The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.

The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.

Thesis (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: David L. Dickensheets. Includes bibliographical references (leaves 69-70).

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Rebecca Richards-Kortum. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Rebecca Richards-Kortum. Vita. Includes bibliographical references. Also available from UMI.

Thesis (M.E.E.)--University of Delaware, 2005. / Principal faculty advisor: Balaji Panchapakesan, Dept. of Electrical & Computer Engineering. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Made available in DSpace on 2015-12-06T23:07:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2005 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Introdução: O tecido adiposo armazena energia e secreta várias moléculas endócrino/parácrina como a angiotensina II (AII) e expressa receptores como de insulina e de AII (AT1 e AT2). Esses hormônios estão envolvidos em várias patologias tais como a obesidade, hipertensão, diabetes e aterosclerose. Objetivos: Caracterização morfológica da cultura primária de adipócitos humanos e da resposta ao estímulo dos hormônios AII e insulina. Métodos: Para caracterização morfológica fez-se a coloração com Oil red O, gotículas de lipídios, MitoTracker red, mitocôndrias e ERTracker, retículo endoplasmático. Determinaram-se também as fases do ciclo celular, tamanho e complexidade das populações das células cultivadas e dispersas através da citometria de fluxo. O cultivo das células foi realizado de acordo com Hauner et al. (1989). Para as medidas do Ca2+ intracelular foi utilizada a análise por microscopia de fluorescência de alta resolução. As células adiposas foram caracterizadas por imunocitoquímica utilizando-se anticorpos para insulina, AT1 da AII, receptor de insulina e lectina conjugados com um marcador fluorescente. Resultados: Por microscopia confocal verificou-se a presença de várias gotículas lipídicas, citoesqueleto, mitocôndrias, retículo endoplasmático e membrana celular preservados, e próprios dos pré adipócitos, Os pré-adipócitos apresentaram receptores e grânulos de insulina e o receptor AT1. Também se verificou por citometria de fluxo a amostra de células rescém-dispersas que pôde ser classificada como pré- adipócito. A avaliação temporal dos níveis de Ca2+ nas células de pré-adipócitos humanos estimuladas com a AII e insulina induzem respostas transientes no caso da AII o aumento imediato das [Ca2+]i seguido da diminuição desta resposta e no caso da insulina apresentou oscilações nas [Ca2+]i na presença deste agonista. Conclusões: Determinou-se a caracterização morfológica da cultura primária de adipócitos humanos como pré-adipócitos por:apresentarem morfologia semelhante a fibroblastos e inúmeras gotículas de lipídios; evidenciado a presença dos receptores de angiotensina II (AT1) e insulina (RI) por imunofluorescência. As células presentes na amostra do tecido adiposo apresentaram tamanho e complexidade de pré-adipócitos; Nos pré-adipócitos humanos cultivados verificou-se o aumento dos níveis intracelulares de Ca2+ que foi transiente para o estímulo com angiotensina II e oscilatório para a insulina. / Introduction: The adipose tissue stores energy and secretes several endocrine/paracrine molecules as the angiotensin II (AII) and expresses receptors as of insulin and of AII (AT1 and AT2). These hormones are involved in several pathologies such as the obesity, hypertension, diabetes and atherosclerosis. Objectives: Morphologic characterization of the primary culture of human adipocytes and of the answer to the incentive of the AII hormones and insulin. Methods: For morphologic characterization the coloration with Oil red O, lipids droplets, MitoTracker red, mitochondrias and ERTracker, endoplasmic reticulum was made. It also determined the phases of the cellular cycle, size and the populations complexity of the cultivated cells and disperse through the flow cytometry. The cells cultivation was accomplished in agreement with Hauner et al. (1989). For the Ca2+ intracellular measures the analysis by fluorescence microscope of high resolution was used. The adipose cells were characterized by immunocytochemistry using antibodies for insulin, AT1 of AII, insulin receptor and lectina conjugated with a fluorescent marker. Results: For confocal microscope was verified the presence of several lipidics droplets, cytoskeleton, mitochondrias, endoplasmic reticulum and cellular membrane preserved, characteristic on preadipocytes. The preadipocytes presented receptors and insulin granules and AT1 receptor It was also verified by flow cytometry the sample of newly-dispersed cells that could be classified as preadipocyte. The temporary evaluation of the Ca2+ levels in the human adipocytes cells which were stimulated with AII and insulin showed that AII induced transient answers, by the immediate increase of the [Ca2+]i followed by the decrease of this response. The insulin presented oscillations in the [Ca2+]i during the presence of this agonist. Conclusions: For confocal microscopy was determined the morphologic characterization of the primary culture of human adipocytes as preadipocytes and evidenced the presence of the receivers of angiotensina II (AT1) and insulin (RI) for imunofluorescência. The present cells in the sample of the fatty fabric presented size and preadipocytes complexity; In the cultivated human preadipocytes the increase of the levels intracelulares of Ca2+ was verified that was transient for the incentive with angiotensina II and oscillatory for the insulin. / BV UNIFESP: Teses e dissertações

Septic arthritis has the potential to be a highly destructive joint disease. Although numerous bacterial species are capable of inducing septic arthritis, Staphylococcus aureus is most commonly implicated, accounting for up to 65% of cases. Whilst this organism is known to produce a diverse array of potential virulence factors, studies investigating a variety of S. aureus-related infections have implicated alpha(Hla)-, beta(Hlb)- and gamma(Hlg)-haemolysins as key damaging toxins, with the ‘pore-forming’ Hla considered to be the most potent. The work presented in this study focused on gaining further insight into the interaction between S. aureus toxins and in situ chondrocytes during an episode of septic arthritis. An in vitro bovine osteochondral explant model of S. aureus-induced septic arthritis was developed in this study. Utilising fluorescence-mode confocal laser scanning microscopy (CLSM), the model, which avoided the complexities of a host immune response, permitted an assessment of the following: (1) the spatial and temporal quantification of in situ chondrocyte viability following exposure to both a laboratory ‘wild-type’ (S. aureus 8325-4) and clinical strains of S. aureus; (2) the influence of Hla, Hlb and Hlg on in situ chondrocyte viability through the use of specific ‘haemolysin-knockout’ mutant strains; (3) the influence of altered culture medium osmolarity and extracellular Ca2+ on Hla-induced in situ chondrocyte death; and (4) dynamic changes in intracellular Ca2+ within in situ chondrocytes following Hla exposure. S. aureus 8325-4 and S. aureus clinical strains rapidly reduced in situ chondrocyte viability ( > 45% chondrocyte death at 40hrs). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) of cartilage and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins (achieved with the use of subchondral bone-free explants) demonstrated that SZ chondrocytes were more susceptible to the toxins than DZ chondrocytes. When explants were cultured in the presence of a selection of isogenic S. aureus mutants, with varying Hla, Hlb and Hlg production capabilities (all originating from S. aureus 8325-4), Hla-producing mutants induced significant in situ chondrocyte death compared to toxin deficient controls (Hla-Hlb-Hlg-). In contrast, mutants producing Hlb and Hlg in the absence of Hla were unable to induce significant chondrocyte death. Hla alone was therefore identified as the key damaging toxin to in situ chondrocyte viability. Raised culture medium osmolarity had no influence on Hla-induced in situ chondrocyte death. In the absence of Hla, a high extracellular Ca2+ concentration (20mM) had no influence on chondrocyte viability during the experimental period. Hla-induced chondrocyte death increased in the presence of raised extracellular Ca2+ concentrations thereby confirming a role of Ca2+ in the chondrocyte death pathway. There was no significant difference between S. aureus growth in high and low Ca2+ culture media. Finally, when live osteochondral explants stained with the Ca2+-sensitive fluorophore Fluo-4 were cultured with an Hla-containing S. aureus supernatant (S. aureus 8325-4 (Hla+Hlb+Hlg+)) there was a significant rise in intracellular Ca2+ in comparison to those explants exposed to a non-Hla-containing supernatant (S. aureus DU5938 (Hla- Hlb-Hlg-)). The Hla-induced Ca2+ transients were always followed by chondrocyte death. Thus, it is likely that Hla-induced chondrocyte death was associated with a rise in intracellular Ca2+. These findings are of translational relevance. Firstly, toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes, thereby advocating the prompt and thorough removal of bacteria and their toxins during the treatment of septic arthritis. Secondly, the identification of Hla alone as the key damaging toxin to in situ chondrocyte viability, with its destructive action being associated with a rise in intracellular Ca2+, may enable the development of future targeted therapeutic strategies in order to reduce the extent of cartilage destruction during and after an episode of septic arthritis.

Orientador: Carlos Lenz Cesar / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-11T10:58:51Z (GMT). No. of bitstreams: 1 Thomaz_AndreAlexandrede_M.pdf: 10062018 bytes, checksum: 1e19c55cb5a4e709c2015e2d90f3ac13 (MD5) Previous issue date: 2007 / Resumo: A pesquisa em fotônica biomedica está claramente tomando a direção do entendimento de processos biológicos a nível celular. A resolução necessária para atingir esse objetivo requer praticamente ferramentas fotônicas. Contudo, uma integração de diferentes ferramentes fotônicas e uma aproximação funcional serão necessárias para acessar os processos biomecânicos e bioquímicos celulares. Deste modo nós podemos observar eventos bioquímicos disparados mecanicamente ou eventos mecânicos disparados bioquimicamente, ou até mesmo observar simultâneamente eventos biomecânicos e bioquímicos disparados por outros meios, entre outros, eletricamente. Uma das grandes vantagens das ferramentas fotônicas é a sua facilidade de integração. Nós desenvolvemos uma ferramenta integrada incorporando pinça óptica simples com Microscopia Confocal "Single-photon" e Multifóton. O sistema consegue realizar microscopias de fluorescência excitada pela absorção de dois fótons e geração de segundo harmônico em conjunto com manipulações ópticas. Medidas de força, elasticidade e viscosidade de membranes esticadas podem ser monitoradas em tempo real pelas microscopias confocais, bem como protozoários capturados opticamente, como, por exemplo, Trypanosoma cruzi. Nós mostraremos vários exemplos do uso de tal ferramenta integrada e seu potencial para observar processos mecânicos e bioquímicos a nível celular / Abstract: The research in biomedical photonics is clearly evolving in the direction of the understanding of biological processes at the cell level. The spatial resolution to accomplish this task practically requires photonics tools. However, an integration of different photonic tools and a multimodal and functional approach will be necessary to access the mechanical and biochemical cell processes. This way we can observe mechanicaly triggered biochemical events or biochemicaly triggered mechanical events, or even observe simultaneously mechanical and biochemical events triggered by other means, e.g. electricaly. One great advantage of the photonic tools is its easiness for integration. Therefore, we developed such integrated tool by incorporating single Optical Tweezers with Confocal Single and Multiphoton Microscopies. This system can perform 2-photon excited fluorescence and Second Harmonic Generation microscopies together with optical manipulations. Force, elasticity and viscosity measurements of stretched membranes can be followed by real time confocal microscopies. Also opticaly trapped living protozoas, such as Trypanosoma cruzi. Integration with CARS microscopy is under way. We will show several examples of the use of such integrated instrument and its potential to observe mechanical and biochemical processes at cell level / Mestrado / Física / Mestre em Física