Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27Bitstream added on 2014-06-13T19:08:59Z : No. of bitstreams: 1 sodre_lp_me_botfm.pdf: 1339623 bytes, checksum: ed335773fb6c0b581fc33a31e3612a6e (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Anticorpos monoclonais (AcMm) são imunoglobulinas produzidas por engenharia celular através da fusão de células tumorais com linfócitos B provenientes de organismos previamente imunizados contra o antígeno de interesse. As células recém criadas recebem a denominação de hibridomas, crescem indefinidamente em cultura por agregar características de imortalidade das células tumorais. A tecnologia de monoclonais compreende etapas seriadas, incluindo imunização, fusão, screening, clonagem, caracterização de especificidade dentre outras. O CD45 é um marcador expresso na superfície de todos os leucócitos humanos: linfócitos, eosinófilos, monócitos, basófilos e neutrófilos sendo, o maior componente da membrana de linfócitos, o que aumenta a probabilidade de obtenção de AcMm com este perfil em protocolos de imunização com linfócitos. Este marcador é amplamente utilizado em diagnóstico por citometria de fluxo, no acompanhamento de pacientes HIV+, na classificação de leucemias e linfomas, quantificação de células CD34+ para transplantes de medula óssea e, também em imunohistoqu ímica auxiliando na subtipagem das neoplasias, principalmente linfomas (Hodgkin, não Hodgkin e outras categorias) e leucemias de células B e T. Assim, este trabalho teve por objetivo a caracterização de anticorpo monoclonal anti-CD45 desenvolvido in house e validação deste em aplicações diagnósticas envolvendo citometria de fluxo e imunohistoqu ímica, comparando-o com anticorpos monoclonais comerciais, visando minimização dos custos dessas técnicas. Foram utilizadas 50 amostras de sangue excedente de doadores para o grupo controle e 48 amostras de sangue de pacientes HIV+ para validação do AcMm em citometria de fluxo e analisados 114 fragmentos em lâmina de Tissue Microarray - TMA provenientes de 38 amostras de amígdalas para validação do AcMm em imunohistoqu ímica... / Monoclonal antibodies (mAb) are immunoglobulins produced by cell engineering through fusion of tumor cells and B lymphocytes from organisms previously immunized against the antigen of interest, generating thus hybridomas that indefinitely grow in culture since they aggregate immortality characteristics of tumor cells capable of secreting antibodies. The production of such antibodies consists of sequential steps, including immunization, fusion, screening, cloning, and specificity characterization. CD45 is a marker expressed in the surface of all human leukocytes: lymphocytes, eosinophils, monocytes, basophils, and neutrophils. Besides, CD45 is the major component of lymphocyte membranes, which increases the probability of obtaining mAb presenting such profile in immunization procedures using lymphocytes. This marker has been widely used in diagnosis through flow cytometry, besides monitoring of HIV+ patients, classification of leukemia and lymphomas, quantification of CD34+ cells for bone marrow transplantation, and also in immunohistochemistry, helping in subtyping of neoplasms, mainly lymphomas (Hodgkin s, non-Hodgkin and other categories) and B- and T-cell leukemia. Thus, this work aimed to characterize the in-house-developed anti-CD45 monoclonal antibody besides its validation in diagnostic applications involving flow cytometry and immunohistochemistry through comparison with commercial monoclonal antibodies in order to reduce the costs of such techniques. Fifty samples of extra blood from donors were used in the control group, and 48 blood samples from HIV+ patients were used for mAb validation in flow cytometry. For mAb immunohistochemical validation, 114 fragments from 38 amygdala samples were analyzed in a Tissue Microarray - TMA slide. In flow cytometry, the commercial anti-CD45 and the likely in-house-obtained anti-CD45 (LINB5 clone) did not significantly differ concerning cell... (Complete abstract click electronic access below)

Made available in DSpace on 2018-08-01T22:57:27Z (GMT). No. of bitstreams: 1 tese_10051_Dissertação Final Lucas Mesquita Barreto.pdf: 1926946 bytes, checksum: 21145320a1a7d898eb87b86e37b7dd01 (MD5) Previous issue date: 2016-07-18 / Previous cytogenetic studies in Dorstenia mention that the species may have 24 to 72 chromosomes, and suggested a conserved chromosome number 2n = 32 for the Neotropic species. However, some information reported in the literature are dubious or insufficient to assess the potential of cytogenetic data to the better understand of systematics and evolution issues within this genus. Here, eight species of Neotropical Dorstenia had their karyotypes characterized, and the nuclear DNA content measured. Dorstenia bahiensis, D. cayapia, D. grazielae, D. hirta and D. turnerifolia had their karyotypes characterized and the DNA nuclear content measured for the first time. Morphological plant characters and morphometric data were submitted to cluster analysis, followed by a test of group sharpness, and ordination analysis, aiming to support the discussion about the potential of cytogenetic data to infrageneric systematic of Dorstenia. The species showed chromosome number of 2n = 32, varying in chromosomes morphology. The karyotypes least asymmetric were observed in Dorstenia elata, and the more asymmetric were registered in D. bahiensis and D. bonijesu. The 2C value ranged from 3.21 picograms (pg) D. bahiensis to 5.47 pg in D. arifolia. Morphologically similar species, like D. hirta and D. turnerifolia, grouped together based on morphometric data. The sharp groups based on morphometric data correspond to species circumscribed under the sections Dorstenia, Lecania and Emygodia, previously established based on the plant morphology. Our results supports that the chromosome number 2n = 32 is possible conserved in the Neotropical species of Dorstenia, and indicate the potential of cytogenetic data to the systematics of this genus.

Made available in DSpace on 2018-08-01T22:57:30Z (GMT). No. of bitstreams: 1 tese_9189_Dissertação Final Paulo Marcos Amaral Silva20160628-103338.pdf: 832244 bytes, checksum: de4cc3d0c60078e1f76f3d4f95f3b534 (MD5) Previous issue date: 2015-08-14 / The dioecy occurs in 6% of the angiosperms, including all the Myrsine species (Primulaceae) that show male and female individuals distinguishable based in sexual organs and morphology of sepals and petals. The sexual system fixed in this genus, was the motivation in research if the existence of sexual chromosomes culminate in dioecy for Myrsine. Employing tissue culture, flow cytometry and cytogenetic tools, the aimed this study was characterize the karyotype of Myrsine coriacea (Sw.) R.Br. ex Roem & Schult., Myrsine umbellata Mart., Myrsine guianensis (Aubl.) Kuntze and Myrsine parvifolia A.DC. From leaves of male and female individuals collected in the field and leaves and roots of unknown sex obtained of in vitro culture, were found mean values of DNA content statistically identical. These data were attributed to the presence of secondary metabolites reported for the genus. However, an intraspecific variation was observed in cytogenetic analysis in the four Myrsine species. Slides exhibited metaphases with 2n = 45 and 2n = 46 chromosomes in different individuals. These results were observed consistently from different times of exposition to anti-mitotic agent and distinct treatments of enzymatic maceration. Thus, the chromosome number variation found, associated with the sexual system well defined, can be concerning to the sexual chromosomes in Myrsine genus. These data suggest a chromosomal system of sex 9 determination with multiple X and/or Y described in some plant species. The system ZW also is possible, as well as X0 or Z0, systems still not reported in vegetal groups. The present work provided the first data about the nuclear DNA content by flow cytometry in Myrsine and supplied cytogenetics evidences that indicate the existence of sexual chromosomes.

No description available.

During bio-technical processing it is important to monitorbiological parameters such as cell growth, viability andproduct formation. Many of the analyses traditionally used areslow to perform and provide only average data for thepopulation. Flow cytometry is a laser-based technique, whichmeasures physical properties of a cell in a flowing stream, ata rate of several thousand cells per second. It offers theprospect of an at-line, multi-parameter analysis of individualmicroorganisms in a population. In this project several methods for at-line measurements ofbioprocesses were developed such as protocol's for measuringcell concentration, viability and product formation. Theprimary focus was on prokaryotic organisms (E. coli) but eukaryotic organisms (P. pastoris) were included. The possibility to use volumetric cell counting to measurecell concentration (cell number) was evaluated. It was shownthat the method was applicable for high cell density processesof bothE. coliandP. pastoris. The combination of Bis- (1,3-dibutylbarbituric acid)trimethine oxonol (depolarised membranes) and propidium iodide(loss of membrane integrity) as fluorescent markers was usefulto measure viability at-line of cells in high cell densityprocesses. The protocol was shown to be reproducible forE. coliandP. pastoris. The viability staining was used to study the kinetics ofweak organic acids (food preservatives). The protocol provideddata about cell functions such as membrane depolarisation andloss of membrane integrity caused by introducing weak organicacids to shake flask cultures ofE. coli. Labeling inclusion bodies with fluorescent antibodiesprovided a method, which could specifically monitor theincreased accumulation of recombinant promegapoetin proteinwith process time. This technique was further developed forintracellular staining by application of a permeabilising stepbefore labeling with antibodies. Staining of inclusion bodiesdirectly inside permeabilised cells gave information about thedistribution of protein expression in the cell population. In conclusion, flow cytometry provides an at-line, singlecell technique for measurement of several biological parametersin bioprocesses. Key words: flow cytometry, Partec PAS, propidium iodide(PI), bis- (1,3-dibutylbarbituric acid) trimethine oxonol(BOX), Alexa fluor 488, bioprocess,E. coli,P. pastoris, inclusion body, food preservatives,viability, membrane potential

Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-α. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (Tm(A)) and resting (Tm(R)) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of Tm(A) (but not Tm(R)) was capable of blocking monocyte apoptosis and the ability of Tm(A) to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both Tm(A) and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.

No description available.

Field evaluation of frozen-thawed stallion semen has been limited to tests such as post-thaw motility and morphology that are not only subjective but also evaluate only a small population of cells. Flow cytometry has provided a quick, repeatable, objective method of evaluating a large number of cells, including spermatozoa. Two experiments were designed to first validate the use of several flow cytometric tests on frozen-thawed stallion semen and then determine a model that may best explain variation in fertility. Comparing samples that were live and freeze-killed validated the flow cytometric tests. In experiment one, six ejaculates were collected from each of three stallions. The semen from each ejaculate was centrifuged and frozen in 0.5 ml polyvinyl chloride straws. Two straws from each ejaculate were thawed and evaluated. Semen was evaluated for post-thaw motility, morphology, mitochondrial activity using Rhodamine 123 (R123), plasma membrane integrity using propidium iodide (PI) and ethidium monoazide (EMA), and chromatin structure using the sperm chromatin structure assay (SCSA). Data was recorded as percentages for all but the SCSA for both experiment one and two. The extent of chromatin denaturation was calculated using the SCSA and the alpha-t population [at = red/(red +green) fluorescence]. From the alpha-t population, statistics were calculated such mean (Xat), standard deviation (SDat), percentage of cells outside (COMPat) the main alpha-t population and the mean green fluorescence (mean green) of the population. Results from experiment one demonstrated that all flow cytometric tests except EMA were able to distinguish between live and freeze-killed samples (p < 0.0001). Also the stallion accounted for most of the variation in samples when compared to ejaculate and straw within an ejaculate. Therefore two straws could be chosen at random from a stallion and evaluated in experiment two. In experiment two, twenty-nine stallions were evaluated using the same tests as experiment one excluding EMA. Fertility data was obtained from the 1998 or 1999 breeding season. Multiple linear regression was used to determine the best-fit model to predict overall pregnancy rate. SCSA and R123-PI assays accounted for the largest amount of variation in fertility (R² = 0.65, p < 0.0004). Within SCSA and the R123/PI assays Xat and PI staining had the highest contribution to this variation in fertility (R² = 0.11, R² = 0.47) respectively. The best-fit model for predicting fertility included the assay combination listed above and the interactions between SDat and mean green staining as well as R123 and mean green staining. Post-thaw motility and morphology did not account for significant variation in fertility (p = 0.22, p = 0.46) respectively. Based on this project post-thaw motility and morphology are poor predictors of fertility in frozen-thawed stallion semen. However, through the addition of SCSA and R123-PI to the routine evaluation of frozen-thawed stallion semen time and money may be saved in advance by identifying those stallions with poor post-thaw fertility. / Master of Science

Due to the lead spike and its related health concern in the DC area, Point-of-Use (POU) filters were installed at public schools to reduce lead concentrations in water. However, the installation of POU filter could possibly lead to the growth of bacteria inside the filters, which could lead to health concerns. Therefore, the potential effects of POU filters on microbial growth was investigated. To explore the cause of filter effects on microbial growth, a sampling campaign was carried out between July and December 2017 from 25 outlets within 5 elementary schools in the DC area. The applicability of flow cytometry results as a quantification method was validated and then used to quantify the biological growth. Our results revealed that the installation of POU filters may lead to nitrification and an increase in microbial growth. Along with the increase in microbial growth, the microorganism community "fingerprints" based on flow cytometry data showed that the installation of filter could also shift the community distribution of bacteria based on their morphology. This study serves as a preliminary study to investigate the mechanics of microbial colonization on POU filters. / Master of Science / Due to the lead spike and its related health concern in the DC area, Point-of-Use (POU) filters were installed at public schools to reduce lead concentrations in water. POU filters are usually installed at sinks and fountains and the Water Quality Association reported that 41% of American homes used POU filters by 2000. However, the installation of POU filter could possibly lead to the growth of bacteria inside the filters, which could lead to health concerns. Therefore, the potential effects of POU filters on microbial growth was investigated. To explore the cause of filter effects on microbial growth, a sampling campaign was carried out between July and December 2017 from 25 outlets within 5 elementary schools in the DC area. Flow cytometry is an optical technology that can measure and then analyze multiple physical characteristics of a single particle as it flows in a fluid stream. The applicability of flow cytometry results as a quantification method was validated and then used to quantify the biological growth. Our results revealed that the installation of POU filters may lead to nitrification and an increase in microbial growth. Along with the increase in microbial growth, the microorganism community “fingerprints” based on flow cytometry data showed that the installation of filter could also shift the community distribution of bacteria based on their morphology. This study serves as a preliminary study to investigate the mechanics of microbial colonization on POU filters.

Introduction: Viruses, extracellular vesicles (EVs) and endogenous retroviruses (ERVs) are types of sub-micron particles which are known to be released from a vast range of cell types, across many species. There are many medically relevant sub-micron particles which can enter healthy cells and enable the intercellular delivery of functional host-derived and foreign products, through their enclosed lipid layers. While multiple particle subsets have been identified, many of the properties, behaviors and biochemical functions have not been fully described and have yet to be characterized. Materials and Methods: CD4⁺ naïve T-cells were isolated from female C57BL6/N mice and stimulated with varying concentrations of PMA/I. In addition to concentration, the length of PMA/I activation was assessed. Supernatants and cells were harvested, filtered, and stained to be subsequently analyzed by Nanoscale Flow Cytometry, Nanoparticle Tracking Analysis and Flow Cytometry. Particle populations were quantified and sorted by size, by NTA. Labelling dye CFSE was used in conjunction with fluorescently conjugated CD81 and CD9 antibodies to separate EVs, including exosomes, from background signal. Naïve T-cell purity, viability and levels of activation were assessed by flow cytometry using CD3, CD4 and CD62L antibodies and viability staining. Results: Increasing PMA concentration led to a global increase in particles by T-cells and a specific increase in smaller particle production and were demonstrated to be significant by Welch’s T-test, when compared to non-activated and DMSO controls (p<0.0001). In addition to concentration, activation length also correlated with increases in total particle counts and a specific increase in the secretion of smaller particles in comparison to non-activated and DMSO controls (p<0.0001). Labelling techniques by NFC revealed an increased presence of CFSE-CD81 positive and CFSE-CD9 positive particles secreted by T-cells, treated for 24 hours, compared to the 0- and 12-hour timepoints. Conclusion: This work demonstrates preliminary steps and outlines methods to begin assessing discrete particle populations and subsets secreted by murine naïve T-cells. Being able to identify patterns of particle secretions by naïve T-cells, especially under immune-stimulated conditions, may be the solution to uncovering the necessary information on EV physiology, that is required to understand the roles EVs play in pathology and how these conserved pathways may lead conditions to become exacerbated. This knowledge is essential to uncovering the roles EVs play in pathophysiology, and in the development of novel rapid diagnostic tests, to screen for cancers, infections, autoimmune disorders, and numerous other pathological conditions.