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Flow cytometric assessment of T cell activation in asthmaMadden, Jacqueline January 1998 (has links)
No description available.
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A novel method of generating Dendritic cells in vitro using the KG-1 cell line and its use as a model for testing effects of lactic acid bacteriaVidya, Parimala 01 August 2011 (has links)
Dendritic cells (DCs) are prime mediators of innate and adaptive immunity. In humans the DC population comprise only 0.1% of all leukocytes, making their isolation and ex vivo manipulation difficult. Since study of DC activity in vitro requires large numbers of DCs to be readily available, a cell line model, KG-1, was selected. KG-1 cells are a cytokine-responsive human CD34+ myelomonocytic cell line and can be induced to differentiate to a DC phenotype. A range of differentiation agents and protocols were compared, and differentiation efficiency was determined using both morphological features and cell surface marker expression. Expression of CD83, CD11c, CD123, CD86, HLA-DR and DC-SIGN was assessed by immunofluorescence and flow cytometry. KG-1 cells stimulated with 10 ng/ml PMA and 100 ng/ml Ionomycin were found to be the ideal model for obtaining Dendritic Like Cells (DLCs) in vitro. The effect of lactic acid bacteria on KG-1 differentiation was also tested using two immunomodulatory strains, Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052. After 5 days of incubation with R0011 the KG-1 cells expressed DC-specific surface markers CD83, CD86, CD11c, CD123, DC-SIGN and HLA-DR. Lactobacillus rhamnosus R0011 induced a marked rise in CD83 expression with a mean fluorescence intensity of 115.3 after 5 days, suggesting this strain promoted KG-1 differentiation to DLC. Analysis of cytokine by KG-1 DLC indicated that constitutive production of pro-inflammatory cytokines TNF-α and IL-12 was minimal. However IL-10 and TGF-β were detected after TLR-agonist stimulation of R0011-differentiated KG-1s. This study aimed to develop and assess the KG-1 cell model for screening effects of mediators and microbes on DC. / UOIT
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Molecular mechanisms of normal erythropoiesis / Mécanismes moléculaires de l’érythropoièse normaleCico, Alba 25 September 2017 (has links)
Un être humain adulte produit environ deux millions d’érythrocytes par seconde, à travers un processus connu sous le nom d’érythropoïèse. L’érythropoïèse est contrôlée par une balance entre prolifération et différenciation finement régulée. L’expression des gènes impliqués dans ces deux processus distincts, est régulée extrinsèquement (cytokines) et intrinsèquement (microenvirennement métabolique, facteurs de transcription). Les facteurs de transcription, fonctionnent sous forme de complexes multiprotéiques et contrôlent l’activité transcriptionnelle des cellules. Parmi eux, le complexe LDB1 joue un rôle clé dans la régulation de la balance prolifération/différenciation pendant l’érythropoïèse, puisqu’il contrôle l’expression des gènes impliquées dans ces deux processus. Au cours de mon doctorat, nous avons d’abord caractérisé les mécanismes moléculaires de la “pré-activation” des gènes de différenciation, également nommés marqueurs erythroides, dans les progéniteurs erythroides immatures. La pré-activation, est un état dans lequel, les gènes sont exprimés à un niveau basal très bas, permissif pour une activation significative pendant la différenciation. Nous avons ainsi montré que les répresseurs : ETO2, IRF2BP2 et NCOR1, interagissent avec le complexe LDB1, et lient ensemble les gènes des marqueurs erythroides et les répriment. Au cours de l’érythropoïèse, ces corépresseurs sont déstabilisés et LDB1 agit alors comme un activateur. En ce qui concerne les gènes de prolifération, nous avons observé que le complexe LDB1 est déstabilisé au niveau de ces loci pendant l’érythropoïèse. Afin d’étudier les mécanismes moléculaires de la répression génique des gènes de prolifération au cours de l’érythropoïèse, nous avons choisi d’étudier Myb, une cible du complexe LDB1, étudié auparavant dans le laboratoire. Nous avons testé trois facteurs : ZEB1, OGT et RNF12, en tant que candidats dans la répression de Myb. Nous avons montré que RNF12 est le seul facteur intervenant dans la transcription de Myb. RNF12 régule Myb probablement par une modification de complexes épigénétiques. / Every second about 2 million erythrocytes are produced in the adult human body, through a process called erythropoiesis. Erythropoiesis is controlled by a highly regulated balance between proliferation and differentiation. Expression of genes responsible for cell proliferation and differentiation is controlled external (such as cytokines) and internal (such as metabolic microenvironment and transcription factors). Transcription factors bind DNA and recruit co-factors generating transcriptional complexes. The LDB1 complex has a key role in the balance between erythroid proliferation vs. differentiation, since it regulates genes involved in both processes. During my Ph.D., we investigated the molecular mechanisms that LDB1 employs to regulate genes with divergent function. We first showed that in erythroid progenitors, differentiating genes, also known as erythroid markers, are primed. Gene priming consists of genes expressed in low basal but significant levels in progenitors, which can rapidly be activated during differentiation. We showed that in progenitors, ETO2, IRF2BP2 and NCOR1, bind the LDB1 complex therefore generating a priming complex. During differentiation, binding of the repressive (ETO2-IRF2BP2-NCOR1) co-factors to the LDB1 complex, is destabilized and genes become active. In genes involved in erythroid proliferation, we observed that LDB1 is destabilized, a feature leading to gene silencing. We used Myb, as a model of gene silencing in the context of regulation by the LDB1 complex. We tested three transcription factors: ZEB1, OGT and RNF12, as candidates in gene silencing. Among these factors, only RNF12 regulates Myb expression, probably through modifications of epigenetic silencers (Polycomb/MLL).
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Vliv elektrických pulzů na lidské krevní fagocyty / Influence of electrical pulses on human blood phagocytesChorvátová, Michaela January 2019 (has links)
The phagocytic cells circulating in the bloodstream play a key role in both the defense of the body and the pathology of inflammatory diseases. Thus, targeting their functions has potential to modulate an immune response, especially during the inflammatory phase. This master's thesis was focused on the influence of electric pulses on the most abundant phagocyte population in human peripheral blood, namely neutrophils. The theoretical part describes the role of neutrophils in the development of the immune response and the effects of the electric field on various cells. Consequent part of the thesis was the optimization of the electrical stimulation of neutrophils using a unique platform with a network of gold electrodes. In stimulated cells by electrical pulses, activation of selected signaling pathways, degranulation, ROS production, citrullination of histone H3 and expression of surface markers were monitored. Overall, electrical stimulation was observed to induce neutrophil activation but only electrical pulses of size 1 V were found to be statistically significant in the case of ROS production and 10 mV and 100 mV electrical pulses in the case of metalloproteinase MMP8 degranulation. The absence of significant effects in the most observed parameters was probably due to unwanted activation of neutrophils in control samples.
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Characterising the reprogramming dynamics between human pluripotent statesCollier, Amanda January 2019 (has links)
Human pluripotent stem cells (hPSCs) exist in multiple states of pluripotency, broadly categorised as naïve and primed states. These provide an important model to investigate the earliest stages of human embryonic development. Naïve cells can be obtained through primed-to-naïve reprogramming; however, there are no reliable methods to prospectively isolate unmodified naïve cells during this process. Moreover, the current isolation strategies are incompatible for enrichment of naïve hPSCs early during reprogramming. Consequently, we know very little about the temporal dynamics of transcriptional changes and remodelling of the epigenetic landscape that occurs during the reprogramming process. To address this knowledge gap, I sought to develop an isolation strategy capable of identifying nascent naïve hPSCs early during reprogramming. Comprehensive profiling of cell-surface markers by flow cytometry in naïve and primed hPSCs revealed pluripotent state-specific antibodies. By compiling the identified state-specific markers into a multiplexed antibody panel, I was able to distinguish naïve and primed hPSCs. Moreover, the antibody panel was able to track the dynamics of primed-to-naïve reprogramming, as the state-specific surface markers collectively reflect the change in pluripotent states. Through using the newly identified surface markers, I found that naïve cells are formed at a much earlier time point than previously realised, and could be subsequently isolated from a heterogeneous cell population early during reprogramming. This allowed me to perform the first molecular characterisation of nascent naïve hPSCs, which revealed distinct transcriptional changes associated with early and late stage naïve cell formation. Analysis of the DNA methylation landscape showed that nascent naïve cells are globally hypomethylated, whilst imprint methylation is largely preserved. Moreover, the loss of DNA methylation precedes X-chromosome reactivation, which occurs primarily during the late-stage of primed-to-naïve reprogramming, and is therefore a hallmark of mature naïve cells. Using the antibody panel at discrete time points throughout reprogramming has allowed an unprecedented insight into the early molecular events leading to naïve cell formation, and permits the direct comparison between different naïve reprogramming methods. Taken together, the identified state-specific surface markers provide a robust and straightforward method to unambiguously define human PSC states, and reveal for the first time the order of transcriptional and epigenetic changes associated with primed to naïve reprogramming.
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Desenvolvimento e caracterização de células-tronco mesenquimais derivadas do tecido adiposo e seu potencial de diferenciação / Development, characterization and differentiation potential of adipose tissue-derived mesenchymal stem cellsBraunig, Patricia 09 March 2016 (has links)
Mesenchymal stem cells (MSCs) have demonstrated significant potential for clinical use due to their convenient isolation, lack of significant immunogenicity, lack of ethical controversy and their potential to differentiate into tissue-specific cell types. MSCs reside in almost all tissues including the adipose tissue. Adipose tissue has main advantages as wide distribution in the organism, suitable isolation and considerable amount of resident multipotent stem cells. Therefore, in this study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from BALB/c mice omentum and epididymis fat pats. During AT-MSCs maintenance and expansion in vitro, they were characterized for the expression of antigenic surface markers and for osteogenic, chondrogenic, and adipogenic differentiation potential. AT-MSCs form both sources expressed mesenchymal surface markers, CD73, and CD105 and were negative for a hematopoietic marker, CD45. The cultures derived from both adipose tissues differentiated into all three lineages. However, differences were observed in mesenchymal surface marker expression profiles as well as in the differentiation potential of AT-MSCs from different fat sources. Furthermore, AT-MSCs isolated from omentum fat depot were cultured with differentiation medium containing retinoic acid and testicular cell conditioned medium. After treatment periods, AT-MSCs showed Gdnf gene expression, this gene is a marker for Sertoli cells. The results showed that AT-MSCs from distinct fat depots have different characteristics related to stem cell surface marker expression profiles and differentiation potential. / Células-tronco mesenquimais têm demonstrado significativo potencial para aplicação terapêutica devido ao seu fácil isolamento, baixa imunogenicidade, ausência das implicações éticas e sua ampla plasticidade. Essas células estão nos mais diversos tecidos, destacando-se o tecido adiposo devido á sua ampla distribuição no organismo, conveniente obtenção e o considerável número de células-tronco mesenquimais multipotentes que podem ser isoladas desse tecido. Assim sendo, no presente estudo, células-tronco mesenquimais derivadas do tecido adiposo (AT-MSCs) foram isoladas do tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos BALB/c. Durante a manutenção e expansão das AT-MSCs in vitro, elas foram caracterizadas quanto à presença de marcadores antigênicos de superfície e potencial de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. AT-MSCs de ambas as fontes expressaram os marcadores mesenquimais de superfície, CD73 e CD105, assim como foram negativas para o marcador de linhagens hematopoiéticas, CD45. Quanto ao potencial de diferenciação, os cultivos provenientes das duas origens de tecido adiposo apresentaram capacidade de diferenciar nas três linhagens acima citadas. Porém, foram observadas discretas diferenças tanto nos padrões de expressão dos marcadores mesenquimais de superfície quanto nos potenciais de diferenciação entre as AT-MSCs provenientes dos diferentes locais de deposição de gordura. Além disso, as AT-MSCs isoladas do tecido adiposo depositado em contato com o omento quando cultivadas com meios de diferenciação, contendo ácido retinóico e meio condicionado testicular demonstraram expressão do gene Gdnf o qual é reconhecidamente expresso pelas células de Sertoli. Portanto, os resultados obtidos demonstram que conforme a origem do tecido adiposo as AT-MSCs possuem diferentes características relacionadas aos marcadores de superfície assim como aos potenciais de diferenciação.
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Characterization of ex vivo expanded human hematopoietic stem and progenitor cellsAnsari, Unain 04 1900 (has links)
Les cellules souches hématopoïétiques (CSH) sont des cellules souches adultes, responsables du maintien du système sanguin tout au long de la vie des vertébrés. Les CSH sont des cellules multipotentes spécialisées qui possèdent deux propriétés principales : leur capacité à se différencier en de multiples lignées et leur capacité à créer d'autres cellules souches (c'est-à-dire l'autorenouvellement). Grâce à ces caractéristiques, les CSH ont un énorme potentiel thérapeutique. En effet, la transplantation de CSH constitue à ce jour une option de choix pour le traitement de plusieurs maladies et troubles hématologiques. Les CSH ne se retrouvent que dans certains échantillons biologiques comme la moelle osseuse, les cellules mobilisées de la moelle osseuse dans le sang périphérique ou les cellules de sang de cordon ombilical. Les applications cliniques des CSH sont souvent limitées en raison de leur faible fréquence dans les échantillons biologiques, c’est pourquoi leur expansion ex vivo est un domaine de recherche en plein essor. Des approches basées sur des petites molécules pour amplifier le nombre les cellules couches ex vivo ont été testées avec succès pour permettre la prolifération des cellules et freiner leur différentiation. Notre groupe a contribué à ce domaine en identifiant la petite molécule UM171 qui peut amplifier les CSH ex vivo par reprogrammation épigénétique. Dans le cadre des efforts d’expansion ex vivo des CSH, un obstacle majeur est la caractérisation des cellules qui ont proliféré ex vivo afin d’évaluer de façon exhaustive le potentiel des greffons pour des applications ultérieures. La caractérisation phénotypique des CSH amplifiées ex vivo est une approche prometteuse pour aider à isoler et à purifier les cellules souches. Les travaux de cette thèse explorent l'association de l'immunophénotype à la fonctionnalité des cellules souches pour nous aider à définir l'hétérogénéité des cellules amplifiées. Au chapitre 2, en utilisant un profilage de cellules amplifiées basée sur le transcriptome, nous avons pu identifier CEACAM1 comme un nouveau marqueur fonctionnel des CSH. Concomitamment, au chapitre 3, nous appliquons une approche alternative basée sur le protéome de la surface cellulaire pour aider à caractériser le phénotype des cellules souches et progénitrices hématopoïétiques (CSPH) amplifiées ex vivo afin d'identifier GPA33 en comme marqueur probable de CSH. Les marqueurs de surface compatibles avec la culture constituent un excellent outil pour un isolement prospectif rapide et des manipulations in vitro et in vivo supplémentaires pour permettre une meilleure compréhension de la biologie des cellules souches. La caractérisation des HSPC expansées ex vivo est donc une tentative de combler le fossé et de permettre des stratégies thérapeutiques améliorées. / Hematopoietic stem cells (HSCs) are responsible for maintaining the blood system throughout the lifespan of vertebrates. HSCs are specialized multipotent cells that have two main properties – their ability to differentiate into multiple lineages and their ability to create more stem cells (i.e. self-renewal). Due to these special abilities, HSCs have tremendous therapeutic potential. HSCs thus to date are the best curative measure against most hematological malignancies and disorders. HSCs occur in limited frequency and can be found only from certain conserved sources like the bone marrow or mobilized cells from the bone marrow in the peripheral blood or umbilical cord blood cells. Clinical applications of HSCs are often restricted due to their low occurring frequencies, therefore ex vivo expansion is a growing research field. Small molecule-based approaches to expand stem cells ex vivo have been successfully tested to allow for proliferation of cells by curbing their differentiation. Our group has contributed to this field by the identification of the small molecule UM171 which can expand hematopoietic stem and progenitor cells (HSPCs) ex vivo via epigenetic reprogramming. To expand HSPCs ex vivo a major hurdle is the proper characterization of the ex vivo expanded cells to evaluate the full potential of grafts for further downstream applications. Phenotypic dissociation of ex vivo expanded HSPCs is a prospective tool to help isolate and purify stem cells. Identification of culture-compatible surface markers is therefore the first step to help characterize the ex vivo expanded cells. The work in this thesis explores the association of immunophenotype to the functionality of stem cells to help us delineate the heterogeneity of expanded cells. In Chapter 2, using transcriptome-based interrogation of expanded cells, we were able to identify CEACAM1 as a novel functional marker of HSCs. Whereas, in Chapter 3 we apply an alternative cell surface proteome-based approach to help characterize the phenotype of ex vivo expanded HSPCs to identify GPA33 as a probable HSC marker. Culture-compatible surface markers make for an excellent tool for rapid prospective isolation and additional in vitro and in vivo manipulations to allow a better understanding of stem cell biology. Characterization of ex vivo expanded HSPCs is thus an attempt to help bridge the gap and allow for enhanced therapeutic strategies.
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Phänotypische Charakterisierung humaner Monozyten von Blutspendern mit chronischer Toxoplasmose und nicht-infizierten Kontrollen / Phenotypic characterization of human monocytes from blood donors with chronic toxoplasmosis and non-infected controlsEhmen, Hauke Gerhard 17 November 2020 (has links)
No description available.
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