Orientadores: Fernando Ferreira Costa, Nicola Conran / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T10:27:56Z (GMT). No. of bitstreams: 1 Gambero_Sheley_M.pdf: 2005109 bytes, checksum: faec483bdfe41c20b920e16808d8eb59 (MD5) Previous issue date: 2006 / Resumo: A anemia falciforme é um distúrbio genético da hemoglobina causado por uma mutação de ponto produzindo hemoglobina S (HbS) que quando desoxigenada causa, entre outros sintomas, eventos vaso-oclusivos. Um dos mecanismos indicados como causador da vaso-oclusão é a adesão de hemácias falciformes ao endotélio dos vasos. Eritrócitos falciformes e normais aderem ao endotélio vascular utilizando moléculas de adesão como, CD 36 e integrina VLA-4, entre outras moléculas de adesão. Hidroxiuréia (HU) é um agente quimioterápico que diminui a freqüência de crises vaso-oclusivas, síndrome torácica aguda e necessidade de transfusão. Longos tratamentos com HU levam a uma redução global das proteínas de superficie dos neutrófilos, monócitos e linfócitos, além de aumentar os níveis de Hemoglobina fetal (Hb F), que inibe a polimerização da célula falciforme desoxigenada. O NO é um importante vaso-dilatador responsável por inúmeros efeitos benéficos durante as crises vaso-oclusivas. Estudos demonstram que o NO pode diminuir a expressão endotelial de moléculas de adesão, mas estudos diretos sobre os níveis de expressão dessas moléculas em anemia falciforme na presença de NO não tem sido encontrados na literatura. Deste modo objetivamos neste trabalho, analisar a expressão, gênica e protéica, e a função das moléculas de adesão VLA-4 (CD49d ou cadeia a. e CD29 ou cadeia 13) e CD 36 em células vermelhas de pacientes com anemia falciforme com e sem terapia com HU e os efeitos do tratamento in vitro com HU e agentes doadores de NO na adesão dessas células. Analisando a adesão das células vermelhas normais e de pacientes com e sem terapia com HU confirmamos que as células vermelhas de pacientes falciformes são mais aderentes que as células vermelhas normais e que a terapia com HU provoca uma diminuição dessa aderência. A citometria de fluxo comprovou a maior presença de células CD36+ e CD49d+ além de maior índice de expressão dessas moléculas nos pacientes falciformes em relação ao controle e a diminuição da expressão e positividade dessas moléculas em células de pacientes em terapia com HU quando comparadas com pacientes sem terapia. Adicionalmente, a análise por Real Time PCR demonstrou que a expressão gênica de CD36, CD49d e CD29 em reticulócitos falciformes é significativamente menor em pacientes em terapia com HU quando comparado com pacientes que não recebem essa terapia. Em resumo, nossos resultados sugerem que: as propriedades adesivas à fibronectina (FN) são aumentadas em células SS e que estas propriedades diminuem nos pacientes que tomam a terapia de HU; a terapia com HU diminui a expressão protéica e a positividade, das células vermelhas falciformes, hemácias e células jovens, para os receptores CD36 e CD49d; e a expressão gêniea das moléculas de adesão CD36 e CD49d em reticulócitos de pacientes com anemia falciforme que recebem terapia com HU é diminuída em relação ao grupo de pacientes falciformes que não recebem essa terapia / Abstract: Sickle cell vaso-occlusion constitutes a complex process involving interactions between SS red blood cells (RBC), endothelial cells, leukocytes, platelets, coagula tive factors and plasma proteins. Propagation ofthe vaso-occlusive process in sickle cell anemia (SCA) is a complex process involving the adhesion of SS red cells and reticulocytes to the vascular endothelium. The effect of hydroxyurea therapy (HUT) on the adhesive properties of sickle red cells and the expression of adhesion molecule genes by erythroid cells of SCA individuals is not yet fully understood. The expressions of the CD36 gene and the VLA-4 integrin subunit genes, CD49d (a-subunit) and CD29 (J3-subunit), were compared in the reticulocytes of steady-state SCA patients and patients on HUT using Real-Time PCR Basal adhesion of red cells ftom these subjects was also compared using static adhesion assays, as was surface protein expression, using flow cytometry. Basal sickle red cell adhesion to fibronectin was significantly greater than that of normal cells (P<O.OI); in contrast, HUT significantly decreased (P<O.OI) red cell adhesion to levels similar to those of control cells and this decrease could not be justified solely by alterations in reticulocyte numbers in this population. Accordingly, flow cytometry demonstrated that HUT significantly decreased CD36 and CD49d surface expression (P<O.Ol) and, importantly, significant reductions in the expressions of the CD36, CD49d and CD29 genes were seen (P<O.O5) in the reticulocytes of SCA patients on HU. Taken together, data support the hypothesis that HUT reduces the adhesive properties of sickle cells and that this decrease appears to be mediated, at least in part, by a decrease in the gene and, consequently, surface protein expression of adhesion molecules such as VLA-4 and CD36 / Mestrado / Patologia Clinica / Mestre em Ciências Médicas

Orientadores: Joyce Maria Annichino-Bizzacchi, Nicola Amanda Conran Zorzetto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T04:29:01Z (GMT). No. of bitstreams: 1 Zapponi_KiaraCristinaSenger_M.pdf: 3766938 bytes, checksum: e4531f39f9f53ff883ad33f684d56647 (MD5) Previous issue date: 2012 / Resumo: Tromboembolismo venoso (TEV) é uma doença multifatorial que afeta 1-3:1000 indivíduos mundialmente. A formação do trombo venoso na superfície endotelial é um processo multicelular, de estrutura laminar composta por camadas de plaquetas, leucócitos, eritrócitos e fibrina, originando uma resposta inflamatória loco-regional. A relação entre inflamação e coagulação é bidirecional. e tem sido principalmente avaliada através de interações de proteínas entre citocinas próinflamatórias e elementos da cascata de coagulação. As células inflamatórias tais como neutrófilos, não foram previamente correlacionadas com os processos trombóticos ou pró-coagulantes. Objetivo: Avaliar as propriedades adesivas de neutrófilos, eritrócitos e plaquetas, assim como a expressão das moléculas de adesão de superfície dos neutrófilos em pacientes com TEV, correlacionando-os com marcadores sistêmicos da resposta inflamatória, presença de trombo residual e D-dímero aumentado. Pacientes e Métodos: Foram incluídos neste estudo 10 pacientes com TEV agudo atendidos no Hospital de Clínicas da Unicamp (HCUNICAMP), 30 pacientes com TEV crônico (1 a 6 anos após o evento agudo), atendidos no Hemocentro de Campinas-UNICAMP, e controles normais pareados aos pacientes por idade, gênero e etnia. A adesão de neutrófilos, eritrócitos e plaquetas foram determinados através de ensaio estático usando fibronectina (FN) e fibrinogênio (FB) como ligantes. A expressão das moléculas de adesão dos neutrófilos (CD11a, CD11b, CD18) foi avaliada por citometria de fluxo. Os níveis dos marcadores inflamatórios (IL-6, IL-8, TNF-_ e PCR) foram avaliados por ELISA e nefelometria. O trombo residual (TR) por ultrassom com Doppler e o Dímero-D (DD) por método coagulométrico. Resultados: A adesão de plaquetas ao fibrinogênio com e sem estímulo de trombina em pacientes dos grupos agudo e crônico foi semelhante ao observado nos controles normais. Da mesma forma, não houve diferença da adesão de eritrócitos à fibronectina nesses mesmos grupos analisados. No subgrupo de pacientes com alto risco de recorrência de TEV definido pela presença de altos níveis de D-dímero (>0,55mg/L) e trombo residual observou-se um aumento significativo da adesão de neutrófilos em relação aos controles (24.68% vs 19.07%, P<0.05). A atividade inflamatória também estava aumentada neste subgrupo em comparação aos outros pacientes, demonstrada pelo aumento significativo dos níveis séricos de IL-6, IL-8, TNF-_ e PCR (2.08pg/mL VS 0.90pg/mL, P=0.01; 28.72pg/mL vs 16.46pg/mL, P=0.02; 4.50pg/mL vs 2.11pg/mL, P=0.04; 0.35 pg/mL vs 0.14 pg/mL, P=0.09, respectivamente). Houve uma correlação das propriedades adesivas de neutrófilos com IL-6 (r=0.3815 e P=0.0375), e D-dímero (r=0.3831 e P=0.0367). A quantificação das proteínas de superfície (CD11a, CD11b e CD18) de neutrófilos não foi diferente entre os grupos analisados. Conclusão: Nossos resultados sugerem que pacientes com TEV não apresentam aumento das propriedades adesivas de plaquetas e eritrócitos. Entretanto, as propriedades adesivas dos neutrófilos foram aumentadas em pacientes com D-dímero aumentado e presença de trombo residual, independente da expressão quantitativa das proteínas de superfície em sua membrana. A hipótese para este aumento pode ser devido as alterações na afinidade das moléculas de adesão de superfície aos seus ligantes, como consequência do processo inflamatório associado com a hipercoagulabilidade que é característica desta doença / Abstract: Venous Thromboembolism (VTE) is a multifactorial disease that affects 1-3:1000 individuals worldwide. The venous thrombus develops via a multicellular process on the surface of the endothelium and presents a laminar structure comprised of layers of platelets, leukocytes, erythrocytes and fibrin. The relationship between inflammation and coagulation is bidirectional, and has been mainly evaluated through protein interactions between pro-inflammatory cytokines and elements of the coagulation cascade. Inflammatory cells such as neutrophils, have not been previously correlated with thrombotic or procoagulant processes. Objective: To evaluate the adhesive properties of neutrophils, erythrocytes and platelets, as well as the expression of neutrophil adhesion molecules in patients with VTE, correlating them with markers of the systemic inflammatory response, and with the presence of residual vein obstruction (RVO) and higher D-dimer (DD). Patients and Methods: Study group consisted of 30 chronic VTE patients (1-6 years after the acute episode) followed in our outpatient clinic, and 10 patients with VTE during the acute episode treated at the Hospital of Clinics (HC-UNICAMP) Campinas, as well as age, gender and ethnic background-matched healthy. Adhesive properties of neutrophils, erythrocytes and platelets were determined by a static adhesion assay using ligands such as fibrinogen (FB) and fibronectin (FN). The expression of neutrophils adhesion molecules (CD11a, CD11b, CD18) was evaluated by flow cytometry. Levels of inflammatory markers (IL-6, IL-8, TNF-_, PCR) were evaluated by ELISA and nephelometry. RVO was evaluated by Doppler ultrasound and DD by coagulometric method. Results: No significant difference could be observed in the platelets adhesion (basal: 16.37% vs. 14.59%, p=0.309; and stimulated with thrombin: 33.45% vs. 26.62%, p=0.200) and erythrocytes adhesion (7.28% vs 7.49%, p=0.859) between chronic VTE patients and healthy individuals. Similarly, no statistical differences were observed in the platelets adhesion (basal: 28.36% vs. 21.63%, p=0.109; and stimulated with thrombin: 38.45% vs. 30.15%, p=0.715) and erythrocytes adhesion (6.00% vs 4.62%, p=0.326) in the VTE acute xxii patients when compared to their respective controls. Interestingly, in patients with a higher risk of recurrent VTE (defined by the presence of high levels of DD and RVO), a significant increase in neutrophils adhesion was observed when compared to healthy individuals (24.68% vs. 19.07%, p <0.05). Inflammatory markers (IL-6, IL-8, TNF-_ and CRP) were significantly elevated (2.08pg/mL vs 0.90pg/mL, p=0.01; 28.72pg/mL vs 16.46pg/mL, p=0.02; 4.50pg/mL vs 2.11pg/mL, p=0.04; 0.35 pg/mL vs 0.14 pg/mL, p=0.09, respectively) in this subgroup of patients when compared to the other patients. Adhesive properties of neutrophils were correlated with IL-6 (r= 0.3815 and p= 0.0375) and D-dimer levels (r= 0.3831 and p= 0.0367). Neutrophils adhesion molecules (CD11a, CD11b and CD18) were not altered in any of the groups. Conclusion: Our results suggest that VTE patients do not exhibit increased adhesive properties of platelets and erythrocytes. Neutrophils adhesive properties were increased in patients with higher D-dimer levels and RVO, independently of the expression of neutrophil adhesion molecules. A hypothesis for this increase is alterations in affinity of surface adhesion molecules to their ligands, as a response to inflammatory processes associated with the hypercoagulability demonstrated in this subgroup of patients / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestra em Fisiopatologia Médica

Orientador: Nicola Amanda Conran Zorzetto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T23:21:17Z (GMT). No. of bitstreams: 1 Vital_DaianaMorelli_M.pdf: 1971637 bytes, checksum: 022329e1e83ad1bd2ea02de3d37d61c9 (MD5) Previous issue date: 2015 / Resumo: A inflamação é uma resposta fisiológica normal à infecção ou lesão tecidual que permite a sobrevivência do indivíduo a diversos agentes lesivos e mantém a homeostase dos tecidos sob uma variedade de condições nocivas. Os neutrófilos têm um papel importante em processos inflamatórios e, na presença de estímulos inflamatórios, como citocinas e quimiocinas, são recrutados da circulação para o tecido inflamado por uma sequência de interações adesivas. Recentemente, novas técnicas in vitro têm levado a importantes avanços no entendimento de patologias vasculares e hematológicas e sistemas microfluídicos, que mimetizam a microcirculação humana, demonstrando a utilidade para o estudo de interações adesivas de células inflamatórias. As interações dos neutrófilos e outros leucócitos com a parede vascular tem contribuição importante para as doenças inflamatórias crônicas, como a anemia falciforme (AF) e aterosclerose, pois podem participar em processos de oclusão vascular. O objetivo deste trabalho foi avaliar se a hidroxiureia (HU), uma droga utilizada como terapia na AF, modula as propriedades adesivas de neutrófilos quando incubados in vitro com a droga e frente um estímulo inflamatório. Os neutrófilos, isolados do sangue periférico de indivíduos saudáveis, foram estimulados com a citocina TNF-? (Fator de Necrose Tumoral alfa) e tratados com HU em três concentrações (100, 500, 1000 ?M); as propriedades adesivas das células foram avaliadas por ensaios de adesão estática e por ensaios microfluídicos, utilizando como ligantes proteínas expressas no endotélio (ICAM-1 e E-selectina), proteínas da matriz extracelular (fibronectina - FN) e células endoteliais (HUVEC). Além disso, foi analisada a ativação celular (spreading celular, observado como o espalhamento das células redondas que se tornam achatadas sobre um substrato sólido 2D). Observamos que os neutrófilos, quando estimulados com TNF-?, demonstram aumentos significantes na adesão e spreading celular. O pré-tratamento das células com HU reduziu significativamente o spreading dos neutrófilos nas três proteínas estudas (FN, ICAM-1 e E-selectina). Sob condições estáticas, o pré e o pós-tratamento dos neutrófilos com a HU diminuiu significativamente a adesão à FN quando comparados ao estimulados por TNF-?. Nos ensaios de adesão em fluxo, foi possível observar que o pré-tratamento com HU nas três concentrações (100, 500 e 1000 ?M) diminuiu significativamente a adesão dos neutrófilos a FN e o pós-tratamento com HU diminuiu apenas na concentração de 100 ?M. Avaliamos a adesão em fluxo de neutrófilos aderidos a proteínas de adesão presentes no endotélio, ICAM-1 e E-selectina; o pré e o pós-tratamento de neutrófilos com HU diminuiu a suas propriedades adesivas frente ao estímulo inflamatório de TNF-? à proteína E-selectina, enquanto que o pré-tratamento nas três concentrações diminuiu a adesão dos neutrófilos ao ICAM-1 e ao ICAM-1 e E-selectina adicionados juntos ao chip. A técnica de citometria de fluxo demonstrou que as integrinas LFA-1 (CD11a) e Mac-1 (CD11b), mostraram-se aumentadas na superfície dos neutrófilos após estímulo com TNF-?. Todavia, a expressão da L-selectina, mostrou-se diminuída com este potente estímulo inflamatório, provavelmente devido o mecanismo de "shedding" celular. A incubação dos neutrófilos com HU, após o estímulo com o TNF-?, reduziu significativamente a expressão de CD11a nos neutrófilos tratados com HU nas concentrações de 100 e 1000 ?M para níveis de expressão equivalentes ao grupo de neutrófilos não estimulados com TNF-?. Observamos também que houve aumento da presença da integrina LFA-1 (CD11a) na sua conformação ativada, após estímulo com TNF-? e que a pós-incubação das células estimuladas com HU na concentração de 1000 ?M, reduziu a ativação da subunidade CD11a da molécula de adesão LFA-1, comparado ao grupo apenas estimulado com TNF-?. Diante destes resultados, é possível concluir que o tratamento de neutrófilos com HU foi capaz de proteger ou até reverter algumas das ações inflamatórias do TNF-?. A HU é utilizada como uma terapia de uso crônico em pacientes com AF, mas estes dados indicam que a HU também pode exercer efeitos imediatos que são independentes da elevação de hemoglobina fetal. Dados melhor clarificam o mecanismo de ação de HU na AF e sugerem que a droga pode ter potencial para uso em outras doenças inflamatórias. Ainda será necessário entender como a HU exerce estes efeitos anti-inflamatórios nos neutrófilos / Abstract: Inflammation is a normal physiological response to infection or tissue injury that defends against various damaging agents and maintains tissue homeostasis in a variety of deleterious conditions. Neutrophils play an important role in inflammatory processes and, in the presence of inflammatory stimuli, such as cytokines and chemokines, are recruited from the circulation into the inflamed tissue by a sequence of adhesive interactions. Recently, new in vitro techniques have led to significant advances in our understanding of vascular and hematological conditions and microfluidic systems that mimic the human microcirculation have been shown to be useful for the study of adhesive interactions in inflammatory cells. The interaction of neutrophils and other leukocytes with the vascular wall makes an important contribution to chronic inflammatory diseases, such as sickle cell anemia (SCA) and atherosclerosis, as these may participate in vascular occlusion processes. The aim of this study was to evaluate whether hydroxyurea (HU), a drug used as a therapy in SCA, modulates the adhesive properties of neutrophils in vitro under an inflammatory stimulus. The neutrophils isolated from the peripheral blood of healthy subjects were stimulated with the cytokine TNF-? (Tumor Necrosis Factor alpha) and treated with HU in three concentrations (100, 500, 1000 ?M); the adhesive properties of the cells were evaluated by static adhesion assays and microfluidic assays using ligands such as proteins expressed on the endothelium (ICAM-1 and E-selectin), extracellular matrix proteins (fibronectin - FN) and endothelial cells (HUVEC). Furthermore, cellular activation was evaluated as cell spreading (observed when round cells become flattened and spread on a 2D solid substrate). When stimulated with TNF-?, neutrophils presented a significant increase in cell adhesion and spreading. Pre-treatment of cells with HU significantly reduced neutrophil spreading on all three proteins studied (FN, ICAM-1 and E-selectin). Under static conditions, the pre-treatment and post-treatment of neutrophils with HU significantly decreased adhesion to FN, compared to TNF-?-stimulated cells. In the flow adhesion assays, pre-treatment of neutrophils with HU at three concentrations (100, 500 and 1000 ?M) significantly reduced the adhesion of neutrophils to FN and post-treatment with HU decreased only at the concentration of 100 ?M. We evaluated the in vitro flow adhesion of neutrophils to proteins present on endothelial cells, ICAM-1 and E-selectin; pre-treatment and post-treatment of neutrophils with HU decreased their adhesive properties after TNF-? inflammatory stimulus to the E-selectin ligand, while pre-treatment in the three concentrations decreased neutrophil adherence to ICAM-1 ligand and ICAM-1/E-selectin ligands added together to the chip. Flow cytometry demonstrated that the expressions of the integrins LFA-1 (CD11a) and Mac-1 (CD11b) were elevated on the surface of neutrophils after a TNF-? stimulus. However, L-selectin expression was reduced with this potent inflammatory stimulus, probably due to the mechanism of "shedding". Incubation of neutrophils with HU (100 and 1000 ?M), after stimulation with TNF-?, significantly reduced the CD11a expression on neutrophils to levels similar to those found on the neutrophils of the non-TNF-?-stimulated group. We also observed that there was an increased expression of the LFA-1 (CD11a) integrin in its active conformation after stimulation with TNF-? and that, post-incubation with HU (1000 ?M) was able to reduce the activation of the adhesion molecule CD11a (LFA-1 subunit), compared to the group with TNF-? stimulus only. Given these results, it is possible to conclude that treatment of neutrophils with HU was able to protect or even reverse some of the inflammatory actions of TNF-?. HU is used as a chronic therapy in patients with SCA, but our data indicate that HU may also have immediate effects that are independent of the increase in fetal hemoglobin usually observed in patients in therapy. Our results further clarify the mechanism of action of HU in SCA and suggest that this drug may have potential for use in other inflammatory diseases. Further studies will be needed to better comprehend how these HU anti-inflammatory effects act on neutrophils / Mestrado / Fisiopatologia Médica / Mestra em Ciências

Hematopoietic cells exploit the multistep paradigm of cell migration to ultimately enable them to perform their function. This process is dictated by the ability of adhesion molecules on the circulating hematopoietic cells to find their counter-receptors on endothelial cells. Of those molecules, the selectin family and their respective ligands induce the initial transient interactions between circulating cells and the opposing endothelium. In this thesis, I focused on studying E-selectin mediated cellular migration in two hematopoietic cell types, namely human hematopoietic stem and progenitor cells (HSPCs) and human T-lymphocytes. HSPCs derived from pluripotent sources theoretically offers a novel, unlimited source for hematopoietic stem cell transplantation therapy. In vitro pluripotent stem cell derived- hematopoietic stem/progenitor cells (ES/iPS-HSPCs) behave much like somatic HSPCs in that they exhibit clonal expansion and multilineage hematopoietic capacity. However, unlike somatic sources, ES/iPS-HSPCs do not give rise to effective hematopoietic repopulation, which may be due to insufficient HSPCs homing to the bone marrow. HSPCs exploit E- and P-selectin to home and engraft into bone marrow niches. Thus, one of my objectives in this thesis was to study the expression of E-selectin ligands associated with ES/iPS-HSPCs. I showed that ES/iPS-HSPCs lack functional E-selectin ligand(s). In an effort to enhance the interaction between Eselectin and ES/iPS-HSPCs, we decorated the cell surface with sialyl-Lewis x (sLex) using the ex-vivo glycan engineering technology. However, this decoration did not improve the engraftment capacity of ES/iPS-HSPCs, in vivo. Induction of E-selectin expression during inflammation is key to recruitment of immune cells and therefore I also focused on analyzing the expression of E-selectin ligands on activated human T-cells. I identified several novel glycoproteins that may function as E-selectin ligands. Specifically, I compared the role of the known E-selectin ligands, namely PSGL-1 and CD43, to CD44. I showed that CD44 purified from in vitro human activated T-cells or from psoriasis patients acts as a functional E-selectin ligand. Furthermore, our knock-down studies demonstrated that CD44, and not CD43, cooperates with P-selectin glycoprotein ligand-1 (PSGL-1) as a major E-selectin ligand.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第12872号 / 論医科博第1号 / 新制||医科||4(附属図書館) / 31590 / (主査)教授 野田 亮, 教授 楠見 明弘, 教授 瀬原 淳子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Plants are used as a primary food source by humans. Some plants produce edible roots or leaves, but most crops used today are grown to harvest their nutrient-rich seeds which are a product of double fertilization in flowering plants. </p> <p>Cell-cell recognition, adhesion, and fusion are widespread phenomena in many biological processes, where fertilization is an exemplary process. Many players have been identified to mediate sperm-egg fusion in both animals and plants. Interestingly several of these components were shown to be structurally and functionally conserved across kingdoms. In animals Tetraspanins act as facilitators of sperm-egg fusion. Tetraspanins are known to associate in clusters in the plasma membrane of cells, where they recruit diverse signaling proteins, forming the so called Tetraspanin-enriched microdomains (TEMs). TEMs are therefore recognized as major signaling platforms mediating specific cellular processes in the plasma membrane of cells. Two <i>Arabidopsis</i>-expressed tetraspanins, <i>TET11</i> and <i>TET12</i>, are highly expressed in the sperm cells (SCs), however their function in fertilization are unknown. Using fluorescence microscopy, we quantified the expression of TETs in SCs and found evidence for the existence of a Tetraspanin-enriched microdomain (TEM) at the SC-SC adhesion interface. Sperm cell factors which are necessary for fertilization were found to accumulate at the TEM, suggesting that plant SC TEMs may function as protective platforms for fertilization factors. Sperm-expressed TETs directly interact with members of a novel, plant-specific family of unknown proteins, <i>DMP8/9</i>. DMP8/9 function as negative regulators of SC-SC adhesion and are required for double fertilization. Structural and functional analysis suggest that these two proteins may perform unique functions as membrane remodelers in SCs. In addition, we provide evidence of a new GEX2 function as a SC-SC adhesion factor and potential partner of TET-DMP complexes at the SC-SC interface.</p>

Capillary Morphogenesis Gene 2 protein (CMG2) is a type 1 transmembrane receptor known as the anthrax toxin receptor 2 (ANTXR2). While it is documented that the cell surface receptor CMG2 mediates anthrax toxin entry into the cell via endocytosis, the physiological role of CMG2 is not well understood. Others have suggested that CMG2 may have a role in mediating ECM homeostasis and angiogenesis. Additionally, both anthrax protective antigen (PA) and a furin protease-resistant mutant, PASSSR, inhibit corneal neovascularization in a mouse model, and interestingly PASSSR has a greater affinity to CMG2 receptor. PASSSRalso has a more potent antiangiogenic effect than wild-type PA. However, a mechanism for PASSSR inhibition of the putative CMG2 role in angiogenesis is not yet elucidated. The experimental results in this thesis provide evidence that CMG2 is the key receptor for regulating adhesion, migration, and actin dynamics in cells, and 200-pM PASSSR inhibits cell adhesion, migration, and actin localization at the cell leading edge. Furthermore, we observed that PASSSR remains bound to CMG2 under acidic conditions similar to the lysosome (pH 4). This observation suggests that the PASSSR-CMG2 complex remains intact following internalization and traffic to lysosomes, different from previous reports for PA, which likely results in CMG2 recycling. Together, these results suggest that following PASSSR treatment, CMG2 traffics to the lysosome for degradation; hence, we predict fewer CMG2 receptors are available at the cell surface to function in their native role in signaling angiogenic processes such as adhesion and chemotaxis towards vascular growth factors.

When there is a possibility of a low-template sample being processed in a forensic laboratory, it becomes important to retrieve all cells possible from the substrate they are collected on. The most common form of evidence received by forensic laboratories is epithelial cells collected on cotton material, swab or fabric, which may contain inhibitors. Data shows a likely mechanism for cellular adherence is the denaturation of surface proteins to expose residues hidden within. Proteins on cotton’s cell surface hydrogen bond to these residues, forming a strong attachment. An epithelial cell preparation was pipetted onto ISO adjacent cotton swatches. These swatches were incubated in 10mM Tris, 0.1 mM EDTA (TE) buffer with a constant temperature and agitation from a Thermal Mixer. The swatch was removed from the liquid and placed in a separate tube and digest separately. Each was quantified and used to calculate the percentage of cellular release. Variations of this baseline procedure were used to help determine the most efficient cellular release process. These variables included different temperatures and agitation speeds, sonication, resuspension and the addition of disaccharides. Results showed that the addition of a disaccharide is the most efficient method to achieve cellular release from cotton fabric. Specifically, drying 0.75 M D-(+)- Trehalose Dihydrate onto a cotton fabric swatch before the addition of the epithelial cell preparation. This procedure produced an average of 65.5% cellular release compared to a 26.0% release from our baseline procedure.

No description available.

Purpose. These experiments were designed to characterize tight junction formation by retinal pigment epithelial (RPE) cells in vitro and to compare the effects on this process of hormonally defined medium (HDM) and serum- containing medium. Methods. Formation of RPE tight junctions was analyzed in freshly isolated rat RPE cells maintained either in HDM or serum-containing medium. Junctions were evaluated functionally by measuring transepithelial electrical resistance (TER) and permeability and structurally by immunolocalization of the junction-associated actin microfilaments. Calcium dependency of the junction was determined by reducing media calcium concentration. Results. RPE cells cultured in serum-free HDM developed calcium-dependent tight junctions, which exhibited TER levels > 150 Ω · cm 2 and low paracellular permeability. Serum-containing media inhibited tight junction formation as indicated by significant reductions in TER and increases in permeability. Junction-associated actin microfilaments and cell density were unchanged. Conclusions. Tight junction formation by RPE cells is inhibited by serum. This activity may play an important role in responses of the RPE layer to injury, contributing to the pathologic progression of blood- retinal barrier dysfunction.