorations into a series of diphenylacetylene-containing oligoester ion channels. Eighteen final compounds were synthesized and tested for ion transport activity utilizing both vesicle and planar bilayer assays. The oligomers varied in length, hydrophobicity and the nature of the aromatic moiety. Compounds incorporating a modified diphenylacetylene (‘Dip’), or a novel phenyl-extended fluorophore (‘Trip’) were made using a reliable, modular synthesis. The final compounds were prepared in a total of 5 to 11 steps from commercial materials in yields ranging from 10 to 40%. The compounds’ activity varied considerably; both highly active and completely inactive compounds were discovered. The differences in activity are controlled by structure via the influence of structural variables on the aqueous phase aggregation and the ability of the compound to insert into the bilayer membrane. These structure-activity studies uncovered two highly-active ion transporters, HO2C-Hex-Dip-Hex-Hex-OH and –OPO32- (Hex = 6-hydroxyhexanoyl) which exhibited activity almost 10-fold higher than the fully-saturated oligoesters developed in previous work. In some cases, the transport activity is initially high but declines over a period of 20-30 minutes following compound addition. This suggests that the compound slowly transitions to an environment where it cannot form active channels. In the bilayer clamp, a variety of behaviours including highly-conducting openings were observed. An apparent voltage-gated response was exhibited by one of the Trip compounds (HO2C-Trip-G(E3)-OH), a property rarely seen for synthetic ion channels. The Dip and Trip molecules exhibited environment-sensitive fluorescence. The observed Dip excimer-like emission is the second reported instance of this in solution. The Trip compounds are solvatochromic; this property was used to infer their location in the membrane. Partitioning into the membrane was followed by a blue-shifting and increased intensity of the fluorescence emission for both series of compounds. For the Trip isomers, which are significantly more emissive than the Dip molecules, this enhancement in intensity could be visualized by eye. For the Dip oligomers, the excimer emission is a broad band with variable shape and intensity; it is time-dependent under some conditions. The excimer emission has a sub-nanosecond lifetime in homogenous solution that is significantly prolonged in the presence of vesicle bilayers, in which a number of lifetimes could be detected. Both monomer and excimer emissions can be quenched by aqueous copper, the excimer emission is more efficiently quenched than is the monomer. The photophysical characteristics of these molecules allowed for a variety of experiments designed to probe their membrane partitioning and localization behaviours. The results indicate the formation of a complex mixture of interconverting monomeric and aggregate species as the compounds move from water to the bilayer. The slow evolution of the mixture is consistent with the times noted for loss of membrane activity in transport assays. From these data a new model that describes the transport process is proposed. The key feature of this model is that transport must occur via a species that forms quickly upon the mixing of the components. Possible structures of the intermediates formed are discussed. / Graduate

The synthesis and biological sensing applications of novel water soluble poly(para-phenyleneethynylene)s (PPEs) are presented. The ease of synthesis, synthetic variability, and dramatic chromicity of PPEs makes them well suited for biological and sensing applications. Molecular recognition and signal transduction can be achieved by using PPEs as sensory materials. By incorporating biological functional groups (e.g. sugars), PPEs can efficiently detect the presence of toxic heavy metals, proteins, and bacteria through either fluorescence quenching or enhancement. Rapid, precise, and convenient sensory arrays for the detection of biological analytes are possible through the formation of gold nanoparticle-PPE constructs.

Pesticides are an important component of New Zealand’s primary production sectors. Infestation of pests and diseases can affect crop yield, crop value and damage the country’s export reputation, resulting in economic losses. Repeat applications of pesticides, however, can result in contamination of land and water. Therefore, it is important to understand the fate of pesticides in the environment. Factors which can affect pesticide persistence include soil properties (pH, SOM, CEC), leaching and run-off, volatilisation and co-contamination with heavy metals. Many soils in New Zealand contain high levels of copper from historical applications of copper-based pesticides. Co-contamination of soils may lead to the persistence of some synthetic organic pesticides. An investigation was undertaken to determine the effects of co-contamination with copper on the biodegradation of atrazine and indoxacarb in a New Zealand soil. A Templeton sandy loam soil was spiked with CuSO₄ to achieve concentrations of 0, 100, 250, 500 and 1000 mg kg⁻¹ Cu. The spiked soils were field aged for six months prior to pesticide spiking with either atrazine or indoxacarb. The aged Cu-spiked soils were spiked with either atrazine or indoxacarb at a rate of 2 mg kg⁻¹. A glasshouse study was conducted to determine if copper inhibited the degradation of the pesticides. The pesticide-spiked soils were sampled at the time of spiking (t₀), at the estimated half-lives (t₁) and at twice the estimated half-lives (t₂) of the individual pesticides. The estimated half-lives were based on literature values. The bioavailability and subsequent adverse effects of copper on the soil microbial community was investigated. Total and bioavailable copper concentrations, phosphatase and urease enzyme activities, microbial biomass, and pesticide residue concentrations were all measured in the experimental soil. Methods were developed for the extraction of atrazine, atrazine metabolite and indoxacarb residues from the experimental soil. Total copper concentrations extracted ranged from 4–1060 mg kg⁻¹ in the experimental soils and were consistent throughout the pesticide degradation studies. The bioavailability of copper was a maximum of 2% of the total copper concentration. Bioavailable copper concentrations were positively correlated to total copper (p<0.01). Soil biological properties were investigated to determine the effects of copper on the soil microbial community. Phosphatase and urease enzyme activities, as well as microbial biomass concentrations, were negatively correlated with total copper (p<0.05). Total copper was a better indicator of effects on microorganisms than bioavailable copper. The soil biological properties began showing adverse effects above a total copper concentration of 100 mg kg⁻¹. This concentration also corresponds to New Zealand’s copper limit in biosolids, which is protective of human, plant and microorganism health. Phosphate buffer extraction methods were developed for the analysis of atrazine and indoxacarb residues in the experimental soil by HPLC-UV. Elevated copper concentrations did not inhibit the degradation of atrazine or indoxacarb in the experimental soil. The half-lives of both atrazine (≤19.4 d) and indoxacarb (≤18.8 d) were lower in the spiked experimental soils than the means reported in previous New Zealand and international studies, but were within the reported ranges. This study provided the first data on the fate of indoxacarb in New Zealand. Hydroxyatrazine was the only metabolite detected in the atrazine-spiked experimental soils. Significant differences between the control (Cu-1) and copper levels above 100 mg kg⁻¹ were observed for hydroxyatrazine at t₂. Significant negative correlations were observed between hydroxyatrazine and the microbiomass at t₁ and phosphatase activity at t₂ (p<0.05). These significant relationships suggest that elevated copper concentrations may alter the degradation of this metabolite in the experimental soils due copper toxicity of the soil microbial community. The results of this thesis indicate that elevated levels of copper above 100 mg kg⁻¹ negatively impact the soil microbial community and may reduce the overall health of the soil. Biodegradation is a key mechanism for the degradation of atrazine and indoxacarb in the soil, so it is important that the health of the soil microbial community is maintained. Therefore, it is recommended that atrazine and indoxacarb are only applied to soils with a total copper concentration less than 100 mg kg⁻¹. This will protect the health of the soil microbial community and prevent the potential adverse effects of copper on the degradation of pesticide metabolites in the soil.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 3 uppsatser.

Os esteróides gonadais exercem importantes influências no direcionamento da resposta imune do hospedeiro frente à infecção. Alterações decorrentes de sua ausência ou reposição podem significar em uma evolução distinta de determinada parasitose. Existe também uma relação entre a produção de melatonina e o ritmo de atividade do sistema imune. A resposta imune depende de uma interação entre inúmeros hormônios que irão agir sinergicamente com distintas células imunes. O objetivo desse trabalho foi investigar o efeito dos hormônios sexuais masculinos, através da orquiectomia, e da administração oral de melatonina sobre a resposta imune de ratos Wistar machos infectados com a cepa Y de Trypanosoma cruzi, durante a fase aguda da doença de Chagas experimental. Inúmeros parâmetros foram avaliados, entre eles: parasitemia, histologia cardíaca, peso dos animais e órgãos, linfoproliferação de timócitos e esplenócitos, quantificação de macrófagos, dosagem de óxido nítrico, citometria de fluxo para avaliação das populações de CD3+CD4+ e CD3+CD8+; e ensaios imunológicos como IFN-gama, IL-2, IL-6, IL-12, IL-10. Observou-se em todos os parâmetros analisados a ação sinérgica da melatonina e da orquiectomia sobre o sistema imune, favorecendo uma resposta positiva contra o parasita na fase aguda da infecção. Dessa forma, concluiu-se que a melatonina e a redução dos hormônios sexuais masculinos exerceram um papel imunoestimulador importante, que se refletiu em uma resposta mais efetiva do hospedeiro durante a infecção experimental. / Gonadal steroids exert important influence in the host immune response during infection. Changes resulting from the absence or replacement of these hormones may represent a distinct evolution of a particular parasite. There is also a relationship between melatonin production and the rhythmic activity of the immune system. The immune response depends on the interaction of many hormones that will act synergistically with other immune cells. The objective of this research was to evaluate the effect of male sex hormones through orchiectomy, and oral administration of melatonin on the immune response of male Wistar rats infected with the Y strain of Trypanosoma cruzi, during the acute phase of experimental Chagasdisease. Several parameters were evaluated among them: quantification of blood parasites, heart histology, body weight of animals and organs, lymphoproliferation of thymocytes and splenocytes, quantification of peritoneal macrophages, nitric oxide concentration, alterations in CD3+CD4+ and CD3+CD8+ lymphocyte populations by flow cytometry and immunological assays, as IFN-gama, IL-2, IL-6, IL-12, IL-10. It was observed in all parameters examined the synergistic action of melatonin and orchiectomy on the immune system, promoting a positive response against the parasite in the acute phase of infection. Thus, it was concluded that melatonin and the absence of male sex hormones exerted an important immunostimulating role, which reflected a more effective hosts response during the experimental infection.

Made available in DSpace on 2014-06-11T19:28:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-19Bitstream added on 2014-06-13T20:58:45Z : No. of bitstreams: 1 costa_cc_me_arafo.pdf: 3083928 bytes, checksum: 2605dd91339dbf0d4e3c26e02afc6696 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi determinar a citotoxicidade in vitro de duas resinas reembasadoras experimentais autopolimerizáveis contendo alta concentração de agentes de ligação cruzada, etilenoglicol dimetacrilato (EGDMA) e 1,4-butanodiol dimetacrilato (BDMA) comparadas com uma resina reembasadora comercial (Tokuyama Rebase Fast II). Foram confeccionados corpos-de-prova em forma de discos das três resinas acrílicas reembasadoras rígidas sob condições assépticas. Os fibroblastos (L929) foram cultivados em placas com 96 compartimentos e incubadas por 24 h em meio de cultura Eagle. Os extratos foram preparados, colocando-se três amostras de cada resina em frascos de vidro estéreis com 9 mL de meio de Eagle e incubadas a 37º C por 24 h. O efeito citotóxico dos extratos foram avaliados utilizando os testes de incorporação do 3H-timidina e MTT, que refletem os níveis de síntese de DNA e metabolismo celular, respectivamente. Os dados foram analisados estatisticamente, utilizando análise de variância (P<0.05). Para o teste de incorporação do 3H-timidina, não foram observadas diferenças significativas (P>0.05) entre os materiais. A média da quantidade do isótopo incorporado no DNA celular para todas as resinas acrílicas foi estatisticamente menor do que para o controle. Todas as resinas acrílicas foram classificadas pelo teste de incorporação do 3Htimidina como moderadamente citotóxicas. Da mesma forma, para o teste do MTT, não foi encontrada diferença significativa para a citotoxicidade entre os materiais (P>0.05). Quando a citotoxidade foi comparado com o grupo controle, todas as resinas foram classificadas entre não citotóxicas e discretamente citotóxicas pelo teste do MTT. Assim, conclui-se que a incorporação dos agentes de ligação cruzada, EGDMA e BDMA não influenciaram significativamente sobre a citotoxicidade observada. Não houve diferença... / The aim of this study was to determine the cytotoxicity in vitro of two trials autopolymerizing reline systems made with higher concentration of cross-linking agents, EGDMA and BDMA compared to a commercial reline resin. Sample disks of the three hard reline acrylic resins were fabricated under aseptic conditions. The Fibroblast L929 cells were cultured in 96-well plates and incubated for 24 h in Eagle's medium. Eluates were prepared by placing three samples into a sterile glass vial with 9 mL of Eagle's medium and incubating at 37ºC for 24 h. The cytotoxic effect from the eluates was evaluated using the 3H-thymidine incorporation and MTT assays, which reflect DNA synthesis levels and cell metabolism, respectively. The data were statistically analyzed by using the one-way analysis of variance (P<.05). With the 3H-thymidine incorporation assay, no significant differences (P > .05) were found between the materials. The mean quantity of isotope incorporated into cellular DNA for all acrylic resins was statistically smaller than for the control. All acrylic resins were graded by the 3Hthymidine incorporation assay as moderate cytotoxic. Similarly, with the MTT assay, no significant difference in cytotoxicity was found between the materials (P > .05). When the cytotoxicity was compared with control group, all resins assay were between non cytotoxic and slight cytotoxic by the MTT. The incorporation of cross-linking agents, EGDMA and BDMA had no significant influence on the cytotoxicity observed. There was no cell viability difference between all materials in both assays.

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Genetic testing for this pathogenic mutation is challenging due to its GC rich, repetitive nature. I developed PCR based assays to detect the presence of the pathogenic variant, which were used in screening an archival cohort of Scottish ALS patients, and have also been implemented within a diagnostic setting. These PCR assays allow amplification of larger repeat expansions than have previously been reported, and can determine whether a C9orf72 expansion of greater than 100 repeats is present or not. It is not well understood how the repeat expansion leads to disease, but several potential mechanisms have been hypothesised, including reduced expression, RNA toxicity and protein toxicity via dipeptide repeat proteins produced through repeat associated non-AUG translation. Motor neurons are an understandably well studied target in amyotrophic lateral sclerosis, however the role of glia, particularly oligodendrocytes, in the pathogenesis of the disease has recently been highlighted from studies on rodent models and post mortem tissue. To investigate the effect of the C9orf72 repeat expansion on oligodendrocytes, we have applied a differentiation protocol to hiPSCs with the expansion and controls, including an isogenic control which has been generated in the lab. There was no difference in the production of neuronal and glial cell types between these cell lines. I went on to look for evidence of the main proposed pathological mechanisms of C9orf72 repeat expansions: loss of function or gain of function through either RNA or protein toxicity. hiPSC derived oligodendrocytes from both carrier and control showed low expression of C9orf72 mRNA, and there was no difference due to the presence of a repeat expansion. Carrier hiPSC derived oligodendrocytes displayed sense RNA foci, which did not appear to have an effect on cellular morphology. The detection of dipeptide repeat proteins proved challenging, and the results were inconclusive as to their presence in hiPSC derived oligodendrocytes. I went on to show there was no evidence of mislocalisation of TDP-43 in C9orf72 carrier oligodendrocytes. Finally, the study showed similar levels of cell death in basal conditions in carrier and control cells, and no clear difference in the response to endoplasmic reticulum stress. Further research will be required to elucidate the role of oligodendrocytes in C9orf72 related amyotrophic lateral sclerosis.

Embryonic haematopoiesis is a complex process under intensive research. Murine definitive Haematopoietic Stem Cells (HSCs) originates from the Aorta-Gonad-Mesonephros (AGM) region of E10.5 embryo. It is thought that definitive HSCs arise from endothelial lining of dorsal aorta. However, detail of HSC specification in the developing embryo remains elusive. One way to deciphering events occurred during HSC specification is to derive cell lines from the developing HSC niche. Previous work by Oostendorp et al. showed the AGM and fetal liver derived lines could maintain HSCs in vitro (Oostendorp, Harvey et al. 2002). In this study, I established a more robust immortalization system using normal SV40 large T antigen delivered via Neon™ electroporation system. The new immortalization system achieved direct immortalization without going through crisis. And it is compatible with small number of primary cells dissected from different haematopoietic niches. With my new system, multiple cell lines from different haematopoietic sites at different developmental points are derived. Moreover, some of these lines demonstrated ability to mature precursors from E9.5 embryo (pro-HSCs) to definitive HSC without help of growth factors. This result is better compared to OP9 stromal lines. Such data proved usefulness of using stromal cell lines to study haematopoietic specification.

As condições de trabalho em laboratórios de ensaios de materiais e equipamentos, utilizados em redes de energia elétrica, expõem os operadores a importantes riscos de acidente. No entanto, existem poucos estudos com abordagem ergonômica para analisar as condições de trabalho e os riscos em laboratórios deste tipo. Assim, o objetivo deste estudo foi elaborar uma sistemática para avaliar estas condições, visando reduzir os riscos de acidentes e doenças ocupacionais nestes laboratórios. A abordagem elaborada foi aplicada em quatro etapas: a primeira correspondeu à análise da demanda, com o levantamento de indicadores de segurança e saúde; a segunda etapa compreendeu a construção e aplicação de uma ferramenta de análise das condições gerais de segurança com base nas normas brasileiras existentes; na terceira etapa deu-se a construção e aplicação de uma ferramenta de identificação de riscos inerentes às atividades; a quarta e última etapa contemplou a aplicação do método Deparis para diagnosticar riscos nas atividades, a partir da percepção do trabalhador. Como resultado, a sistemática mostrou-se eficaz, evidenciando situações passíveis de causar danos à saúde e à integridade física dos trabalhadores do laboratório analisado. A abordagem se mostrou de entendimento e aplicação simples e permitiu a confrontação de visões distintas (analista e operadores), evidenciando com maior precisão os riscos analisados. / The conditions of work in laboratories of test of materials and equipment, used in nets of electric energy, expose the workers to serious risks of accident. However, there are few studies about this matter with ergonomic approach. Thus, the purpose of this study was to elaborate a systematics to evaluate these conditions, being aimed to reduce the risks of occupational accidents and illnesses in these laboratories. The approach of this work was applied in four stages: the first one corresponded to the analysis of the demand, with the survey of security and health indexes; the second stage was based on the construction and application of a tool of analysis of the general conditions of security the existing Brazilian norms; the third stage was given by the construction and application of a tool to identificate the risks of the activities; the fourth and last stage used the application of the Deparis Method to diagnosis risks in the activities, from the perception of the worker. As result, the systematics revealed efficient, evidencing situations that must cause damages to the health and the physical integrity of the workers of the analyzed laboratory. The approach seemed to be simple to understand and to be applied and allowed the confrontation of distinct sides (analyst and operators), evidencing with most precision the analyzed risks.

Made available in DSpace on 2015-07-22T20:50:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-27 / Objetivo: Investigar a imuno-expressão do c-kit e sua correlação com o prognóstico em pacientes portadores de Osteossarcoma e o efeito do Mesilato de Imatinibe (STI571) na proliferação e invasão de linhagens de células humanas de osteossarcoma. Material e Método Estudo retrospectivo com realização de imunohistoquímica dos blocos de parafina de 52 pacientes com osteossarcoma de extremidades de alto grau, tratados no Instituto de Oncologia Pediátrica e diagnosticados no Departamento de Patologia da UNIFESP. Somente espécimes pré-quimioterapia foram analisados. Imunoexpressão forte e citoplasmática foram considerados como positivos. A linhagem cellular MG 63 foi incubada e o efeito inibitório do STI571 na proliferação e invasão dessas células tumorais in vitro foi estudada. Resultados: 24 casos (46,15%) expressaram o c-kit, sendo que esses tumores positivos tiveram pior resposta à qumioterapia pré-operatória. Não foi encontrada correlação entre os casos c-kit positivos e sobrevida global e livre de doença. STI571 inibiu a proliferação de células de osteossarcoma in vitro, em baixas doses e a invasão dessas células, em altas doses. Conclusões: Osteossarcomas expressam c-kit e esses tumores c-kit positivos são maus respondedores (têm menos necrose) à quimioterapia pré-operatória. O Mesilato de Imatinibe inibe a proliferação de células de osteossarcomas que expressam o c-kit, mas não inibem a invasão. Esses achados permitem sugerir que o STI571 pode ser uma nova estratégia no tratamento dos pacientes portadores de osteossarcomas. / Purpose: To investigate the immunoexpression and its prognostic relevance of KIT in patients with osteosarcomas and the effect of Imatinib mesylate (STI571) on proliferation and invasion of human cell osteosarcoma line. Material and Methods: A retrospective immunohistochemical study was performed on archival formalin fixed paraffin-embedded tissue obtained from Department of Pathology of Federal University of São Paulo of 52 high-grade patients with primary osteosarcomas of extremities treated at the Instituto de Oncologia Pediátrica. Only pre-chemotherapy specimens were analysed. Strong cytoplasmic and membranous staining cases were taken as positive. The human cell line MG 63 was incubated and inhibitory effect of STI571 on cell proliferation and invasion was studied. Results: Twenty four cases (46,15%) expressed c-kit and tumours ckit positive had lower necrosis pos chemotherapy. No correlation was found between ckit expression and overall and disease free survival. STI571 inhibited the rates of cell growth of osteosaroma cells in low doses and invasion in high doses Conclusions: Tumours c-kit positives had worse response to chemotherapy and STI571 plays a role in blocking or slowing the rate of growth of osteosarcoma cells expressing ckit, but not the invasive capacity of these neoplastic cells. These data suggested that Imatinib Mesylate could be a therapeutic target of strategies against Osteosarcoma that express c-kit. / TEDE / BV UNIFESP: Teses e dissertações