Orientadores: Silvana Pereira Barros, Decio dos Santos Pinto Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-09T16:03:06Z (GMT). No. of bitstreams: 1 Vaz_SoniaAndradeSilveira_D.pdf: 1101467 bytes, checksum: b3ab61ba8bad61148a6ab70c57bece91 (MD5) Previous issue date: 2006 / Resumo: Muitas pesquisas a respeito do paratormônio (PTH) têm demonstrado que o efeito anabólico (neoformativo) desse peptídeo sobre o tecido ósseo, quando administrado de um modo intermitente, resulta na indução de nódulos de mineralização em cultura de osteoblastos, deposição óssea e aumento da resistência a fraturas em todo o esqueleto. Frente a esses possíveis benefícios, o PTH foi aprovado recentemente pela US Food & Drug Administration (FDA) com o nome genérico de Teriparatide e com o nome comercial de Forteo® (Lilly) que já está sendo comercializado para o tratamento da osteoporose, mas, no entanto ainda vem suscitando grande interesse na comunidade científica, uma vez que o seu complexo mecanismo de ação não está totalmente esclarecido. Relatos científicos indicam que parte do anabolismo do PTH se deve a diferenciação e proliferação dos osteoblastos, aumentando significativamente o número destas células, e parte a um efeito inibidor da apoptose nos osteoblastos. O presente estudo investigou in vitro a capacidade anti-apoptótica do hPTH(1-34) em osteoblastos induzidos à apoptose pelo TNF-a, elegendo como modelo de estudo o cultivo de osteoblastos de calvária de rato. As células foram cultivadas em MEM- a, suplementado com SFB, ácido ascórbico, ß-glicerofosfato e gentamicina, e foram divididas em grupos experimentais que receberam tratamentos diferentes. No grupo I, as células foram induzidas à apoptose, recebendo tratamento com o TNF-a; no grupo II, as células não foram induzidas à apoptose e receberam tratamento intermitente com o hPTH(1-34); no grupo III, as células foram induzidas à apoptose e receberam tratamento com o hPTH(1-34); e no grupo IV, as células não foram induzidas à apoptose e não receberam tratamento com o PTH (controle). Para a detecção da apoptose foram empregados os métodos de exclusão por marcação com o corante azul de Trypan (câmara de Neubauer) e a citometria de fluxo (FACSCalibur). Os resultados obtidos foram submetidos à análise estatística que demonstrou que o tratamento intermitente com o PTH inibiu a apoptose em torno de 60% nos osteoblastos induzidos pelo TNF-a, aumentando, desta forma, o número de osteoblastos / Abstract: It is still not clear why sustained elevation of parathyroid hormone (PTH) stimulates bone resorption, whereas intermittent administration stimulates bone formation. Intermittent PTH administration was recently approved by the United States Food and Drug Administration as the first form of osteoporosis therapy that increases bone mass de novo, reverses the bone deficit, providing a proof-of-principle that osteoblast-targeted (anabolic) agents can effectively reduce osteoporotic fractures. Considering indications that attenuation of osteoblast apoptosis by daily injections of PTH in mice should account for the increased number of osteoblasts and, thereby, the increased bone formation produced by this treatment regimen, in the present study we aimed to investigate the anti-apoptotic ability of hPTH (1-34) in cultured osteoblastic cells through induction by TNF-a in association with PTH anabolic treatment using a calvariaderived osteoblastic cell line. Cells were cultured in MEM-a, supplemented with FBS, ascorbic acid, ß-glycerphosphate and gentamicin, and were divided in experimental groups that received different treatments. In group I, cells were induced to apoptosis, receiveing treatment with TNF-a; in group II, cells were not induced to apoptosis and received intermittent treatment with hPTH(1-34); in group III, cells were induced to apoptosis and received intermittent treatment with hPTH(1-34); and in group IV, cells were not induced to apoptosis and didn¿t receive intermittent treatment with hPTH(1-34) (control). For determination of cell death, two methods were used: staining with Trypan Blue and flow cytometry. The results were statistically analyzed and demonstrated that intermittent treatment with hPTH(1-34) was able to inhibit apoptosis induction by 60%, thereby increasing osteoblast number even in the presence of TNF-a / Doutorado / Histologia e Embriologia / Doutor em Biologia Buco-Dental

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>Magister Scientiae - MSc / Chromosome doubling has become an important tool in breeding programmes as it offers the ability of introducing novel traits into existing plants. Doubled haploid plants are highly valued by both consumers and breeders as these plants usually show larger flower, leaves and fruit, thus making them more marketable. Marianna open pollinated plum rootstocks’ adaptability to different soil types and moisture conditions has been favoured in polyploidy studies as parental material in breeding programmes. The potential of the microtubule depolymerizing herbicide (oryzalin) for in vitro chromosome doubling were investigated by optimizing the concentration and incubation time of plant shoots to the antimitotic agent. Meristem tissues were treated for two time intervals (24 and 48 h) with five different concentrations of oryzalin (50, 75, 100, 150 or 200 μM) in liquid Murashige and Skoog (MS) medium. After treatment, plants were allowed to grow under a 16/8 h light/dark photoperiod at 24±2˚C for 4 weeks. One and two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) was used to separate, visualise and identify differently expressed proteins. Furthermore, changes in ROS accumulation, photosynthetic pigmentation, lipid peroxidation and antioxidant enzyme activity (SOD, APX and GR) were investigated. Flow cytometry results revealed that treatment of plants with oryzalin concentrations ranging from 75 to 150 μM induced ploidy after 24 h exposure whereas, 200 μM produced mixoploids containing both tetraploid and octoploids plants after 24 h exposure. Longer incubations of 48 h were detrimental to plant tissues as complete mortality was observed in the higher concentration (100 to 200 μM) treatments. Mass spectrometry analysis identified 14 differentially expressed protein spots that were characterized into different functional categories. ROS accumulation, the extent of lipid peroxidation and antioxidant capacity were differentially regulated in response to oryzalin treatment whereas photosynthetic pigments were significantly enhanced. The results suggests that oryzalin-induced proteins may act as potential biomarkers to improve fruit characteristics in future breeding programs whereas antioxidant enzymes play an important role in scavenging ROS in plants to enhance their adaptability to different environmental conditions.

The one-way mixed lymphocyte culture (MLC) is used to assess histocompatibility between donor and recipient. First introduced in 1966, this method involves the co-culture of lymphocytes from the peripheral blood of the donor and the recipient for a period of 6 to 7 days: antigen disparities, primarily in the HLA-DR region, stimulate proliferation of the responding cells, which is detected by addition of 3H-labelled thymidine and subsequent measurement of radioactivity. The lymphocytes of either the donor, used to predict graft-versus-host disease (GVHD) or recipient, used to predict host-versus-graft disease (HVGD)/graft rejection, are inactivated by exposure to radiation or mitomycin C, so that the observed proliferation is that of the other set of lymphocytes, hence the name “one-way” MLC. The amount of measured radioactivity is directly proportional to the amount of DNA synthesized, which is a reflection of the number of disparities at the major histocompatibility complex (MHC).Previous studies have established that inactivation of the lymphocytes by radiation and mitomycin C, has a negative effect on the structure/expression of HLA-DR molecules on the cell surface, which provides the primary stimulus for the MLC reaction. The laboratory research presented in this dissertation was designed i) to compare the viabilities and HLA-DR levels on stimulator cells exposed to cyclosporin A, mitomycin C and ionizing irradiation , in order to determine whether cyclosporin A can be used as an alternative to mitomycin C or radiation as inactivator of the stimulator cells in the one-way MLC; ii) to improve sensitivity and accelerate the MLC reaction by addition of IL-2; iii) establish a flow cytometric mixed lymphocyte assay using the fluorochrome 5,6 carboxyfluorescein diacetate succinimidyl ester (CFSE). Cyclosporin A showed striking similarities to mitomycin C and ionizing radiation in its effect on viability and reduction/structural changes in HLA-DR molecules of the stimulator cells. Exposure of the stimulator cells to 20ìM cyclosporin A, demonstrated a significant loss of both cell viability and HLA-DR molecule cell surface expression. Thus, in evaluating these three methods of inactivation of the stimulator cells in the one-way MLC, it was concluded that a one-way MLC may not in fact be an accurate and qualitative reflection of the histocompatibility between donor and recipient. Instead the two-way MLC , in which neither the donor’s nor recipient’s cells are inactivated, may be a more reliable alternative. The only limitation associated with a two-way MLC is the inability to distinguish between a host-versus-graft-rejection and a graft-versus-host reaction in the observed allogeneic response Addition of 5 and 10 IU/ml IL-2 to the MLC showed the opposite effect to that intended, inhibiting proliferation in the MLC. Previous studies have shown that an excess of IL-2 results in the production of suppressor T cells. The amount of IL-2 produced during the MLC depends on the number of disparities in the MLC between donor and recipient, which will be different for each MLC reaction. Since the number of allogeneic T cells involved in the MLC reaction is not known, the amount of IL-2 produced during the allogeneic immune response in the MLC can not be predicted and addition of exogenous IL-2 may result in production of suppressor T cells and an inhibition of proliferation. The two-way MLC was modified by staining one of the participating set of lymphocytes (donors or recipients) with CFSE and tracking proliferation in this population, using flow cytometry. The two-way CFSE-based MLC analyzed in this study were counterstained with CD25 (IL-2R). An increase in CD25 expression on the cell surface is an indicator of cell activation and proliferation. Proliferation, as indicated by a progressive loss of CFSE fluorescence correlated well with the corresponding increase in CD25 expression and accumulated daughter cells. In addition, by loading only one of the participating donors in the two-way MLC, the responder/stimulator interaction, observed in the one-way MLC, is re-established. Thus the modified, CFSE-based two-way MLC can be used to predict GVHD. To conclude, the use of CFSE labeling and flow-cytometry to measure proliferation in a two-way MLC, together with CD25 counterstaining provides an alternative, reliable and probably superior method to 3H thymidine uptake. / Dissertation (MSc)--University of Pretoria, 2009. / Immunology / unrestricted

Le contrôle de la qualité microbiologique de l’eau constitue une problématique majeure en termes sanitaire et économique. L’efficacité des traitements appliqués est actuellement vérifiée par des méthodes standards basées sur la mise en culture des microorganismes. Cependant, leur mise en œuvre ne permet d’obtenir des résultats ne présentant qu’une vision partielle de la population microbienne présente dans un échantillon et ce dans un délai qui n’est pas compatible avec le niveau de réactivité requis dans le domaine de la gestion de l’eau.Les présents travaux visaient à démontrer que l’application de la cytométrie en flux pour le suivi microbiologique au sein de filières de production d’eau potable et de circuits de tours aéroréfrigérantes représente une approche alternative aux méthodes réglementaires et classiques utilisées dans ce domaine. Une attention particulière a été portée sur le fait d’être en mesure de quantifier les cellules totales et viables et d’être capable d’identifier l’impact de différents traitements chimiques et/ou physiques (ozone, UV, biocides oxydants) au niveau de la cellule microbienne. La démarche de calibration et l’utilisation conjointe de différents marqueurs fluorescents incluant le SYBR Green II, l’iodure de propidium et le Chemchrome V6 a permis d’atteindre ce double objectif. Par ailleurs, 3 systèmes de cytométrie en flux différents ont été utilisés selon les études, comprenant le FACSCaliburTM, le FACSCantoTM et l’ACCURITM C6.Au regard des résultats obtenus, il apparait que la cytométrie en flux représenterait un atout dans le domaine du diagnostic rapide d’installations de par sa simplicité, son délai de résultat court (1 heure) et le niveau d’information apporté sur la population microbienne (impact sur la membrane, le matériel génétique ou le métabolisme). / The water microbiological quality control is a major issue in health and economic terms. The effectiveness of treatments applied is currently tested using culture-based standard methods. However, their application leads to a partial view of the microbial population present in a sample and within a period that is not compatible with the required responsiveness concerning water management.The present work aims to demonstrate that the application of flow cytometry for microbiological monitoring of drinking water treatment plants and cooling tower circuits represents an alternative approach to regulatory and conventional methods used in this field. Particular attention was paid to being able to quantify total and viable cells and be able to identify the impact of different chemical and/or physical treatment (ozone, UV, oxidizing biocides) on a microbial cell. The calibration process and the joint use of various fluorescent dyes including SYBR Green II, propidium iodide and ChemChrome V6 have achieved this double objective. Furthermore, three different flow cytometric systems have been used according to the studies comprising the FACSCaliburTM, FACSCantoTM and ACCURITM C6.Given the results, flow cytometry appears as an asset in the field of rapid diagnostic for treatment efficiency due to its ease of use, short time to result (1 hour) and the information provided related to the microbial population (impact on membrane, genetic material or metabolism).

Picoplankton, cells between 0.2 - 2 μm, play a vital role in the carbon flow and nutrient cycling in marine food webs. Auto- and heterotrophic picoplankton dominate the biomass of oligotrophic tropical and subtropical oceans. However, little is known about their vertical distribution, changes in space and time and their relationships with environmental variables in the central Red Sea. The goal of this Ph.D. dissertation is to obtain baseline knowledge about their abundance, cellular characteristics (cell size, relative pigment and nucleic acid content) and biomass at seasonal and high-frequency temporal resolution (every 2 hours). This dissertation also aims at assessing picoplankton responses to separate and joint effects of nutrients additions (inorganic, organic and mixed) and temperature in order to be able to predict the relative contribution of eutrophication and warming in the future standing stocks of picoplankton in the Red Sea. I conducted a total of 63 vertical profiles (15 at around noon plus 48 more from the high-frequency diel samplings) from the surface down to the bottom (ca. 700 m) at a station situated 6 km off the coast of King Abdullah Economic City (KAEC) in the central Red Sea and performed 4 nutrient and temperature experiments lasting each 6 days with surface waters from the harbor of King Abdullah University of Science and Technology (KAUST). Flow cytometry allowed me to consistently identify five groups of autotrophs (Prochlorococcus, two populations of Synechococcus separated by their relative phycoerythrin fluorescence, and two differently-sized groups of picoeukaryotes) and two groups of heterotrophic prokaryotes characterized by their different relative nucleic acid content. One of the most surprising findings is the relatively lower abundances and to a lesser extent also growth rates of picoplankton compared with other tropical and subtropical oceans. Seasonality in environmental conditions emerged as an important factor in the response of picoplankton to nutrient additions and temperature. Picoplankton mostly responded to inorganic and mixed nutrient additions rather than warming. Overall, the information provided in this dissertation fills the gap of a critical component of Red Sea pelagic ecosystems and expands the information available on picoplankton communities in tropical waters.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22737号 / 医博第4655号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊佐 正, 教授 林 康紀, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Barbora Hadámková Determination the mechanism of entry F. tularensis into B lymphocytes Diploma thesis Charles University, Faculty Of Pharmacy in Hradec Králové Study program: Pharmacy Background: Besides processing the research with basics knowledge of the problem, the main aim of the study was the analysis of mechanism of entrance of intracellular bacteria Francisella tularensis into B cells. Methods: The B cells, which we obtained through peritoneal lavage from mice Balb/c, we blocked using antibodies individual complement receptors, B cell receptor and Fcƴ receptor. The population of the cells was infected by bacteria F. tularensis LVS/GFP opsonized by complement and/or by antibodies. Using flow cytometry we measured the percentage of infection of individual subpopulations of B cell B1a, B1b and B2 and we evaluated the influence of blocking and opsonization on the infection. Results: From the measured data, we can say that the percentage of infected B cells after infection by F. tularensis opsonized by complement is increased. This increase was more distinct in subtype of B cells B1b and B2. On the other hand, the opsonization F. tularensis by antibodies did not affect the infection. We also found out, that blocking of Fcƴ receptor has decrease the infection, if we used for infection of B cells...

Stem cell transplantation is a known treatment for various cancers. Currently most cells transplanted are collected via apheresis. An injection of growth factor is given to the patient to start the proliferation and mobilization of stem cells. Apheresis can be initiated when the patient has a stem cell count of 15 to 20 stem cells/µL of peripheral blood. The standard method with which stem cells are analysed is immune flow cytometry where CD34+ and CD45+ are identified with targeted fluorescent antibodies. This analysis takes more than 45 minutes to perform.     Sysmex XN-9000 analyses samples with flow cytometry by lysing erythrocytes and platelets and staining the leukocytes with fluorescent dye. Analysis of the hematopoietic progenitor cells (HPC) takes less than 4 minutes. The purpose of this study was to investigate ifit is possible to predict the start of the apheresis using XN-9000.     For this study, 43 samples were analysed using both methods. Using the sign test, a p-value was calculated to <0.05, which indicates a significant difference between the results received by the two methods. Spearman’s rank correlation gave an observed ρ-value > the critical ρ-value which revealed a correlation between the methods, although not linear according to Pearson’s correlation coefficient. PPV and NPV were calculated with cut-off at 20, 30 and 40 HPC/µL blood where 20 HPC/µL gave an NPV at 100 %. According to the test made, there is correlation between the two methods, but further samples must be analysed to investigate how the results should be compared.

Several cytochemical studies of the DNA content and ploidy status of neuronal cell nuclei in the central nervous system have reported the occurrence of hyperdiploid amounts of DNA in Purkinje cells and suggest the existence of some type of ‘extra’ DNA, the biological significance of which is, as yet, unknown. To explore this phenomenon further, the DNA content of glial and Purkinje cell nuclei was determined in several vertebrate species, using the DNA‐specific fluorochrome 4′,6‐diamidino‐2‐phenylindole (DAPI) to stain isolated cerebellar nuclei for analysis with a single parameter flow cytometer. The Feulgen reaction for DNA was used to stain liver and cerebellar tissue imprints for the measurement of individual nuclei with a Vickers M86 integrating microdensitometer. In both types of analyses, chicken erythrocyte nuclei served as an internal reference standard of 2.5 pg DNA per cell. The mean DNA content of Purkinje cells and glial or granule cells was essentially the same as that found for diploid (2C) non‐neuronal cells, such as hepatocytes, in rainbow trout, Amazon molly fish, salamander (Plethodon), mouse, rat, rabbit, cat, dog, monkey and human. Although Purkinje cell nuclei with 4C DNA levels were found in all of these species, except salamander and rabbit, the frequency of such cells was low (1–7%) and varied with the species. There was a low incidence of Purkinje cell nuclei with interclass DNA amounts in all species examined. Our data show that most neuronal cell nuclei in the cerebellum contain 2C levels of DNA.