Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-28Bitstream added on 2014-06-13T18:53:48Z : No. of bitstreams: 1 carrenho_lca_me_araca.pdf: 1197310 bytes, checksum: 3d5470911a8b6fe2c7996a8ec61a673a (MD5) / A endotoxemia é um importante distúrbio sistêmico que se origina da resposta do hospedeiro a um componente das bactérias Gram-negativas, o lipopolissacarídeo (LPS) ou endotoxina, que é liberado após bacteriólise ou rápida multiplicação. A ativação do sistema imune inato pelo LPS é um fator chave para o disparo da resposta inflamatória pelo hospedeiro e que acarreta a produção de mediadores inflamatórios, responsáveis pelos eventos patológicos da endotoxemia. A interação dos receptores Toll-like (TLRs) com antígenos específicos deflagram a resposta inflamatória, sendo que o receptor Toll-like-4 (TLR-4) é ativado pela ação das endotoxinas, enquanto o receptor Toll-like-2 (TLR-2) interage com uma variedade de componentes microbianos. Uma exposição prévia a baixas concentrações de LPS pode tornar os cavalos “tolerantes” a um desafio letal subsequente, acarretando uma diminuição na produção de citocinas inflamatórias por um período transitório. Pouco se sabe a respeito do mecanismo celular deste fenômeno em equinos, supondo-se o envolvimento dos receptores Toll-like semelhante ao encontrado em outras espécies. Com este estudo investigaram-se os mecanismos celulares da tolerância à endotoxina em um modelo ex vivo com sangue total. Foi demonstrado redução na síntese de citocinas pró-inflamatórias (TNF-α, IL-1 e IL-6), aumento da expressão gênica da citocina anti-inflamatória IL-10, e ausência de expressão do TGF-β, após o desafio secundário com LPS. A maior expressão dos receptores TLR-2 e -4 após o segundo estímulo de LPS demonstrou que a tolerância à endotoxina não acarreta diminuição da expressão de ambos os receptores em equinos. / Endotoxemia is an important systemic disease originated from host response to a component of Gram-negative bacteria, lipopolysaccharide (LPS) or endotoxin, which is released after bacteria death or quick replication. The innate immune recognition of LPS has a key role triggering host inflammatory answer and is due to inflammatory mediator’s synthesis, which are responsible for pathologic events of endotoxemia. Signs initiated by interaction of Toll-like receptors (TLRs) with specific products induce the inflammatory response. Toll-like receptor-4 (TLR- 4) is activated by endotoxin action while Toll-like receptor-2 (TLR-2) interacts with a range of microbial compounds. Some studies demonstrate that both can act like LPS receptors, although by independent pathways. It was demonstrated that a previous exposition to low concentrations of LPS can render horses “tolerant” to a lethal subsequent challenge with endotoxin, leading to a diminished release of inflammatory cytokines during a transient period. However, little is known about the cellular mechanisms involved in this phenomenon in horses, suspecting that there is involvement of cell surface receptors, similarly to other species. This study investigated cellular mechanisms of endotoxin tolerance in a whole blood ex vivo model, demonstrating a reduction on pro-inflammatory cytokines synthesis (TNF- α, IL-1 and IL-6), increased gene expression of anti-inflammatory cytokine IL-10 and no alteration in TGF-β expression, after a secondary stimulus with LPS. The Toll-like receptors-2 and -4 increased expression after a second stimulus with LPS showed that endotoxin tolerance does not lead to a decreased expression of both receptors in horses.

Made available in DSpace on 2014-06-11T19:27:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T20:17:33Z : No. of bitstreams: 1 moco_np_me_botfm.pdf: 454787 bytes, checksum: e1174a4016333b9ca192548a5638a090 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Acute chorioamnionitis is a response to microbial infection of the amniotic fluid. The innate immune system constitutes the host’s first line of defense against pathogens and, in this regard, Toll-like receptors (TLRs) are important regulators of such nonspecific response. However, the expression of these receptors in chorioamniotic membranes in pregnancies complicated by chorioamnionitis has not been well established. Objective:The purpose of this study was to examine the localization of TLR-1, TLR-2, TLR-4 and TLR-6 in fetal membranes and determine whether histologic chorioamnionitis is associated with changes in gene expression of these receptors. One hundred and fifteen chorioamniotic membranes were included in the study. They were collected at the Obstetrics Service of the Botucatu Medical School, São Paulo State University, UNESP, from pregnant women with preterm delivery or term delivery with or without labor. Both groups were stratified on the basis of the presence of histologic chorioamnionitis. Fragments of the chorioamniotic membranes were sent for histopathologic analysis in order to confirm histologic chorioamnionitis. Other membranes fragments measuring 1cm2 were placed into RNA later and submitted to total RNA extraction. After RNA extraction, the samples with concentration between 0.02 and 0.2 g/L of RNA were submitted to cDNA collection for later use in quantifying the expression of TLR-1, TLR-2, TLR-4 and TLR-6 by the real-time PCR technique using the TaqMan® Gene Expression Assays System. All membranes analyzed expressed TLR-1 and TLR-4, whereas 99.1% expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expression were statistically higher in the membranes of preterm pregnancies in the presence of chorioamnionitis as compared with preterm membranes in the absence of the inflammatory infiltrate. Among the membranes of term pregnancies, there was... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-28Bitstream added on 2014-06-13T20:50:02Z : No. of bitstreams: 1 almeidajr_op_me_araiq.pdf: 464542 bytes, checksum: cd4d79303894163a1d9272d7c7a86590 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A infecção produzida pelo HIV leva à debilidade do sistema imune e confere ao indivíduo infectado maior vulnerabilidade às infecções oportunistas. Neste aspecto a tuberculose e o HIV estão intimamente relacionados, uma vez que a infecção pelo vírus contribui significativamente para o aumento na incidência da tuberculose. Por outro lado, a co-infecção do HIV com outros patógenos é um importante fator exógeno que influencia a gravidade e a taxa de progressão da doença em indivíduos soro-positivos. O sistema imune pode reconhecer padrões moleculares associados à patógenos (PAMPs) através dos receptores toll-like, sendo que esta sinalização resulta na ativação de fatores de transcrição fundamentais para as respostas imune e inflamatória. A indução do HIV como conseqüência da ativação imunológica produzida por ligantes microbianos tem sido implicada como o mecanismo responsável pela elevada expressão viral observada em indivíduos co-infectados. Polimorfismos nos genes tlr, bem como nos vários componentes de seus caminhos de sinalização, têm grande importância na resposta imunológica do hospedeiro frente a vários patógenos. Neste estudo foi avaliado se a do hospedeiro infecção pelo Mycobacterium tuberculosis tem influência na replicação do HIV e na imunidade celular relacionada aos polimorfismos nos genes tlr2 e 4. Foram analisados 37 pacientes de ambos os sexos com média de idade de 41 anos. Os pacientes foram divididos em 2 grupos: grupo A (17 indivíduos portadores de HIV) e grupo B ( 20 indivíduos portadores de HIV co-infectados com M. tuberculosis); o grupo B foi subdividido em pacientes com histórico de infecção pregressa pelo M. tuberculosis (12 indivíduos designados por B’) e aqueles que apresentavam manifestações clínicas da tuberculose durante as coletas (8 indivíduos designados por B’’)... / The infection caused by the HIV virus lead to debility of the immune system and confers to the infected individual a greater vulnerability to opportunist infections. In this aspect the tuberculosis and the HIV are closely related, once that the infection by the virus significantly contribute for the increase of tuberculosis incidence. The co-infection with others pathogens is an important exogenous factor that influence the severity and the rate of progression of the disease at serum-positive patients. The Immune System can recognize pathogens associated molecular patterns (PAMPs) through the toll-like receptors, this signalization results in the activation of fundamental transcriptional factors to the immune and anti-inflammatory responses. The induction of HIV like a consequence of immunological activation produced by microbial ligands has been implicated as the responsible mechanism of the elevated viral expression observed at co-infected individuals. Polymorphisms at the tlr genes, as well as at various components of its signaling pathways, has a great importance on the immunological response of the host front various pathogens. In this study was evaluated the influence of the Mycobacterium tuberculosis at the replication of HIV and at cellular immunity related to polymorphisms at the genes tlr2 and 4. Thirty seven patients, from both sex and with age ratio of 41 years old, were analyzed. The patients were divided into two groups: group A (17 individuals HIV positive) and group B (20 coinfected HIV – M. tuberculosis individuals); the group B was divided into patients with historic of infection by M. tuberculosis (12 individuals designated by B’) and those that was manifesting the symptoms of tuberculosis during the reaps (8 individuals designated by B’’). The immunological profile was evaluated by the quantitative determination of phenotypes CD3+/CD4+ and CD3+/CD8+ of lymphocytes ...(Complete abstract click electronic access below)

Orientador: Juliana Regina Peiró / Coorientador: Sérgio Ribeiro Aoki / Banca:Lina Maria Wehrle Gomide / Banca: Flávia Lombardi Lopes / Banca:José Paes de Oliveira Filho / Banca:Glenda Nicioli da Silva / Resumo: A resposta imune inata é a principal responsável pela defesa do hospedeiro contra endotoxinas de bactérias Gram-negativas. Tal resposta se dá através dos receptores Toll-like-4 e -2, seja pela via MyD88-dependente, TRIF-dependente ou ambas. Entretanto, uma resposta exagerada pode resultar em muitos danos para o organismo e levar até mesmo ao choque séptico. Uma das formas de controle desta resposta se dá através da tolerância à endotoxina (TE). Por isso o presentes estudo tem por objetivo investigar a indução diferencial dos receptores TLR-4 e 2, suas vias de sinalização MyD88 e TRIF-dependentes e avaliar outros possíveis genes relacionados a estas vias após indução da TE. Foi coletado sangue total de cavalos saudáveis (n=6), o qual foram estimulados com diferentes doses (0, 1 ou 10ng de LSP/mL) LPS de E.coli no momento 0 e 4 horas após o primeiro estímulo e 4 horas após o segundo estímulo. O "pool" (n=6) de RNA das amostras, coletado 0, 2, 4 e 8 horas, foi utilizado para a transcrição do cDNA. A hibridização do cDNA marcado com Cy-3 foi realizada em uma lâmina 4x44 contendo sequências específicas da espécie equina. Foi identificado durante a TE, nas diferentes doses de LPS, um aumento de transcritos dos receptores TLR-4 e -2, TNFAIP3 e IL-10 e uma diminuição de outros transcritos de genes importantes, tais como: MyD88, IL1-B, TNF-a, TRAM2, o que caracteriza e esclarece, de certa forma, as alterações na sinalização do TLR nas condições de tolerância à endotoxina. / Abstract: The innate immune response is the main responsible for the host's defense against endotoxins from Gram-negative bacteria. Such response occurs via Toll-like-4 and -2 receptors, MyD88-dependent pathway, TRIF-dependent pathway or both. However, an exaggerated reponse can induce many damages to the organism, including septic shock. One way to control this exacerbated response is through endotoxin tolerance (ET). The goals of the present study were to investigate TLR-4 and 2 receptors, their signaling through MyD88 e TRIF-dependent pathways and evaluate other possible genes related to this ways after ET induction. Blood samples were collected from healthy horses (n=6) which were stimulated with different doses (0, 1 or 10ng of LSP/mL) of LPS from E.coli, for 0 and 4 hours after the first stimulus and 4 hours after second stimulus. The pool (n=6) of RNA, extracted from the samples collected at 0, 2, 4 e 8 hours, was used for the transcription of the cDNA. The hybridization of the cDNA marked with Cy-3 was done in one slide 4x44 containing specific sequences from the equine species. We identified during the ET, on the different doses of LPS, an increase of transcripts for the receptors TLR-4 and -2, TNFAIP3 and Il-10 and a reduction of transcripts of other important genes, such as: MyD88, IL1-B, TNF-a, TRAM2, what characterizes and enlightens the changes in TLR signaling during endotoxin tolerance. / Doutor

Orientador: Paulo Inácio da Costa / Banca: Clarice Queico Fujimura Leite / Banca: Cleni Mara Marzocchi Machado / Resumo: A infecção produzida pelo HIV leva à debilidade do sistema imune e confere ao indivíduo infectado maior vulnerabilidade às infecções oportunistas. Neste aspecto a tuberculose e o HIV estão intimamente relacionados, uma vez que a infecção pelo vírus contribui significativamente para o aumento na incidência da tuberculose. Por outro lado, a co-infecção do HIV com outros patógenos é um importante fator exógeno que influencia a gravidade e a taxa de progressão da doença em indivíduos soro-positivos. O sistema imune pode reconhecer padrões moleculares associados à patógenos (PAMPs) através dos receptores toll-like, sendo que esta sinalização resulta na ativação de fatores de transcrição fundamentais para as respostas imune e inflamatória. A indução do HIV como conseqüência da ativação imunológica produzida por ligantes microbianos tem sido implicada como o mecanismo responsável pela elevada expressão viral observada em indivíduos co-infectados. Polimorfismos nos genes tlr, bem como nos vários componentes de seus caminhos de sinalização, têm grande importância na resposta imunológica do hospedeiro frente a vários patógenos. Neste estudo foi avaliado se a do hospedeiro infecção pelo Mycobacterium tuberculosis tem influência na replicação do HIV e na imunidade celular relacionada aos polimorfismos nos genes tlr2 e 4. Foram analisados 37 pacientes de ambos os sexos com média de idade de 41 anos. Os pacientes foram divididos em 2 grupos: grupo A (17 indivíduos portadores de HIV) e grupo B ( 20 indivíduos portadores de HIV co-infectados com M. tuberculosis); o grupo B foi subdividido em pacientes com histórico de infecção pregressa pelo M. tuberculosis (12 indivíduos designados por B') e aqueles que apresentavam manifestações clínicas da tuberculose durante as coletas (8 indivíduos designados por B'')...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The infection caused by the HIV virus lead to debility of the immune system and confers to the infected individual a greater vulnerability to opportunist infections. In this aspect the tuberculosis and the HIV are closely related, once that the infection by the virus significantly contribute for the increase of tuberculosis incidence. The co-infection with others pathogens is an important exogenous factor that influence the severity and the rate of progression of the disease at serum-positive patients. The Immune System can recognize pathogens associated molecular patterns (PAMPs) through the toll-like receptors, this signalization results in the activation of fundamental transcriptional factors to the immune and anti-inflammatory responses. The induction of HIV like a consequence of immunological activation produced by microbial ligands has been implicated as the responsible mechanism of the elevated viral expression observed at co-infected individuals. Polymorphisms at the tlr genes, as well as at various components of its signaling pathways, has a great importance on the immunological response of the host front various pathogens. In this study was evaluated the influence of the Mycobacterium tuberculosis at the replication of HIV and at cellular immunity related to polymorphisms at the genes tlr2 and 4. Thirty seven patients, from both sex and with age ratio of 41 years old, were analyzed. The patients were divided into two groups: group A (17 individuals HIV positive) and group B (20 coinfected HIV - M. tuberculosis individuals); the group B was divided into patients with historic of infection by M. tuberculosis (12 individuals designated by B') and those that was manifesting the symptoms of tuberculosis during the reaps (8 individuals designated by B''). The immunological profile was evaluated by the quantitative determination of phenotypes CD3+/CD4+ and CD3+/CD8+ of lymphocytes ...(Complete abstract click electronic access below) / Mestre

Submitted by Isaac Francisco de Souza Dias (isaac.souzadias@ufpe.br) on 2015-05-19T16:34:38Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO Carolina da Rocha Simões.pdf: 2694652 bytes, checksum: 6446d4fa347e53fe42ce7e769f745124 (MD5) / Made available in DSpace on 2015-05-19T16:34:38Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO Carolina da Rocha Simões.pdf: 2694652 bytes, checksum: 6446d4fa347e53fe42ce7e769f745124 (MD5) Previous issue date: 2014-02-20 / CAPES / O aumento da informação proveniente do sequenciamento de alta performance (NGS), e de projetos como o 1000 genomas e HapMap, permitiram a descoberta de milhões de variações. Entretanto, o maior desafio é a identificação da relação entre o genótipo e o fenótipo, proporcionando informações que possam ajudar a definir os polimorfismos que podem ou não causar doenças. Ferramentas computacionais tem auxiliado na predição das modificações estruturais geradas pelos polimorfismos, e as consequentes alterações funcionais sofridas pelas proteínas. Os receptores Toll Like (TLR) são proteínas do sistema imunológico que estão envolvidas na regulação da inflamação e em alguns casos no desenvolvimento do câncer. O objetivo deste projeto foi analisar, através de ferramentas in silico, os polimorfismos de base única nos genes das TLRs, buscando por polimorfismos que possam estar relacionados com a predisposição ao câncer e com alterações da via de sinalização das TLRs. Foram encontrados 37 genes que estão envolvidos na via de sinalização e podem ser utilizados como marcadores genéticos (biomarcadores) para o diagnóstico e predição das alterações na expressão dos genes relacionados à esta via. Estes genes, se regulados, podem ser utilizados como inibidores. Em relação aos polimorfismos foram coletados no banco de dados dbSNP/NCBI 5.839 SNPs entre os 10 genes das TLRs. Destes, 1.017 variações foram classificadas como missense e analisadas para avaliar as consequências estruturais pela troca dos aminoácidos. Para isso quatro ferramentas preditoras (SIFT, Polyphen, MutationAssessor e SDM) foram utilizadas gerando informações sobre as modificações e associando-as com possíveis danos nas proteínas. Dos polimorfismos analisados 223 foram classificados como danosos baseados na troca de aminoácido e podem causar uma desregulação funcional na proteína. Entre eles está o rs5743708 (TLR2), rs3775291, (TLR3) e rs11466653 (TLR10) que já foi estudado in vitro e tiveram associação com câncer colorectal (TLR2 e 3) e carcinoma da tireóide (TLR10). A predição prévia, in silico, das alterações funcionais pode auxiliar na interpretação das variações gênicas, neste caso associadas com o câncer, e também na caracterização precisa dos fatores que levam a estas alterações, contribuindo no diagnóstico, na prevenção e em melhores respostas aos tratamentos oferecidos.

Orientadores: Raquel Franco Leal, Maria Lourdes Setsuko Ayrizono / Dissertação (Mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T16:08:44Z (GMT). No. of bitstreams: 1 Paiva_NielceMariade_M.pdf: 3729371 bytes, checksum: 4077b1681124ea203fae5904b5fd8254 (MD5) Previous issue date: 2011 / Resumo: A retocolectomia total com anastomose do reservatório ileal (RI) ao canal anal é a cirurgia de escolha para doentes com retocolite ulcerativa inespecífica (RCUI) refratária ao tratamento clínico e para a polipose adenomatosa familiar (PAF). Entretanto, a ileíte primária do reservatório é uma das complicações mais comuns após a cirurgia do RI em pacientes com RCUI, sendo rara na PAF; e somente ocorre após o fechamento da ileostomia de proteção. Neste sentido, há necessidade de estudos que avaliem a forma como as bactérias, por meio de receptores específicos, possam participar no processo inflamatório do RI. Desta forma, foi analisada a expressão dos Toll-like receptors (TLR) em mucosa do RI endoscópica e histologicamente normal em pacientes operados por RCUI e PAF, a fim de se detectar alguma anormalidade nesta via em indivíduos assintomáticos, que poderia contribuir com processo inflamatório no RI. Casuística e Método: Doze pacientes (seis com RCUI e seis com PAF) submetidos à retocolectomia total e confecção do RI em "J", foram estudados após a reconstrução do trânsito intestinal. Foram obtidas biópsias da mucosa do RI por meio de endoscopia do mesmo. O grupo controle foi constituído por seis doentes com íleo-colonoscopia normal. As biópsias foram congeladas em nitrogênio líquido e as expressões de TLR-2, TLR-4 (receptores de reconhecimento de antígenos bacterianos) e p- JNK (fator de sinalização nuclear) foram avaliadas por meio de imunoblot de extrato protéico total. A ausência de ileíte do RI foi determinada por parâmetros clínicos, histológicos e endoscópicos, de acordo com o Índice de Atividade da Ileíte do RI (PDAI). Os pacientes não estavam em uso de medicações. O presente estudo foi aprovado pelo Comitê de Ética em Pesquisa Local e os participantes assinaram o termo de consentimento informado. Utilizou-se análise de variância (ANOVA), seguida por análise de significância (Teste de Tukey-Kramer) para a análise estatística. Nível de significância adotado foi p<0,05. Resultados: Houve maior expressão de TLR-4 em mucosa de RI de doentes operados por RCUI, quando comparada aos grupos Controle e PAF (p<0,05). Não houve diferença estatística das expressões de TLR-2 e p-JNK entre os diferentes grupos (p>0.05). Conclusão: Pacientes com RCUI apresentaram maior expressão de TLR4 na mucosa do RI, mesmo sem evidência clínica, endoscópica e histológica de inflamação no RI. Estes achados podem explicar a tendência de ativação de vias intracelulares deflagradas por antígenos bacterianos em pacientes com RCUI, o que pode contribuir com a produção de mediadores pró-inflamatórios, sendo coadjuvante do processo inflamatório inicial na mucosa do RI / Abstract: Introduction: Ileal pouch-anal anastomosis (IPAA) is the preferred surgical procedure for patients with refractory ulcerative colitis (UC) and familial adenomatous polyposis (FAP). However, pouchitis is the most common complication after ileal pouch-anal anastomosis (IPAA) in UC patients, being rare in FAP; and only occurs after ileostomy closure. Therefore, it is important to get more information about the role of the ileal pouch microbiota and mucosa susceptibility to inflammation. Therefore, we evaluated Toll-like receptors (TLRs) expression in normal endoscopic and histological mucosa of the ileal pouch in patients with UC and FAP, in order to find any abnormality in this pathway in asymptomatic patients, which may contribute to pouchitis. Patients and Method: Twelve patients (six with UC and six with FAP) who underwent to total rectocolectomy and "J" pouch reconstruction, were studied. Biopsies were obtained from the mucosa of the pouch by endoscopy. Normal ileum biopsies were obtained from six patients submitted to ileocolonoscopy with no abnormalities. The specimens were snap-frozen and the expressions of TLR2, TLR4 (bacterial recognition receptors) and JNK (nuclear signalization factor) were determined by immunoblot protein extract. The absence of pouchitis was assessed by clinical, histological and endoscopic parameters, according to the Pouchitis Disease Activity Index (PDAI). The patients were not under any medication. The committee approved the study and informed consent was signed by all participants. The ANOVA and Tukey-Kramer Tests were applied for statistical analysis. The level of significance was p<0.05. Results: Patients with UC had significantly higher protein levels of TLR4 than controls and FAP (p<0.05). The expressions of TLR2 and JNK were similar in the groups (p>0.05). Conclusion: Patients with UC had higher levels of TLR4, even in the absence of clinical, endoscopic and histological pouchitis. These findings may explain a tendency towards the up-regulation of intracellular pathways activated by bacterial antigens in UC patients, which could contribute to the production of proinflammatory mediators, being coadjuvant of the inflammatory process in the ileal pouch mucosa / Mestrado / Fisiopatologia Cirúrgica / Mestre em Ciências

Class of 2015 Abstract / Objectives: Two characterized peptide substrates were assayed with human Polo-like kinase 4 to determine phosphorylation activity. A pilot library of Type-II kinase inhibitors designed to fit into the ATP-binding pocket will be screened to determine HsPlk4 inhibition activity, which will help characterize a novel drug compound. Methods: Two peptide substrates of varying concentrations (2 uM, 1 uM, and 0.5 uM) were each combined with serial dilutions of HsPlk4 (1.25 uM, 0.625 uM, 0.313 uM, 0.156 uM, 0.078 uM, and 0.039 uM). EZ Reader detected phosphorylation activity by measuring fluorescence of both substrate and product, which separated at respective time points based on electrophoresis. The subsequent part of the experiment will be to inhibit the kinase activity with molecular inhibitors. Results: The results showed HsPlk4 activity with the modified PLKtide, (5FAM)KKKTPSDSLYDDGLSKK(CONH2). All reactions with the various concentrations of substrate 1 and HsPlk4 showed phosphorylation activity. The reaction started within the first 10 minutes, quickly reaching maximal phosphorylation of substrate. No p-values were calculated due to lack of data. Conclusions: No overall conclusions can be drawn based on the current results. Results showed the reaction reached its saturation point, so methods need to be refined to obtain data within the first 10 minutes. HsPlk4 phosphorylation of PLKtide confirmed the presumption that PLK family is a conserved family of Ser/Thr kinases. There are practical limitations for obtaining good kinetics data depicting enzyme activity, such as having EZ Reader quickly sample the reaction.

Magister Artium - MA / Dawes (2011) claims that there are more than 5.2 million children who were in both institutional and foster care in South Africa by 2011, which was increasing by 6.2% every year due to the HIV epidemic and high levels of poverty affecting the community. In the light of the large number of children in foster care, this study investigates the family-like roles displayed by caregivers and experienced by children within three selected Children's homes in Cape Town, South Africa. In this study, the term "family-like roles" was used interchangeably with "family values" displayed or instilled by caregivers within the children's home. Relatively a number of studies in child care have advocated for the benefit of family-like roles in children’s homes. The study established the importance of family-like roles, and it is evident that children's homes with a large group of children often prevent the creation of a nurturing environment with the characteristics of a family. The study employed interviews and observations to collect data, which were analysed according to the research questions by making codes and themes. The study involved the caregivers, children and managers of the three selected children's homes from Khayelitsha Township, Sothern and Northern suburbs of Cape Town. Furthermore, data were examined from both structural functionalism and eco cultural perspective, which helped to understand that caregivers display or instill the family values in a formal rather than in an informal way. This is because of the structure and functioning of the institutions of care, which makes the caregivers not to act naturally when taking care or displaying the family-like roles for vulnerable children to experience. Despite the negative declarations made in various scholars' studies about children’s homes, this study confirms the opinion of the caregivers that if the children living in the children's homes are shown respect, love, care, trust and kind treatment, they are able to extend these values to other people around them. Yet, this is not realised should the houses-units/cluster or cottages be overcrowded. In addition, the family-like roles displayed by caregivers in these particular children's homes may lead the children to develop a certain kind of phobia. This is a situation where the vulnerable children may develop a total fear and anxiety of certain social conditions in the communities

The induction of an innate immune response upon infection is dependent on the detection of the invading organism and the generation of a signalling cascade leading to the production of inflammatory mediators. Toll-like receptors are expressed on multiple cell types and induce the activation of a complex network of signalling pathways containing numerous branches with multiple interactions and cross-talk between the different branches. The TLR system is integral to the generation of a protective immune response and as such is an important target for pathogen-associated modulation. Many bacterial and viral pathogens employ strategies for interrupting or modulating TLR signalling to evade the host immune response. The obligate intracellular bacterial pathogen, F. tularensis, successfully invades and replicates within immune and epithelial cells. However, despite significant research the exact mechanisms used by this pathogen to successfully evade the host immune response remain elusive. To establish the exact signalling events that occur within a host upon infection with F. tularensis, the activation of specific signalling proteins was characterised using in vitro and in vivo models. The MAPKs, ERK and p38, were identified as critical in generating the host response. Furthermore, the temporal regulation of these signalling proteins was found to be bi-phasic with an early transient activation of both ERK and p38 followed by a sustained activation of ERK and a suppression of p38 activation at later time points. The role of ERK was investigated further using a specific inhibitor (PD0325901). Although there was no decrease in bacterial burdens in vitro and no increase in survival in mice treated with PD0325901, the inhibition of ERK activation reduced the secretion of TNF and IL-6 and reduced systemic bacterial proliferation in vivo. The induction of immune signalling cascades requires the activation of one or more receptors. The contribution of TLR2, TLR4 and TLR9 to the immune response to F. tularensis infection was examined using KO cell lines and specific antagonists. TLR2 was confirmed as a receptor for F. tularensis and was observed to play a role in the translational regulation of TNF. A role for TLR4 was also identified and further characterisation identified a potential priming relationship with TLR9. Sub-stimulation of 13 TLR4 by LPS enhanced the response induced by a subsequent stimulation of TLR9 by purified F. tularensis DNA. Overall, this study has provided evidence that, during infection, F. tularensis interacts with innate immune signalling pathways. By simultaneously suppressing p38 activation and prolonging ERK activation F. tularensis is able to regulate cytokine secretion and the induction of host-cell death mechanisms. Furthermore, this work has demonstrated that the activation of TLR9 by F. tularensis genomic DNA can be primed by a prior sub-stimulation of TLR4, although more research is required to fully understand the contribution of this interaction to the pathogenesis of F. tularensis.