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Accurate measurement of stallion spermatozoal concentration is important to equine breeding operations. The hemacytometer is considered the standard for measuring spermatozoal concentration but is time consuming and may be imprecise. The flow cytometer is considered precise and accurate, but only practical for research purposes due to sample preparation time and high cost. Photometric systems are commonly used but can be inaccurate outside a relatively narrow concentration range and can be rendered inaccurate in the presence of contaminants. A new instrument, the NucleoCounter SP-100 is reported to enumerate spermatozoa at wider concentration ranges and can identify spermatozoa in opaque semen extenders. Epididymal, neat (raw) ejaculates, and ejaculates diluted in various semen extenders were analyzed with the NucleoCounter, the Densimeter, the Spermacue, flow cytometric and hemacytometric methods. Results were compared statistically by: 1) regression analysis, 2) the agreement of two instruments, whereby the difference in values between two instruments was plotted on the y-axis against the mean of those values on the x-axis [26] and 3) a modified method that measured the percentage deviation, whereby the percentage (of the difference in values between two instruments divided by the mean) of the same two values was plotted on the y-axis against the mean value of the two instruments on the x-axis. The NucleoCounter showed more agreement with both the flow cytometer and hemacytometer for epididymal, neat ejaculated and extended spermatozoa over a range of concentrations than the Densimeter or the Spermacue. The NucleoCounter showed more agreement with the flow cytometer for epididymal and neat ejaculated spermatozoa and more agreement with the hemacytometer for spermatozoa diluted in semen extenders. The Spermacue showed the least agreement with both standards for all spermatozoal comparisons. All coefficients of variation for the flow cytometer, hemacytometer and NucleoCounter were >10% for all spermatozoal comparisons. This study indicates that the NucleoCounter shows more agreement with the flow cytometer and hemacytometer than photometric systems when evaluated with epididymal, neat ejaculated and extended spermatozoa. The instrument is also more repeatable than either photometric system, but may be cost-prohibitive for some operations.

Orientador: Rui Seabra Ferreira / Resumo: O lobo-guará (Chrysocyon brachyurus) é encontrado em grande parte do território da América do Sul, mas 90% de seus exemplares se distribui principalmente no cerrado brasileiro, sendo o maior canídeo sul americano, e como tal, demanda grandes áreas em suas atividades. Consequentemente, ele sofre severamente os efeitos da perda de habitat pelas ações antrópicas. Isto o faz estar na lista vermelha pela União Internacional de Conservação da Natureza, como espécie ameaçada de extinção da fauna brasileira. Tendo em vista a necessidade de desenvolver estratégias para a conservação do lobo-guará, o objetivo deste projeto foi caracterizar e padronizar o isolamento, o cultivo e a criopreservação de células tronco mesenquimais derivado da medula óssea do lobo-guará, além de utilizar o selante heterólogo de fibrina derivado de veneno de serpente como arcabouço tridimensional possibilitando procedimentos de terapia celular para a espécie. Também foi realizada uma revisão bibliográfica sobre a espécie, terapia celular e selantes de fibrina. Para tanto, realizou-se a coleta de medula óssea de animais provenientes de CETAS e zoológicos; o cultivo das células tronco mesenquimais (CTMs); a caracterização a partir do uso de marcadores de superfície (CDs) por citometria de fluxo; o potencial de diferenciação das CTMs em linhagens celulares osteogênica, adipogênica e condrogênica; a análise da viabilidade celular pré e após a criopreservação; e por fim a observação e avaliação do selante de fibri... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by ANDRÉA SEBELIM SESSO null (sessoandrea@hotmail.com) on 2017-09-05T18:54:27Z No. of bitstreams: 1 Mestrado-Andréa Sesso-homologação.pdf: 37681624 bytes, checksum: f7073a1b3ee92b81750c17a746355135 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-09-05T19:39:09Z (GMT) No. of bitstreams: 1 sesso_as_me_bot.pdf: 37681624 bytes, checksum: f7073a1b3ee92b81750c17a746355135 (MD5) / Made available in DSpace on 2017-09-05T19:39:09Z (GMT). No. of bitstreams: 1 sesso_as_me_bot.pdf: 37681624 bytes, checksum: f7073a1b3ee92b81750c17a746355135 (MD5) Previous issue date: 2017-08-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O lobo-guará (Chrysocyon brachyurus) é encontrado em grande parte do território da América do Sul, mas 90% de seus exemplares se distribui principalmente no cerrado brasileiro, sendo o maior canídeo sul americano, e como tal, demanda grandes áreas em suas atividades. Consequentemente, ele sofre severamente os efeitos da perda de habitat pelas ações antrópicas. Isto o faz estar na lista vermelha pela União Internacional de Conservação da Natureza, como espécie ameaçada de extinção da fauna brasileira. Tendo em vista a necessidade de desenvolver estratégias para a conservação do lobo-guará, o objetivo deste projeto foi caracterizar e padronizar o isolamento, o cultivo e a criopreservação de células tronco mesenquimais derivado da medula óssea do lobo-guará, além de utilizar o selante heterólogo de fibrina derivado de veneno de serpente como arcabouço tridimensional possibilitando procedimentos de terapia celular para a espécie. Também foi realizada uma revisão bibliográfica sobre a espécie, terapia celular e selantes de fibrina. Para tanto, realizou-se a coleta de medula óssea de animais provenientes de CETAS e zoológicos; o cultivo das células tronco mesenquimais (CTMs); a caracterização a partir do uso de marcadores de superfície (CDs) por citometria de fluxo; o potencial de diferenciação das CTMs em linhagens celulares osteogênica, adipogênica e condrogênica; a análise da viabilidade celular pré e após a criopreservação; e por fim a observação e avaliação do selante de fibrina como arcabouço celular mostrando sua interação com as CTMs pela microscopia de varredura e viabilidade por microscopia confocal. A técnica empregada para coleta de medula óssea e posterior cultivo foi eficiente, sendo possível sua expansão até a sexta passagem. Os marcadores de superfície celular utilizados foram capazes de reconhecer as células possibilitando a sua caracterização por citometria de fluxo. As CTMs apresentaram potencial de diferenciação em linhagens osteogênica, adipogênica e condrogênica. O processo de criopreservação foi eficaz pois manteve alta a viabilidade celular após o seu descongelamento. A interação com o selante de fibrina foi adequada e manteve as células viáveis como verificado por microscopia confocal, demonstrando a estrutura tridimensional do selante por microscopia eletrônica de varredura. A terapia celular pode proporcionar para a espécie, um tratamento alternativo e eficaz na agilidade de processos regenerativos, minimizando o estresse em tratamentos convencionais de longa duração. O sucesso na criopreservação das CTMs torna possível a criação de um banco de células tronco que certamente ajudará na conservação da espécie. / Maned wolf (Chrysocyon brachyurus) is found in South America, but 90% of its specimens are distributed mainly in the Brazilian Cerrado, being the largest South American canid, and as such, it demands large areas. Consequently, maned wolf severely undergoes the effects of habitat loss by human actions. This makes it on the red list by the International Union for Conservation of Nature, as a threatened species of extinction of Brazilian fauna. Objective of this project was to characterize and standardize the isolation, cultivation and cryopreservation of mesenchymal stem cells derived from the bone marrow of maned wolf. In addition to using the fibrin sealant derived from snake venom as a three-dimensional scaffold enabling cellular therapy procedures for the species. A literature review on the species, cell therapy and fibrin sealants were also carried out. For that, the bone marrow was collected from CETAS and zoological animals; culture of mesenchymal stem cells (MSCs); characterization by surface markers (CDs) using flow cytometry; differentiation potential of MSCs into osteogenic, adipogenic and chondrogenic cell lines; analysis of cell viability before and after cryopreservation; and finally, observation and evaluation of the fibrin sealant as cellular scaffold showing its interaction with the MSCs by scanning microscopy and viability by confocal microscopy. The technique used for collection of bone marrow and subsequent culture was efficient, being possible to expand until the sixth passage. Cell surface markers used were able to recognize the cells allowing their characterization by flow cytometry. CTMs presented differentiation potential in osteogenic, adipogenic and chondrogenic linages. Cryopreservation process was effective because it maintained high cell viability after thawing. The interaction with the fibrin sealant was adequate and kept the cells viable as verified by confocal microscopy, demonstrating their three-dimensional structure of the sealant by scanning electron microscopy. Cell therapy may provide for the species an alternative and effective treatment in the agility of regenerative processes, minimizing stress in conventional long-term treatments. Success in cryopreservation of MSCs makes it possible to create a stem cell bank that will certainly help in its conservation.

The prevention of waterborne diseases requires rapid detection of pathogens in drinking water, with an ultimate goal of in-line monitoring in real time. Standard cultivation-based methods are too time-consuming and thus not suitable for this purpose. Many technologies were proposed to achieve this goal, such as ELISA, PCR, FISH, FTIR and flow cytometry. However, they still have limitations of non-specificity, complexity and high cost. Therefore, an optical system is proposed and developed towards the in-line monitoring of bacteria, which combines the advantages of FTIR and micro-flow cytometer for bacterial identification and precise quantification. The in-line use requires obtaining IR spectra of bacterial cells directly in water, which is achieved using a CaF2 liquid cell. The spectra of a series of bacterial samples are collected and analyzed using principal component analysis for their differentiation. A preliminary study on fabricating a CaF2 concentrator is conducted, in which a novel phenomenon on stress release of silicon nitride film on CaF2 substrate is discovered and studied. To determine the concentration of bacteria in drinking water, a micro-flow cytometer is built based on a micro-fabricated device that integrates on-chip beam-shaping optics and microfluidic channels. With this micro-flow cytometer and optimized data analysis for counting particles in real time, linearity with correlation coefficient of over 0.99 is achieved for the dependences of throughput on both volumetric flow rate and concentration of sample. With a one-dimensional hydrodynamic focusing, no degradation of the counting efficiency is demonstrated when the focused sample stream expands. The high accuracy of counting makes this micro-flow cytometer a promising candidate for low concentration applications. Counting of E. coli DH5α cell suspensions in phosphate buffered saline is performed using the micro-flow cytometer. Side-scattered light signals are used to count the E. coli cells. A detection efficiency of 92% is achieved when compared with the expected count from a haemocytometer. It is demonstrated that E. coli can be easily distinguished from beads of similar sizes (2-4µm) as their scattering intensities are different. / Thesis / Doctor of Philosophy (PhD)

As technology improves, scientists are able to generate high-throughput data faster and cheaper. Consequently, the field of biological sciences is progressively becoming more reliant on data science tools like machine learning methods for analysis and sorting of big data. The Complex Object Parametric Analyzer and Sorter (COPAS) is a large particle flow cytometer that can perform high-throughput fluorescence screens on small animals, like Caenorhabditis elegans. The outputs of the COPAS are extinction coefficient (EXT), Time of Flight (TOF, arbitrary length unit) and fluorescence. However, the COPAS outputs include unwanted objects like bubbles or bacteria and some animals pass the flow cell in a non-straight manner producing abnormal profiles leading to inaccurate developmental staging. In this thesis, I have created an R package, named COPASProfiler, that generates experiment-specific supervised machine learning (ML) classification models which can detect and remove abnormal profiles enabling standardized fluorescence quantification and analysis. I used COPASProfiler to develop a pipeline to automate fluorescence analysis of high-throughput COPAS data sets. Using R shiny, I created a web program with a graphical user interface that allows users to view, annotate, quantify fluorescence, and classify COPAS-generated datasets. The COPASProfiler is available on GitHub and can be installed using one single R command. Lastly, the COPASProfiler comes with multiple tutorials and examples, and was designed to accommodate users with minimal programming experience. COPASProfiler should enable robust high-throughput fluorescence studies of regulatory elements (e.g., enhancers, promoters, and 3’UTRs) and long-term epigenetic silencing in C. elegans.

Chelonia mydas, denominada tartaruga verde, é uma tartaruga marinha que freqüenta o litoral brasileiro para alimentação e nidificação e é considerada em perigo de extinção pela IUCN (2006). A fibropapilomatose, doença caracterizada por tumores cutâneos benignos (fibropapilomas), é uma das mais importantes ameaças à sobrevivência dessa espécie. Pesquisas sugerem o envolvimento de agentes infecciosos virais em associação com fatores ambientais e genéticos. Foram estudadas 47 tartarugas provenientes do litoral do estado de São Paulo, sendo 18 sem fibropapilomas e 29 acometidas. Dados de biometria das tartarugas, quantidade, localização e tamanho dos tumores foram anotados. O objetivo do trabalho foi avaliar a função dos leucócitos sangüíneos e o perfil hematológico das tartarugas acometidas ou não. As células receberam estímulo de Saccharomyces cerevisiae e Staphylococcus aureus para avaliação da fagocitose e miristato-acetato de forbol (PMA) para avaliação do burst oxidativo. Foram identificadas três populações celulares: heterófilos, monócitos e linfócitos. Os monócitos foram as células responsáveis pela fagocitose e pelo burst oxidativo. Animais com fibropapilomas apresentaram intensidade de burst oxidativo basal e induzido por PMA maior. No entanto, animais sem fibropapilomas apresentaram maior intensidade de fagocitose (estimulada por Saccharomyces cerevisiae). Foi realizado hemograma completo e a análise estatística demonstrou que houve diferença significativa (p<0,05), entre animais com e sem tumores, apenas para HCM (Hemoglobina Corpuscular Média) e os animais com fibropapilomatose apresentaram a menor média para esse parâmetro. A patogenia ainda é pouco elucidada, fato preocupante porque esta doença acomete muitas tartarugas jovens. Doenças relacionadas à poluição são um fator relevante para a redução populacional de animais e para o desequilíbrio dos ecossistemas. / Chelonia mydas, called green turtle, is a sea turtle that feeds and nests in brazilian coast and is considered endangered (Red List - IUCN, 2006). The fibropapillomatosis, disease characterized as benign cutaneous tumor (fibropapillomas), is a threat to Chelonia mydas. Studies suggest that fibropapillomatosis cause may be an association of virus, environmental and genetic factors. We studied 47 turtles from São Paulo north coast, twenty nine with fibropapillomas and 18 without this disease. The biometry of the turtles, quantity, localization and size of the tumors was registered. The goal of this work was to evaluate the leukocytes function and blood profile of the turtles with and without fibropapillomatosis. The cells were stimulated with Saccharomyces cerevisiae and Staphylococcus aureus to phagocytosis evaluation and phorbol miristate-acetate (PMA) to oxidative burst evaluation. Three populations cells were identified: heterophils, monocytes and lymphocytes. The monocytes were the responsible cells to phagocytosis and oxidative burst. The monocytes of the turtles with fibropapillomas displayed the higher oxidative burst fluorescence intensity (basal and stimulated).However, the monocytes of the turtles without fibropapillomas displayed the higher phagocytosis fluorescence intensity. The blood count and the statistics analysis were significant (p<0,05) only for MCH (Mean Corpuscular Hemoglobin) among turtles with and without fibropapillomas, the animals with fibropapillomatosis showed the minor average. The pathogenesis of fibropapillomatosis is still unclear and is a threat to green turtle survive because this disease affect young green turtles. Diseases related with pollution are a relevant factor for the reduction of animal population and ecosystem unbalance.

Chelonia mydas, denominada tartaruga verde, é uma tartaruga marinha que freqüenta o litoral brasileiro para alimentação e nidificação e é considerada em perigo de extinção pela IUCN (2006). A fibropapilomatose, doença caracterizada por tumores cutâneos benignos (fibropapilomas), é uma das mais importantes ameaças à sobrevivência dessa espécie. Pesquisas sugerem o envolvimento de agentes infecciosos virais em associação com fatores ambientais e genéticos. Foram estudadas 47 tartarugas provenientes do litoral do estado de São Paulo, sendo 18 sem fibropapilomas e 29 acometidas. Dados de biometria das tartarugas, quantidade, localização e tamanho dos tumores foram anotados. O objetivo do trabalho foi avaliar a função dos leucócitos sangüíneos e o perfil hematológico das tartarugas acometidas ou não. As células receberam estímulo de Saccharomyces cerevisiae e Staphylococcus aureus para avaliação da fagocitose e miristato-acetato de forbol (PMA) para avaliação do burst oxidativo. Foram identificadas três populações celulares: heterófilos, monócitos e linfócitos. Os monócitos foram as células responsáveis pela fagocitose e pelo burst oxidativo. Animais com fibropapilomas apresentaram intensidade de burst oxidativo basal e induzido por PMA maior. No entanto, animais sem fibropapilomas apresentaram maior intensidade de fagocitose (estimulada por Saccharomyces cerevisiae). Foi realizado hemograma completo e a análise estatística demonstrou que houve diferença significativa (p<0,05), entre animais com e sem tumores, apenas para HCM (Hemoglobina Corpuscular Média) e os animais com fibropapilomatose apresentaram a menor média para esse parâmetro. A patogenia ainda é pouco elucidada, fato preocupante porque esta doença acomete muitas tartarugas jovens. Doenças relacionadas à poluição são um fator relevante para a redução populacional de animais e para o desequilíbrio dos ecossistemas. / Chelonia mydas, called green turtle, is a sea turtle that feeds and nests in brazilian coast and is considered endangered (Red List - IUCN, 2006). The fibropapillomatosis, disease characterized as benign cutaneous tumor (fibropapillomas), is a threat to Chelonia mydas. Studies suggest that fibropapillomatosis cause may be an association of virus, environmental and genetic factors. We studied 47 turtles from São Paulo north coast, twenty nine with fibropapillomas and 18 without this disease. The biometry of the turtles, quantity, localization and size of the tumors was registered. The goal of this work was to evaluate the leukocytes function and blood profile of the turtles with and without fibropapillomatosis. The cells were stimulated with Saccharomyces cerevisiae and Staphylococcus aureus to phagocytosis evaluation and phorbol miristate-acetate (PMA) to oxidative burst evaluation. Three populations cells were identified: heterophils, monocytes and lymphocytes. The monocytes were the responsible cells to phagocytosis and oxidative burst. The monocytes of the turtles with fibropapillomas displayed the higher oxidative burst fluorescence intensity (basal and stimulated).However, the monocytes of the turtles without fibropapillomas displayed the higher phagocytosis fluorescence intensity. The blood count and the statistics analysis were significant (p<0,05) only for MCH (Mean Corpuscular Hemoglobin) among turtles with and without fibropapillomas, the animals with fibropapillomatosis showed the minor average. The pathogenesis of fibropapillomatosis is still unclear and is a threat to green turtle survive because this disease affect young green turtles. Diseases related with pollution are a relevant factor for the reduction of animal population and ecosystem unbalance.

This research mainly develops a new z-position measurement based on the chromatic aberration effect. An objective-type dark-field illumination scheme is built to produce diascopic chromatic aberration light, and aimed to enhance the signal-to-noise ratio. The xenon lamp is adapted to create white light with continuous spectrum, besides, lens with low Abbe number is needed to extend the degree of chromatic aberration, so lens made of PMMA is as a chromatic aberration component. In the proposed system, the depths of samples in micro-channel is illuminated by the dispersed light and scatter the optical signals, which are captured by a low numerical aperture (N.A.) objective lens. After the simple normalization, the intensity ratio of two selected wavelengths 450 nm (blue light) and 670 nm (red light) from the scattered spectrum becomes a reliable index for the depth information of the detecting objects. By means of establishing the relationship between depth and intensity ratio, every object flowing through diagnosed spot is able to be determined the depth level by cross-referencing the database. By using spectrometer as detector, delicate moving components for light filtering or electrical stage for light scanning can be excluded for high-speed z-position detection. Furthermore, in order to identify the depth level of sample with high flowing rate, avalanche photodiodes are adapted to achieve rapid detection. The experimental results show that the relationship between depth and intensity ratio is a parabola curve, but in this research, the region which tends to behavior linearly is adapted. The proposed system provides a linear detection range of ¡Ó15 £gm for particles with a diameter of 20 £gm. The lens with high Abbe number only obtains ¡Ó10 £gm with linear detection range though, the resolution for size is better than PMMA. The BK7 lens is capable to discriminate the depth change of 2 £gm micro-beads, note that there is no limitation of depth discrimination in this system, because of the measurement is achieved by cross-referencing the linear line. The use of UV-Vis-NIR spectrometer enable this system to analyze the depths of the samples in flow rate 0.5 mm/s. To gain the higher performance, the two avalanche photodiodes are utilized, and the short(CWL=450 nm, ¡Ó20 nm) and long(CWL=650 nm, ¡Ó20 nm) band pass filter are also equipped to represent enhancements of blue and red ray. The effective detection range extends to ¡Ó25 £gm and has high linearity(R square=0.99285) after the optimization of light stop. In high flowing rate detection, this system is able to identify the depth of sample when the flow velocity is 4.167 mm/s, the calculated throughput is 126 particles/s. It also successfully analyzes the depth of flowing human erythrocytes under the flow velocity is 2.778 mm/s, the velocity which the developed system is capable to analyze is about 5-8 folds to the conventional micro-PIV system. With this novel and simple approach, there will be the quantified information from z-direction of flowing body for bio-analysis, and also benefits estimating the performance of micro structure or device in the microfluidic chip, also the analysis of flow field. Except for dynamical detection, this system also be capable to apply in a open and static situation, such as cell or tissue proliferation assay.

In this work a Raman flow cytometer is presented. It is a whole new microfluidic device that takes advantage of basic principles of Raman spectroscopy and fluorescent flow cytometry mixed together in a system of particularly shaped channels. These are indeed composed by specific shape and sizes – thanks to which cells can flow one-by-one – and a trap by means of which cells are trapped in order to perform Raman analysis on single ones in a constant and passive way. In this sense the microfluidic device promotes a fast method to look for single cells in a whole multicellular sample. It is a label-free analysis and this means that, on the contrary of what happens with fluorescent flow cytometry, the sample does not need to undergo any particular time-consuming pretreatment before being analyzed. Moreover it gives a complete information about the biochemical content of the sample thanks to the involvement of Raman spectroscopy as method of analysis. Many thought about a device like this, but eventually it is the first one being designed, fabricated and tested. The materials involved in the production of the Raman flow cytometer are chosen wisely. In particular the chip – the most important component of the device – is multilayered, being composed by a slide of calcium fluoride (which gives a negligible signal in Raman analyses), a photosensitive resist containing a pattern with channels and another slide of calcium fluoride in order for the channels to be sealed on both sides. The chip is, in turn, connected to gaskets and external frames. Several fabrication processes are followed to ultimately get the complete Raman flow cytometer and experiments on red blood cells demonstrate its validity in this field.