Orientador: Carlos Lenz Cesar / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-22T08:41:16Z (GMT). No. of bitstreams: 1 Thomaz_AndreAlexandrede_D.pdf: 9291787 bytes, checksum: 9554ec1cfb5b3952506ff59b61aec5f9 (MD5) Previous issue date: 2013 / Resumo: A comunidade científica concorda que há grandes chances que a próxima revolução tecnológica virá do controle dos processos biológicos. Grandes mudanças são esperadas, desde como produzimos alimentos até como combatemos as doenças. O controle dos processos biológicos nos permitirá produzir carne sintética para alimentação, produzir biocombustíveis retirando CO2 da atmosfera, produzir órgãos inteiros para transplante e combater de forma eficiente doenças como câncer, por exemplo. Está claro para o nosso grupo que para se obter esses resultados é necessário entender a biologia na sua unidade mais básica: a célula. A partir do entendimento e domínio das reações químicas que acontecem dentro da célula, e mais especificamente do controle do DNA, é que vamos conseguir atingir essas previsões e revolucionar a maneira como vivemos hoje. Com esse pensamento em mente, o objetivo dessa tese foi desenvolver uma plataforma fotônica integrada para estudos de processos celulares. Nós acreditamos que as ferramentas fotônicas são as ferramentas que preenchem todos os requisitos para os estudos de processos celulares, pois possibilitam o acompanhamento dos processos em tempo real sem causar dano as células. As técnicas presentes são: fluorescência excitada por 1 ou 2 fotons, geração de segundo ou terceiro harmônico, pinças ópticas, imagem por tempo de vida da fluorescência e "fluorescence correlation spectroscopy" (FCS). Nesta tese demonstramos como montar essa plataforma integrada e mostramos sua versatilidade com resultados em várias áreas da biologia e também para o estudo de quantum dots. / Abstract: The scientific community believes there is a great chance that the next technological revolution is coming from the control of biological processes. Great changes are expected, from the way we produce food up to the way we fight diseases. The control of biological processes will allow us to produce synthetic meat as food, to produce biofuels extracting CO2 directly from the atmosphere, to produce whole synthetic organs for transplant and to fight diseases, like cancer, in more efficient ways. It is clear to our group that in order to obtain these results it is necessary to understand biology from its most basic unity: the cell. Only from understanding and controlling chemical reactions inside a cell, and more specifically from the DNA controlling, it will be possible to achieve these predictions and cause a revolution in the way we live nowadays. Bearing these thoughts in mind, the objective of this thesis was to develop an integrated photonic platform for study of cellular processes. We believe that photonic tools are the only tools that fulfill all the requeriments for studies of cellular processes because they are capable to follow processes in real time without any damage to the cells. The techniques integrated are: 1 or 2 photon excited fluorescence, second or third harmonic generation, optical tweezers, fluorescence lifetime imaging and fluorescence correlation spectroscopy. In this thesis we demonstraded how to assemble this integrated plataform and we showed its versatility with results from different areas of biology and quantum dots. / Doutorado / Física / Doutor em Ciências

Submitted by Fernanda Rodrigues de Lima (fernanda.rlima@ufpe.br) on 2018-10-10T20:16:05Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Bernardo Menelau Cavalcanti.pdf: 5192624 bytes, checksum: dd9afbb36d7edcf2451a221593b65396 (MD5) / Approved for entry into archive by Alice Araujo (alice.caraujo@ufpe.br) on 2018-11-21T20:52:03Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Bernardo Menelau Cavalcanti.pdf: 5192624 bytes, checksum: dd9afbb36d7edcf2451a221593b65396 (MD5) / Made available in DSpace on 2018-11-21T20:52:03Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Bernardo Menelau Cavalcanti.pdf: 5192624 bytes, checksum: dd9afbb36d7edcf2451a221593b65396 (MD5) Previous issue date: 2012-11-19 / Introdução: Herpes Zoster Oftálmico (HZO) resulta em perda da sensibilidade corneal e consequente desenvolvimento de ceratopatia neurotrófica. Objetiva-se analisar as alterações bilaterais das células imunes e do plexo nervoso sub-basal (PNS) da córnea em pacientes com HZO, através da microscopia confocal a laser in vivo (LCM), bem como sua correlação com a sensibilidade corneal. Casuística: Vinte e quatro olhos de 24 pacientes com o diagnóstico de HZO, e seus respectivos olhos contralaterais não afetados foram avaliados e comparados com grupo controle (n = 24). Métodos: Os olhos dos pacientes e do grupo controle foram submetidos ao exame de microscopia confocal a laser (Heidelberg Retina Tomograph 3 / Rostock Cornea Module) bem como estesiometria corneal (Cochet-Bonnet) na região central da córnea e em ambos os olhos. As imagens obtidas pela microscopia foram avaliadas e quantificadas para a presença de células dendríticas (DCs) e do plexo nervoso sub-basal. Resultados: Tanto o olho afetado quanto o contralateral dos pacientes com HZO apresentaram um aumento significativo de DCs na região central em comparação com o grupo controle (147,4 ± 33,9; 120,1 ± 21,2; e 23,0 ± 3,6 células/mm²; p=0,001). Em ambos os olhos, foi identificada uma redução importante dos parâmetros do plexo nervoso sub-basal em relação à densidade de nervos totais (9.052,6 ± 1.151,8; 14.959,8 ± 903,2; e 22.851,4 ± 661,4 μm/mm²; respectivamente p<0,001), número de nervos totais (5,8 ± 0,9; 11,9 ± 1,2; e 26,6 ± 1,2 n/imagem; p<0,001), número de troncos principais (2,4 ± 0,3; 3,8 ± 0,3; e 4,4 ± 0,2; p<0,001) e número de ramos secundários (3,4 ± 0,7; 8,2 ± 1,1; e 22,2 ± 1,2; p<0,001) em relação ao grupo controle. A densidade de DCs apresentou correlação negativa com a densidade de nervos totais (R=-0,43; p<0,001). Ademais, a redução do plexo nervoso sub-basal apresentou uma correlação positiva com a sensibilidade corneal (R=0,63; p<0,001). Conclusão: HZO unilateral apresentam significante aumento bilateral de células dendríticas, redução do plexo nervoso sub-basal e da sensibilidade corneal. / Introduction: Herpes zoster ophthalmicus (HZO), thought to be a unilateral disease, results in loss of corneal sensation, leading to neurotrophic keratopathy. This study aimed to analyze bilateral corneal immune cell and nerve changes (PNS) in patients with HZO by laser in vivo confocal microscopy (LCM) and their correlation with corneal sensation. Participants: Twenty-four eyes of 24 patients with diagnosis of HZO and their contralateral clinically unaffected eyes were studied and compared with normal controls (n = 24). Methods: Laser in vivo confocal microscopy (Heidelberg Retina Tomograph 3 / Rostock Cornea Module) and corneal esthesiometry (Cochet-Bonnet) of the central cornea were performed bilaterally in all eyes of the patients and controls. Confocal images were evaluated for the presence of dendritiform immune cells (DC) and subbasal nerve plexus. Results: HZO affected and contralateral clinically unaffected eyes had a significant increase in DC infiltration of the central cornea (147.4 ± 33.9; 120.1 ± 21.2; e 23.0 ± 3.6 células/mm²; p=0.001). A significant decrease of subbasal nerve parameters in both eyes was found for total nerve length (9.052.6 ± 1.151.8; 14.959.8 ± 903.2; e 22.851.4 ± 661.4 μm/mm² respectively; p<0.001), total number of nerves (5.8 ± 0.9; 11.9 ± 1.2; e 26.6 ± 1.2 n/frame; p<0.001), number of main nerve trunks (2.4 ± 0.3; 3.8 ± 0.3; e 4.4 ± 0.2; p<0.001) and the number of branches (3.4 ± 0.7; 8.2 ± 1.1; e 22.2 ± 1.2; p<0.001) as compared to controls. DC density showed a moderate negative correlation with total nerve length (R=-0.43; p<0.001). Moreover, reduced nerve length and number of nerves were strongly correlated with corneal sensation across all subgroups (R=0.63; p<0.001). Conclusions: Patients with unilateral HZO demonstrated a profound and significant bilateral increase in corneal dendritiform cell density and decrease of the corneal subbasal nerve plexus.

Zebrafish is a frequently used model organism with an array of transgenic lines that have been used indevelopmental and physiological studies. We aim to generate novel transgenic zebrafish reporter lines to study subpopulations of spinal neurons in vivo. The gene editing system called CRISPR/Cas9 system was used to knock in reporter genes such as green fluorescent protein (GFP) or Gal4 transcription factor, to generate transgenic fish lines. Zebrafish embryos were injected with gRNAs targeting gabrb1 or nr4a2a and GFP or Gal4 plasmid, respectively. F0 larvae were screened, positive fish were raised until sexual maturity, and founders characterized to verify germline insertion. Three founders were found for gabrb1 and the location and the direction of the insert verified. The GFP expression was studied during development and differential expression patterns were identified whereas all founders had expression in brain and spinal cord. In parallel, positive fish from the Gal4 injections were raised and will be screened. Immunohistochemistry was performed to check if nr4a2a is expressed in the same cells as known neuronal markers. However, no co-localization was detected. The three gabrb1 founders identified in this study highlight the challenges into creating stable transgenic lines recapitulating true expression of the gene of interest. Sequencing, in-situ hybridization and immunohistochemistry should be performed to verify the line. A possible reason for the varying expression may be that through the knock-in we may interfere with regions regulating gene. The nr4a2a-Gal4 line will be used to perform functional studies. Those experiments will be performed using reporter genes, such as opsins or GCaMP, controlled by Upstream Activation Sequence (UAS). These transgenic lines will provide important insights regarding neuronal subpopulations that express gabrb1 and nr4a2a to unravelhow the locomotor network is formed.

Blood vessels mimics (BVMs) are tissue-engineered blood vessels used to test vascular devices in an environment that mimics some simple anatomical factors of native blood vessels. It is important to accurately and consistently assess tissue-engineered blood vessels, although there is currently a lack of standardization in Cal Poly’s Tissue Engineering Lab and in the entirety of the field. The goal of this thesis was to develop and optimize imaging and image quantification techniques for tissue-engineered blood vessels. The first aim of this thesis optimized and compared imaging and assessment techniques for electrospun scaffolds. Images from different SEMs were compared to determine the benefits and drawbacks of each microscope. Several materials were also imaged using these microscopes to characterize polymers at the microscopic scale and to compare the quality of images from different SEMs. The second aim of this thesis validated and implemented a MATLAB-based automatic fiber diameter measurement tool. Fiber measurements were obtained from a manual ImageJ method, a semi-automatic DiameterJ method, and a new automatic MATLAB method and compared to evaluate accuracy and user variability of the MATLAB tool. The results of this aim validated the accuracy of the MATLAB tool and showed that it resulted in lower user variability as compared to other fiber diameter measurement methods. The third aim of this thesis developed imaging techniques for novel silicone BVMs at each stage of development. Evaluation techniques to quantify cell adhesion and coverage on silicone BVMs using SEM, widefield fluorescent imaging, and immunochemistry were developed. After refining those methods, they were applied and adapted to silicone BVMs with deployed devices. BBI, H&E, and PECAM-1 staining were all found to be effective assessment methods for silicone BVMs. Overall, the work described in this thesis increased the consistency, standardization, and accuracy of scaffold and BVM assessment in Cal Poly’s Tissue Engineering Lab.

This thesis focuses on the study of the life of a fluorescent dye. It also examines changes in its emission spectrum determined with the aid of development of fluorescence intensity over the long term sensing in several kinds if of cella. The general part of the thesis with the issues of confocal microscopy, fluorescent radiation and maks. Furthermorer, an analysis of methods for measuring fluorescence lifetime is done. In the thesis there is elaborated a description of the program, whitch was designed for the analysis of the collected output of the used confocal microscope. Sebsequently, the results are evaluated.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19972号 / 薬科博第63号 / 新制||薬科||7(附属図書館) / 33068 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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Elevated eosinophil counts in the circulation and/or tissues is considered a clinical feature and biomarker of several chronic airway diseases including asthma. As such, many therapeutic biologics for asthma developed within the past decade target eosinophil recruitment to and accumulation in the airways to mixed success. Although the nature of adhesive interactions and directional migration of eosinophils has been well studied, there remains a lack of comprehensive understanding regarding the components which modulate eosinophil movement from the blood into respiratory tissues that impacts the efficacy of these clinical studies; therefore, continued research in this area may reveal novel therapeutic targets and ultimately improve clinical outcomes of patients with eosinophilia-mediated diseases. The Janssen lab serendipitously discovered that the mere perfusion of standard media without pharmacological additives over human eosinophils in vitro induced the release of intracellular calcium (Ca2+) reminiscent of chemokine-induced Ca2+ release well documented in the literature. The central focus of my doctoral research was to characterize this novel phenomenon of the perfusion-induced calcium response (PICR), and to determine its physiological role in the eosinophil extravasation process to inflamed tissue sites. In our first research objective, we optimized a protocol of eosinophil isolation directly from whole blood with emphases on maximizing population purity and yield efficiency while minimizing cell activation that could potentially interfere with secondary functional assays. For our latter two studies, we utilized real-time fluorescent confocal microscopy and immunofluorescence staining to investigate the PICR. We observed that the latency to the PICR post-perfusion was significantly shorter in eosinophils subjected to physiological rates of shear stress, suggesting a temporal-regulatory function of eosinophil mechanosensitivity. Furthermore, the disruption of the PICR via pharmacological inhibitors significantly reduced eosinophil motility by increasing the latency to cytoskeletal rearrangements (flattening onto substrate-coated surfaces, formation of membrane protrusions that explore the environment) necessary for cell migration out of the vasculature. Detailing the role of eosinophil sensitivity to the mechanical trigger of fluid shear stress expands upon the current paradigm of eosinophil recruitment and will contribute to the development of clinical strategies. / Dissertation / Candidate in Philosophy