No description available.

Axons are directed along stereotypic pathways to their targets by cues arrayed in the extracellular environment. Identifying the cellular and molecular nature of these signals is of high interest and the developing optic pathway is a useful model system for achieving this. Although previous studies have identified several molecules essential for optic pathway formation, in vivo only subsets of retinal axons rely on them. I focused on the Dscam (Down’s syndrome cell adhesion molecule) and Sidekick (Sdk) cell adhesion molecules for potentially playing crucial roles in this system. In situ hybridisation in the embryonic mouse visual system showed Dscam and Sdk-1 expression in the RGC layer of the retina, along the optic pathway and in the visual targets. Sdk-2 was detected in the glia of the optic nerve and optic chiasm, marking the pathway that RGC axons follow, but not in RGCs. No DscamL1 was detected in RGCs or the optic pathway at the stages investigated and it was discounted from future analysis. In vitro, DSCAM promoted RGC axon outgrowth, whereas SDK 1 was inhibitory. SDK 2 had no effect on RGC axon outgrowth, suggesting it does not play a direct role in their pathfinding. Repeating this assay using retinal explants from the Dscamdel17 mouse mutant, showed that DSCAM enhanced retinal axon outgrowth, at least in part, through homophilic interactions. Analysis of visual system development in Dscam mutants showed DSCAM involvement in RGC axon fasciculation and in enhancing their growth, particularly within the ipsilateral optic tract. Retinal cell counts revealed that DSCAM played diverse roles in controlling cell number. Pre- and postnatal retinas lacking DSCAM contained more RGCs and mitotic cells. Postnatally, Dscam-/- retinas also show decreased cell death. In many cases, defect severity was dose-dependent, with an intermediate phenotype in the heterozygous mice, implicating DSCAM in the neurological defects of Downs’ Syndrome patients.

Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects neutrophils to cause human granulocytic anaplasmosis. Sialyl Lewis x (sLex)-modified P-selectin glycoprotein ligand-1 (PSGL-1) is the confirmed receptor utilized by A. phagocytophilum to bind and invade human neutrophils and myeloid cell lines. As an obligate intracellular pathogen, the binding of A. phagocytophilum to a host cell receptor is a prerequisite step for entry and replication, and thus its survival. However, the bacterial adhesins mediating this process have yet to be identified. In this study, we sought to identify surface proteins of A. phagocytophilum as putative adhesins. A. phagocytophilum undergoes a biphasic developmental cycle, transitioning between a smaller electron dense-cored cell (DC), which has a dense nucleoid, and a larger, pleomorphic electron lucent reticulate cell (RC), which has a dispersed nucleoid. We determined that the respective roles of the A. phagocytophilum DCs and RCs are adherence/infection and vacuolar replication, respectively, which is a finding that is consistent with the life cycles of other obligate intravacuolar pathogens that undergo biphasic development. Most importantly, we demonstrated the A. phagocytophilum DC is responsible for recognizing human PSGL-1. To identify surface proteins as putative adhesins we tested a variety of approaches. Three different computer prediction programs were compared, resulting in identification of 16 to 130 potential membrane proteins. As a more direct means to identify A. phagocytophilum surface proteins as PSGL-1 adhesins, several affinity capture approaches were tested. We used commercially available recombinant human PSGL-1 (rhPSGL-1) to try and capture adhesins by crosslinking and affinity purification. We were unsuccessful, but nevertheless gained insight into the binding properties of A. phagocytophilum. We next chose to take a broader approach to identify outer membrane proteins of the adherent DC by biotinylation. In the process we developed new density-gradient centrifugation approaches which successfully purified an RC-enriched population as well as a mixed population of RC and DC organisms. Results from this work demonstrate that A. phagocytophilum DC organisms are responsible for binding PSGL-1. Additionally, the results obtained thus far of gradient-purified bacteria will serve as a foundation for future experiments in identifying surface and developmental form specific proteins.

Cell adhesion is a critical process that allows the organisation and functioning of tissues in three-dimensions. However, the replenishing of cells, via cell division, within tissues is equally important for functioning complex life. Both cell adhesion and division are tightly controlled processes and rely on a complex network of signals that, as yet, are not wholly understood. This Thesis presents a structural analysis of several proteins involved in these processes. In the case of cell adhesion, we have made use of high-throughput (HTP) cloning and expression screening technologies in the Oxford Protein Production Facility (OPPF) for the study of the Kindlin protein family – a recently discovered set of proteins essential for integrin-mediated cell adhesion. As a direct result of the HTP pipeline used we were able to determine the high resolution crystal structure of a single domain, the Pleckstrin Homology Domain, from the isoform Kindlin-1. Deletion of this domain in the full-length protein resulted in impaired integrin activation in vivo. This structure, in combination with molecular dynamics simulation demonstrated that, unlike other well characterised PH domains, the binding of secondary messenger lipids (phosphoinositides) is dictated by a, previously unseen, salt bridge that occludes the putative binding site. Mutation of the salt bridge alters the binding characteristics of this domain in vitro. In addition to the PH domain, we have also studied and biophysically characterised full-length Kindlin-3, a blood cell specific isoform. By optimising baculovirus-infected Sf9 cell expression systems we were able to obtain, for the first time, sufficient quantities of protein for characterisation. Furthermore, by using small-angle X-ray scattering (SAXS) in solution we were able to determine a low resolution solution structure of Kindlin-3, revealing a linear arrangement of its FERM domain - a novel conformation known only otherwise in talin. We characterised the interaction of full-length Kindlin-3 with β-integrin cytoplasmic tails using nuclear magnetic resonance spectroscopy, which confirmed that a direct interaction with a membrane distal NPxY motif occurs, and demonstrated the importance of a preceding Serine/Threonine rich region in peptide binding. In the case of cell division, we have determined the crystal structure of the cell cycle checkpoint control related protein, Cid1, a terminal uridine tranferase from Schizzosaccharomyces pombe, alone and in complex with UTP. Structural and biochemical analysis of Cid1 identified a novel Uridine selection mechanism that is suggested to be conserved in metazoan ZCCHC enzymes involved in let-7 miRNA biogenesis, which are important for proliferation, differentiation and cell fate. We have also demonstrated that Cid1 is an RNA binding protein, a property essential for activity that employs a novel mechanism of RNA binding in the absence of RNA binding motifs. The structural work undertaken in this thesis has focussed on two distinct, but interwoven, aspects of cell biology and has significantly added to both fields of research. Excitingly, this has opened many new avenues of investigation and, in the case of Cid1, has the strong potential to lead to the development of novel anticancer therapies.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Markéta Marešová Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of alpha-humulene on adhesion of breast cancer cell line MDA-MB-231 α-humulene is a sesquiterpene contained in the essential oil of Chinese Bayberry (Myrica rubra), which has various biological effects. The aim of this thesis was to study the cell adhesion of tumor cell line MDA-MB-231, and the effects of α-humulene on expression of cell adhesion molecules. Furthermore, the cytotoxic effect of α-humulene on this cell line was verified. Effect of α-humulene on cell proliferation was evaluated by neutral red uptake test (NRU test) and by xCelligence system. Cell adhesion was also continuously monitored by xCelligence. Expression ganges of cell adhesion molecules upon α-humulene treatment were determined by Western blotting and by quantitative polymerase chain reaction on the protein and mRNA levels, respectively. The proliferation of the cells was significantly affected by α-humulene after 24 hour treatment (IC50 13.6 µg/ml). α-humulene alone caused slightly increased cell adhesion. Adhesion molecules EpCAM and β-cathenin were almost unaffected. Level of ICAM1 mRNA was increased after 12 hours and...

No description available.

Introdução Aproximadamente 1/3 da população mundial está infectada pelo bacilo M. tuberculosis e a prevalência da infecção é maior entre os homens. As razões para as diferenças na epidemiologia da tuberculose e adesão ao tratamento em relação ao sexo são desconhecidas. Objetivos Verificar diferenças na adesão ao tratamento da tuberculose em relação ao sexo. Identificar aspectos facilitadores e dificultadores para a adesão ao tratamento da tuberculose em relação ao sexo. Analisar as crenças consideradas importantes para adesão ao tratamento da tuberculose. Metodologia Pesquisa quali-quantitativa, baseada na análise de conteúdo e com fundamentação teórica no Modelo de Crenças em Saúde. Foram realizadas entrevistas semi-estruturadas em 6 serviços de saúde do Distrito de Saúde da Freguesia do Ó/Brasilândia, com 28 pacientes em tratamento supervisionado de tuberculose, sendo 11 homens e 17 mulheres. Os dados quantitativos foram obtidos por meio do livro de Registro e Controle de Tratamento dos Casos de Tuberculose de cada serviço de saúde. Resultados Aqueles que falharam na adesão apresentaram o seguinte perfil: mulher, solteira e separada, com atividade remunerada não comprovada, com nível de escolaridade entre fundamental I completo e ensino médio completo; homem: casado, com atividade remunerada comprovada, nível de escolaridade entre ensino fundamental II completo e ensino médio completo. Os aspectos facilitadores encontrados para a boa adesão residem no bom atendimento dos profissionais de saúde e a percepção por parte do paciente na sua melhora de saúde. As crenças para a boa adesão ao tratamento no sexo masculino e feminino foram: bom atendimento do serviço de saúde e bom tratamento (em relação aos medicamentos). Conclusão Homens e mulheres não apresentam diferenças nas crenças de suscetibilidade. Nas crenças ligadas à seriedade percebida houve diferenças. As mulheres apresentaram medo de transmitir a doença aos filhos e associam a tuberculose ao HIV. Os homens possuem medo voltado às questões sexuais. Os benefícios mencionados foram o bom atendimento e bom tratamento. As barreiras para a adesão foram o diagnóstico errado, os efeitos colaterais e questões assistencialistas independente do sexo. As medidas sugeridas relacionam-se a necessidade de trabalhos voltados ao público masculino para que estes utilizem os serviços de saúde, principalmente o PSF, e assim, o diagnóstico precoce da tuberculose ocorra. Sugere-se treinamentos para todo os sistema de saúde para o diagnóstico da tuberculose. A realização de trabalho conjunto do sistema de saúde e educação é necessário para desfazer estigma da doença. / Introduction Approximately one third of worlds population is infected by M. tuberculosis, and the prevalence of infection is higher among men. The reasons for the differences in the epidemiology of tuberculosis and the adhesion to the treatment in relation to sex are unknown. Objectives Verify the differences in the adhesion to the treatment of tuberculosis in relation to sex. Identify facilitating and difficulty aspects for the adhesion to the treatment of tuberculosis in relation to sex. Analyze the belief that is considered important for the adhesion to the tuberculosis treatment. Methodology Quali-quantitative research, based on the content analysis and with theoretical foundation in the Health Belief Model. Semi-structured interviews were carried out in 6 health services from the Health District of Freguesia do Ó/Brasilândia, with 28 patients in supervised treatment for tuberculosis, being 11 men and 17 women. Quantitative data were obtained in the book of Register and Control of Treatment for Tuberculosis Cases of each health service. Results Those who failed to treat tuberculosis showed the following profile: woman, single and divorced, with non-proved paid activity, and scholarship level between complete fundamental I and complete medium education; man, married, with proved paid activity, and scholarship level between complete fundamental II and complete medium education. The facilitating aspects found for the good adhesion are the good attendance of health professionals and the patients perception in his/her health improvement. The beliefs for the good adhesion to treatment both in male and female were: good attendance of health service and good treatment (related to medicines). Conclusion Men and women do not show differences in susceptibility beliefs. In regard to the beliefs related to noticed seriousness there were differences. Women showed fear of transmission of the illness for their children and they associate the tuberculosis to HIV. Men are scared about sexual questions. The benefits mentioned were the good attendance and treatment. Barriers for adhesion were wrong diagnosis, collateral effects and assistance questions independent of sex. The suggested measures are related to the need of specific tasks for the male public, for them to use the health services, principally the PSF, so that the early diagnosis of tuberculosis can occur. Training courses for the diagnosis of tuberculosis are suggested for all the health system. It is necessary to carry out a work joining health and educational systems to dismantle the disease stigma.

O estudo objetivou avaliar a resistência da união de sistemas adesivos aplicados ao esmalte, bem como a morfologia do substrato condicionado e a qualidade da interface de união formada. Após o esmalte das superfícies lisas de molares receberem profilaxia com pedrapomes, abrasão por lixa ou desgaste por ponta de diamante, foram aplicados Scotchbond Multi-Purpose Plus (SBMP), Single Bond (SB), Clearfil SE Bond (CSE); OptiBond Solo Plus Self-Etch (OP) e Tyrian Self Priming Etching (TY). Os semidentes ?restaurados? com resina Filtek Z250 foram armazenados em água deionizada (24h/37°C), secionados no formato de paralelogramo (área transversal aproximada de 0,8 mm2), aleatorizados em grupos (24h, 6 e 12 meses de armazenagem), e testados em microtração (0,5mm/min). Os valores médios de resistência da união (MPa) foram tratados por métodos de ANOVA e Tukey. Os resultados não mostraram diferenças significativas para os fatores Tratamento, Armazenagem, nem para as interações..Porém, houve diferença significante para o fator Adesivo (p<0,05). Os padrões morfológicos do esmalte obtidos após o uso do agente condicionante dos sistemas SBMP e SB não foram influenciados pelo Tratamento. Os fatores Adesivo e Tratamento influenciaram no padrão dos sistemas CSE, OP e TY. Não foram observadas fendas nas interfaces dos sistemas SBMP e SB, independente dos outros fatores. .Os sistemas OP e TY mostraram fendas em todas as condições, e o CSE após 12 meses no esmalte abrasionado por lixa. Com base nos resultados, conclui-se que o desempenho dos sistemas adesivos estudados está vinculado mais às características dos produtos do que ao tratamento realizado no esmalte ou armazenagem. / This study aimed to evaluate the resin-enamel bond strength, the etching pattern and the bonded interfaces provided by self-etch systems by means of scanning electron microscopy. The following adhesives systems (Scotchbond Multi-Purpose Plus [SBMP], Single Bond [SB], Clearfil SE Bond [CSE]; OptiBond Solo Plus Self-Etch [OP] or Tyrian Self Priming Etching [TY] were applied to sound enamel (after tooth prophylaxis), SiC paper or diamond bur-treated enamel. Resin composite buil-dups (Filtek Z250) were bonded to the enamel surfaces. After storage in water (24h/37°C), the bonded samples were sectioned into beams (0.8mm2), randomly assigned into subgroups (24h, 6 and 12 months) and subjected to microtensile bond strength test (0.5 mm/min). The data was analyzed by ANOVA and Tukey?s test. The main factors enamel Treatment and Storage as well as the interactions were not statistically significant. Significant differences were observed only for the main factor Adhesive (p<0.05). The Treatment did not affect the etching pattern of SBMP and SB; for CSE, OP and TY the morphology varied with the Treatment and Adhesive system. Gaps were not observed in the interfaces bonded with SBMP and SB, regardless of the Storage and Treatment. The adhesive CSE showed gaps after 12 months of storage for SiC paper, while this finding was observed for OP and TY in all conditions. Based on the results of the present investigation, it was concluded that the performance of some adhesive systems is influenced by the material´s composition instead of surface treatment or storage.

Introdução Aproximadamente 1/3 da população mundial está infectada pelo bacilo M. tuberculosis e a prevalência da infecção é maior entre os homens. As razões para as diferenças na epidemiologia da tuberculose e adesão ao tratamento em relação ao sexo são desconhecidas. Objetivos Verificar diferenças na adesão ao tratamento da tuberculose em relação ao sexo. Identificar aspectos facilitadores e dificultadores para a adesão ao tratamento da tuberculose em relação ao sexo. Analisar as crenças consideradas importantes para adesão ao tratamento da tuberculose. Metodologia Pesquisa quali-quantitativa, baseada na análise de conteúdo e com fundamentação teórica no Modelo de Crenças em Saúde. Foram realizadas entrevistas semi-estruturadas em 6 serviços de saúde do Distrito de Saúde da Freguesia do Ó/Brasilândia, com 28 pacientes em tratamento supervisionado de tuberculose, sendo 11 homens e 17 mulheres. Os dados quantitativos foram obtidos por meio do livro de Registro e Controle de Tratamento dos Casos de Tuberculose de cada serviço de saúde. Resultados Aqueles que falharam na adesão apresentaram o seguinte perfil: mulher, solteira e separada, com atividade remunerada não comprovada, com nível de escolaridade entre fundamental I completo e ensino médio completo; homem: casado, com atividade remunerada comprovada, nível de escolaridade entre ensino fundamental II completo e ensino médio completo. Os aspectos facilitadores encontrados para a boa adesão residem no bom atendimento dos profissionais de saúde e a percepção por parte do paciente na sua melhora de saúde. As crenças para a boa adesão ao tratamento no sexo masculino e feminino foram: bom atendimento do serviço de saúde e bom tratamento (em relação aos medicamentos). Conclusão Homens e mulheres não apresentam diferenças nas crenças de suscetibilidade. Nas crenças ligadas à seriedade percebida houve diferenças. As mulheres apresentaram medo de transmitir a doença aos filhos e associam a tuberculose ao HIV. Os homens possuem medo voltado às questões sexuais. Os benefícios mencionados foram o bom atendimento e bom tratamento. As barreiras para a adesão foram o diagnóstico errado, os efeitos colaterais e questões assistencialistas independente do sexo. As medidas sugeridas relacionam-se a necessidade de trabalhos voltados ao público masculino para que estes utilizem os serviços de saúde, principalmente o PSF, e assim, o diagnóstico precoce da tuberculose ocorra. Sugere-se treinamentos para todo os sistema de saúde para o diagnóstico da tuberculose. A realização de trabalho conjunto do sistema de saúde e educação é necessário para desfazer estigma da doença. / Introduction Approximately one third of worlds population is infected by M. tuberculosis, and the prevalence of infection is higher among men. The reasons for the differences in the epidemiology of tuberculosis and the adhesion to the treatment in relation to sex are unknown. Objectives Verify the differences in the adhesion to the treatment of tuberculosis in relation to sex. Identify facilitating and difficulty aspects for the adhesion to the treatment of tuberculosis in relation to sex. Analyze the belief that is considered important for the adhesion to the tuberculosis treatment. Methodology Quali-quantitative research, based on the content analysis and with theoretical foundation in the Health Belief Model. Semi-structured interviews were carried out in 6 health services from the Health District of Freguesia do Ó/Brasilândia, with 28 patients in supervised treatment for tuberculosis, being 11 men and 17 women. Quantitative data were obtained in the book of Register and Control of Treatment for Tuberculosis Cases of each health service. Results Those who failed to treat tuberculosis showed the following profile: woman, single and divorced, with non-proved paid activity, and scholarship level between complete fundamental I and complete medium education; man, married, with proved paid activity, and scholarship level between complete fundamental II and complete medium education. The facilitating aspects found for the good adhesion are the good attendance of health professionals and the patients perception in his/her health improvement. The beliefs for the good adhesion to treatment both in male and female were: good attendance of health service and good treatment (related to medicines). Conclusion Men and women do not show differences in susceptibility beliefs. In regard to the beliefs related to noticed seriousness there were differences. Women showed fear of transmission of the illness for their children and they associate the tuberculosis to HIV. Men are scared about sexual questions. The benefits mentioned were the good attendance and treatment. Barriers for adhesion were wrong diagnosis, collateral effects and assistance questions independent of sex. The suggested measures are related to the need of specific tasks for the male public, for them to use the health services, principally the PSF, so that the early diagnosis of tuberculosis can occur. Training courses for the diagnosis of tuberculosis are suggested for all the health system. It is necessary to carry out a work joining health and educational systems to dismantle the disease stigma.

Mantle cell lymphoma (MCL) is a rare lymphoproliferative disorder (LPD) that has very poor survival. Like other LPDs, the neoplastic cells of MCL have an intimate dependence upon accessory cells within haematopoietic tissues. Understanding and exploiting the tissue-relationships of the mantle cells may therefore lead to further new approaches to treatment. This study work has set out to construct an in vitro system to model relevant aspects of the tissue-dependent behaviour of the neoplastic mantle cells, seeking to establish the link between in vitro behaviour and clinical phenotype, and establishing the feasibility of this system to study the effects of different therapeutic interventions. The first experimental chapter employs relevant mouse and human stromal models to mirror the tissue environment of MCL in vivo. Testing relevant agents, the work establishes that the system can identify different behaviour between indolent and aggressive forms of MCL, and demonstrates a particular importance for CD40 ligand both in the proliferation and survival of the neoplastic mantle cells, but shows also how these effects are modulated by the soluble factors interleukin-4 (IL-4) and the toll-like receptor-9 ligand (TLR9-L). The second experimental chapter examines the adhesion molecules expressed on MCL cells. Considerable variation in the level of expression is observed between cases, but overall the cases express particularly high levels of the integrin receptors LFA-1 (detected by alpha chain CD11a) and VLA-4 (detected by alpha chain CD49d). Cases also showed a significant difference in overall adhesion and chemokine-receptor expression between cases that had either a nodal or leukaemic clinical pattern, although no single adhesion molecule was characteristic of clinical phenotype. The final experimental chapter looked at 3-D culture of MCL. Within tissues MCL grows in a 3-D rather than 2-D matrix and it is recognised that cells employ different forms of adhesion and migration within the different spatial environments. This chapter establishes the feasibility of growing cells in 3-D systems and looks at optimal conditions to preserve and examine the cellular characteristics of cells within a 3-D environment. Overall, this thesis demonstrates the feasibility and pathobiological relevance of ex vivo culture of MCL cells giving insights into the factors that drive MCL survival and proliferation and the correlation between in vitro behaviour and clinical phenotype. It is proposed that this work can be expanded to examine therapeutic interventions in the disorder.