by Tang Yan Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 156-168). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.V / ABSTRACT (CHINESE) --- p.VII / TABLE OF CONTENT --- p.1 / GENERAL INTRODUCTION --- p.4 / Chapter 1.1. --- Immunotherapy in Cancer --- p.4 / Chapter 1.1.2. --- Cytokines used in Immunotherapy in Cancer --- p.5 / Chapter 1.1.2.1. --- Interferons (IFNs) --- p.6 / Chapter 1.1.2.2. --- interleukins (ILs) --- p.11 / Chapter 1.1.2.3. --- Tumor Necrosis Factor-Alpha (TNF-α) --- p.14 / Chapter 1.2. --- Immunomodulatory and Anti-tumor Activities of Plant Polysaccharides --- p.17 / Chapter 1.3. --- Previous Studies on the Capsular Polysaccharides (CPS) from Klebsiella pneumoniae --- p.21 / Chapter 1.4. --- Activation of TNF-α Production from Macrophages by Bacterial Polysaccharides --- p.26 / Chapter 1.5. --- Chemotherapy of Cancer --- p.29 / Chapter 1.5.1. --- Types of Chemotherapeutic Drugs --- p.29 / Chapter 1.5.1.1. --- Alkylating Agents --- p.29 / Chapter 1.5.1.2. --- Plant Alkaloids --- p.31 / Chapter 1.5.1.3. --- Anti-metabolites --- p.32 / Chapter 1. 5.1.4. --- Antitumor Antibiotics --- p.32 / Chapter 1.5.2. --- Chemotherapeutic Drugs in this Project --- p.34 / Chapter 1.5.2.1. --- actinomycin D (ACT D) --- p.34 / Chapter 1.5.2.2. --- Cyclophosphamide (CY) --- p.35 / Chapter 1.5.2.3. --- Doxorubicin (Dox) --- p.37 / Chapter CHAPTER TWO --- AIM AND SCOPE OF THIS DISSERTATION --- p.40 / Chapter CHAPTER THREE --- MATERIALS AND METHODS --- p.42 / Chapter 3.1 --- MATERIALS --- p.42 / Chapter 3.1.1. --- Animals --- p.42 / Chapter 3.1.2. --- Klebsiella pneumoniae Serotype 1 and Serotype 24 --- p.42 / Chapter 3.1.3. --- Cell lines --- p.43 / Chapter 3.1.4. --- "Buffers, culture media and chemicals" --- p.43 / "POLYCLONAL RABBIT ANTI-ACTIVE ERK1/2, ANTI-ACTIVE JNK 1/2 AND ANTI-ACTIVE P38WERE FROM PROMEGA. POLYCLONAL RABBIT ANTI-TOTAL JNK1, ANTI-TOTAL P38WERE FROM SANTA CRUZ. MONOCLONAL ANTI-MOUSE TOTAL ERK1 AND POLYCLONAL ANTI-RABBIT TOTAL ERK2 WERE PURCHASED FROM ZYMED" --- p.50 / Chapter 3.2. --- METHODS --- p.51 / Chapter 3.2.1. --- "Extraction, Purification and Characterization of K1 and K24 Capsular Polysaccharides (CPS) from Klebsiella pneumoniae" --- p.51 / Chapter 3.2.1.1. --- Extraction and Purification of K1 and K24 Klebsiella Capsular Polysaccharides (CPS) --- p.51 / Chapter 3.2.1.2. --- Gel filtration of K1 and K24 Klebsiella Capsular Polysaccharides --- p.52 / Chapter 3.2.1.3. --- Characterization of K1 and K24 Capsular Polysaccharides --- p.52 / Chapter 3.2.3. --- Assay for Macrophage Activating Activities of Klebsiella Capsular Polysaccharides --- p.55 / Chapter 3.2.3.1. --- Tumour Necrosis Factor-α (TNF-α) Production by Peritoneal Macrophages --- p.55 / Chapter 3.2.3.2. --- Effect of Capsular Polysaccharides on the activities of MAPK family in murine macrophages --- p.56 / Chapter 3.2.4. --- Assay for Anti-Tumor Activities of Klebsiella Capsular Polysaccharides --- p.63 / Chapter 3.2.4.1. --- Assay of in vivo Suppression of EAT growth by K1 and K24 Capsular Polysaccharides --- p.63 / Chapter 3.2.4.2. --- Assay of the Effect of CPS on the Survival of EAT-Bearing Mice --- p.64 / Chapter 3.2.5. --- "Assay for Anti-Tumor Activities of Combined Treatment of Klebsiella Capsular Polysaccharides and Chemotherapeutic Drugs (Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox)" --- p.65 / Chapter 3.2.5.1. --- "Assay of the Cytotoxic Effect of Chemotherapeutic Drugs： Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox), on wehi-164 cell line" --- p.65 / Chapter 3.2.5.2. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on WEHI-164 cell line in vitro" --- p.66 / Chapter 3.2.5.3. --- Assay for Anti-Tumor Effect of Chemotherapeutic Drugs (Act D，CY and Dox) on EAT-bearing Mice in vivo --- p.67 / Chapter 3.2.5.4. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on EAT-bearing Mice in vivo" --- p.68 / Chapter 3.2.5.5. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the survival of eat-bearing mice in vivo" --- p.72 / Chapter CHAPTER FOUR --- "EXTRACTION, PURIFICATION & CHARACTERIZATION OF KLEBSIELLA K1 & K24 CAPSULAR POLYSACCHARIDES" --- p.76 / Chapter 4.1. --- Extraction and Purification of K1 and K24 Capsular Polysaccharides from Klebsiella pneumoniae Serotype 1 and Serotype 24 --- p.76 / Chapter 4.2. --- Gel Filtration Chromatography of K1 and K24 Capsular Polysaccharides --- p.77 / Chapter 4.3. --- Characterization of K1 and K24 Capsular Polysaccharides --- p.81 / Chapter CHAPTER FIVE --- EFFECT OF THE K1 & K24 CAPSULAR POLYSACCHARIDES ON THE MACROPHAGE ACTIVITIES --- p.83 / Chapter 5.1. --- Effect ofK1 and K24 Capsular Polysaccharides on the in vitro induction of TNF- α Production of Murine Peritoneal Macrophages --- p.83 / Chapter 5.2. --- Effect of Capsular Polysaccharides on the activities of MAPK family in murine macrophages --- p.87 / Chapter CHAPTER SIX --- ANTI-TUMOR ACTIVITIES ON MURINE TUMOR CELL LINES OF KLEBSIELLA K1 &K24 CAPSULAR POLYSACCHARIDES --- p.94 / Chapter 6.1. --- Effect of K1 and K24 Capsular Polysaccharides on the In vivo Anti-tumor Activities on EAT-bearing Mice --- p.94 / Chapter 6.2. --- In vivo Anti-tumor Effect on the Survival of EAT-Bearing Mice by Treatment of K1 and K24 CPS --- p.97 / Chapter CHAPTER SEVEN --- "ANTI-TUMOR ACTIVITIES OF COMBINED TREATMENT OF KLEBSIELLA K1 &K24 CAPSULAR POLYSACCHARIDES AND CHEMOTHERAPEUTIC DRUGS: ACTINOMYCIN D, CYCLOPHOSPHAMIDE AND DOXORUBICIN" --- p.100 / Chapter 7.1. --- "Cytotoxic Effect Chemotherapeutic Drugs： Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox), on WEHI-164 cell line" --- p.100 / Chapter 7.2. --- "Cytotoxic Effect of the Combined Treatment of the CPS and the Chemotherapeutic Drugs (Act D, CY and Dox), on WEHI-164 cell line" --- p.104 / Chapter 7.3. --- "Anti-Tumor Effect of Chemotherapeutic Drugs (Act D, CY and Dox) on EAT-bearing Mice in vivo" --- p.111 / Chapter 7.4. --- "Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the EAT Growth in vivo" --- p.115 / Chapter 7.5. --- "Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the Survival of EAT-bearing Mice in vivo" --- p.122 / Chapter CHAPTER EIGHT --- GENERAL DISCUSSION --- p.135 / Chapter 8.1. --- "Extraction, Purification and Characterization of K1 and K24 Capsular Polysaccharides from Klebsiella pneumoniae Serotype 1 and Serotype 24" --- p.135 / Chapter 8.2. --- Effect of K1 and K24 Capsular Polysaccharides on the Macrophage Activation --- p.140 / Chapter 8.3. --- Anti-tumor Activities on Murine Tumor Cell LInes of Klebsiella K1 and K24 Capsular Polysaccharides --- p.145 / Chapter 8.4. --- "Anti-tumor Activities of Combined Treatment of K1 and K24 Capsular Polysaccharides and Chemotherapeutic Drugs： Actinomycin D, Cyclophosphamide and Doxorubicin" --- p.148 / Chapter CHAPTER NINE --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.152 / BIBLIOGRAPHY --- p.156

INTRODUÇÃO: O Pênfigo Vulgar é uma doença bolhosa crônica que acomente pele e mucosas. A perda de adesão epitelial ocorre por agressão autoimune às desmogleínas presentes nos desmossomos, mediada por anticorpos IgG. Estudos sobre a gênese da autoimunidade no pênfigo indicam associação entre alelos do sistema HLA, especialmente dos loci DR e DQ. A população brasileira apresenta características favoráveis a estudos exploratórios em genética decorrente de sua origem mista e intensa miscigenação. PACIENTES E MÉTODO: O grupo em estudo incluiu trinta e seis pacientes não consanguíneos com diagnóstico de Pênfigo Vulgar comprovado por imunopatologia provenientes do estado de São Paulo, Brasil. Foram tipados para os loci HLA-A, HLA-B e HLA-DR utilizando-se oligonucleotídeos sequência-específica (PCR-SSO). As frequências alélicas e fenotípicas encontradas foram comparadas com as de um grupo controle composto de dados de 712 indivíduos doadores voluntários cadastrados no Registro Nacional de Doadores de Medula Óssea (REDOME) provenientes de São Paulo e tipados pelo mesmo método. O valor de P crítico foi corrigido utilizando-se o método False Discovery Rate. RESULTADOS: Os alelos HLA-DRB1*04:02, DRB1*08:04 e DRB1*14 estiveram associados à doença com riscos relativos de 44,6, 18,6 e 4,8, respectivamente (p<0,001). Não houve diferença estatisticamente significante entre as frequências de nenhum alelo dos loci HLA-A ou HLA-B entre os grupos. DISCUSSÃO: O alelo DRB1*04:02, diretamente, e o alelo DRB1*14, indiretamente por desequilíbrio de ligação com DQB1*05:03, estão associados com Pênfigo Vulgar em diversas populações ao redor do mundo, porém nenhum estudo semelhante observou associação com o alelo DRB1*08:04 em tamanha magnitude. Acreditamos que as associações encontradas em nosso estudo não sejam decorrentes de viés de estratificação populacional. É necessária, no entanto, a tipagem de loci adjascentes ao HLA-DR dos indivíduos do grupo em estudo para diferenciar se o risco à doença é inerente a estes alelos ou a algum outro nas proximidades, com o qual estariam em desequilíbrio de ligação. CONCLUSÕES: Os alelos HLA-DRB1*04:02, DRB1*08:04 e DRB1*14 estiveram associados ao Pênfigo Vulgar em pacientes brasileiros. / BACKGROUND: Pemphigus vulgaris is a chronic blistering disease affecting skin and mucous membranes. Autoimmune aggression to desmoglein in desmosomes, mediated by IgG antibodies, leads to loss of epithelial cell adhesion. Studies indicate association between some alleles of the HLA system and pemphigus vulgaris, mainly at the DR and DQ loci. Brazilian population characteristics are conducive to genetic exploratory studies because of its various origins and intense ethnically admixture. PATIENTS AND METHODS: The study group consisted of thirty-six unrelated patients with clinical and immunopathological diagnosis of pemphigus vulgaris from a tertiary hospital in Sao Paulo - Brazil. HLA allele typing at the A, B and DR loci was performed after DNA extraction using polymerase chain reaction and sequence-specific oligonucleotide probes (PCR-SSO). Allele and phenotypic frequencies were compared to those from a control group composed by 712 individuals volunteer donors registered in a national registry of bone marrow donors (REDOME) from Sao Paulo, typed using the same method. False Discovery Rate method was used to adjust level of critical P values. RESULTS: The HLADRB1* 04:02, DRB1*08:04 and DRB1*14 were associated with pemphigus vulgaris with relative risks of 44.6, 18.6 and 4.8, respectively (p <0.001). There was no significant difference between the frequencies of any allele of loci HLAA or HLA-B among the groups. DISCUSSION: The alleles DRB1*04:02 and DRB1*14 (indirectly through linkage disequilibrium with the DQB1*05:03) are associated with pemphigus vulgaris in several populations worldwide, however, no similar study reported such magnitude of association between pemphigus vulgaris and DRB1*08:04 allele. We consider that the association is not secondary to population stratification bias. HLA typing of nearby loci is required to differentiate if the association with pemphigus vulgaris is inherent to the HLA-DRB1*08:04 allele or to another gene which is in linkage disequilibrium. CONCLUSIONS: The HLA-DRB1*04:02, DRB1*08:04 and DRB1*14 were associated with pemphigus vulgaris in Brazilian patients

Introdução e Objetivos: A maior prevalência de doença celíaca (DC) em indivíduos com diabetes mellitus tipo I (DM1) já é reconhecida. Ambas as doenças tem causa autoimune, em que os genes HLA classe 2 representam o principal fator genético de risco. Porém, existe uma considerável parcela da população que não manifesta tais doenças e são portadores desses genes. Estudo de associação genômica (GWAS) identificaram polimorfismos de susceptibilidade às duas doenças em genes diferentes do sistema HLA, que poderão auxiliar na compreensão da causa e das suas variabilidades clínicas. Os objetivos desse estudo foram avaliar as frequências dos polimorfismos HLA e não HLA em pessoas como DM1 e com DC e relacionar esses dados com a ocorrência de sintomas gastrointestinais, com a idade do diagnóstico da DM1 e com história alimentar. Métodos: Delineamento transversal, com avaliações retrospectivas e prospectivas, em pessoas com DM1 com e sem DC. Foram realizadas entrevista e revisão de prontuário dos pessoas, seguido de coleta de sangue ou saliva. A pesquisa dos genes RGS1, IL2-IL21, BACH2, TLR7/TLR8 e IL18RAP foi realizada por PCR Real-Time. Os alelos DQA1* 0501 e DQB1* 0201 para DQ2.5 e o alelo DQB1*0302 para DQ8 foram identificados a partir da técnica de genotipagem de HLA Tag-single-nuleotide polymorphism (Tag SNP). Resultados: As frequências alélicas e genotípicas entre 273 pessoas com DM1 sem DC e 39 pessoas com DM1 e DC não apresentaram diferença significativa. A presença de sintoma gastrointestinal foi mais frequente nos portadores dos polimorfismos dos genes RGS1 e IL18RAP. O tempo de aleitamento materno, a idade de introdução do glúten e a idade do diagnóstico da DM1 foram semelhantes entre os grupos. A comparação dos cinco polimorfismos com a combinação dos haplótipos para DQ2.5 e DQ8 não apresentou diferença significativa. Nos 312 indivíduos, com DM1 com e sem DC e nos 66 indivíduos portadores de DC sem DM1 foi identificado alelos DQ2.5 e ou DQ8 em 97% dos casos, enquanto que nos indivíduos com DC sem DM1 identificou-se em 76% dos casos. DQ2.5 foi mais frequente entre pessoascom DC e DQ8 foi mais frequentes entre pessoas com DM1. Conclusões: A presença dos polimorfismos dos genes estudados não modificou a chance do indivíduo com DM1 ter ou não DC. Houve associação dos genes RGS1 e IL18RAP com sintomas gastrointestinais. A pesquisa dos alelos DQ2.5 e DQ8, pela técnica Tag-SNP, permitiu determinar um alto valor preditivo negativo no diagnóstico de DC na população com DM1 e com DC, semelhante ao descrito na literatura com a técnica convencional. / Introduction and Objectives: The higher prevalence of celiac disease (CD) in individuals with diabetes mellitus type I (T1D) is already recognized. Both diseases have autoimmune cause, where HLA genes class 2 represent the major genetic risk factor. However, there is a considerable portion of the population that does not manifest such diseases and are carriers of these genes. Genome-wide association studies (GWAS) have identified susceptibility polymorphisms to both diseases in different genes of the HLA system that may assist in understanding the etiology and in its clinical variabilities. The objectives of this study were to evaluate the frequencies of HLA and non-HLA polymorphisms in patients with T1D and CD, related to the occurrence of gastrointestinal symptoms, the age of diagnosis of T1D and food history. Methods: Mixed design with retrospective and prospective evaluations in patients with T1D with and without DC. They were conducted interview and review of medical records of patients, followed by collecting blood or saliva. The search for genes RGS1, IL21-IL2, BACH2, TLR7 / TLR8 and IL18RAP was performed by Real-Time PCR. The alleles DQA1 * 0501 and DQB1 * 0201 for DQ2.5 and DQB1 * 0302 for DQ8 were identified from the Tag-single-nucleotide polymorphism (tag SNP) genotyping HLA technique Results: The allelic and genotypic frequencies between 273 T1D patients without CD and 39 patients with T1D and CD showed no significant difference. The presence of gastrointestinal symptoms were more frequent in patients with polymorphisms of genes RGS1 and IL18RAP. The duration of breastfeeding, the age of introduction of gluten and the age of diagnosis of T1D were similar between the groups. The comparison of the five polymorphisms with the combination of haplotypes for DQ2.5 and DQ8 showed no significant difference. In 312 individuals with DM1 with and without CD and 66 individuals with CD without T1D was identified alleles DQ2.5 and/or DQ8 in 97% of cases, whereas in individuals with CD without T1D was identified in 76% of cases . DQ2.5 was more frequent among patients with CD and DQ8 was more frequent among patients with T1D Conclusions: The presence of polymorphisms of genes studied did not modify the chance of T1D whether or not DC. There was an association of RGS1 and IL18RAP genes with gastrointestinal symptoms. The survey of DQ2.5 and DQ8 alleles by Tag-SNP technique allowed determining a high negative predictive value in the diagnosis of CD in the population of patients with T1D and DC, similar to that described in the literature with the conventional technique.

Bacillus subtilis é uma alternativa ao emprego de Escherichia coli para a produção de proteínas recombinantes. O principal entrave à utilização de B.subtilis para esse fim é a baixa disponibilidade de sistemas de expressão. Nesse trabalho, testamos diferentes plasmídeos e promotores com o objetivo de desenvolver sistemas de expressão heteróloga eficientes. Ao fim do trabalho, propomos dois novos sistemas de expressão baseados do arcabouço do plasmídeo pMTL500E e nos promotores dos genes cdd e gsiB, ambos de B.subtilis. Os dois plasmídeos construídos apresentam expressão constitutiva e demonstraram desempenho superior no tocante à produção de proteínas heterólogas quando comparados ao único sistema comercialmente disponível, conhecido como pHT01. Em uma segunda parte do trabalho, propomos a utilização de Listeria innocua como veículo de entrega para antígenos vacinais. Por meio de ensaios ex-vivo e in vivo, demonstramos que essa bactéria possui potencial promissor para aplicações vacinais, inclusive quando comparada ao bem estabelecido B.subtilis. / Bacillus subtilis is an alternative to the use of Escherichia coli for the production of recombinant proteins. The main bottleneck to the use of B. subtilis for this purpose is the low availability of expression systems. In this study, we evaluated different plasmids and promoters with the aim of developing efficient heterologous expression systems. At the end of this work, we propose two new expression systems based on plasmid pMTL500E and the promoters from cdd and gsiB genes, both of them from B.subtilis. The two plasmids constructed exhibit constitutive expression and demonstrated superior performance regarding the production of heterologous proteins compared to the unique commercially available system, which is known as pHT01. In a second part of the work, we propose the use of Listeria innocua as a delivery vehicle for vaccine antigens. After ex-vivo and in-vivo experiments, we demonstrated that this bacterium has a promising potential for vaccine applications, even when compared to well established B.subtilis.

O objetivo deste estudo consistiu em avaliar a expressão imuno-histoquímica da podoplanina e do CD44v6 pelas células malignas, verificando a associação destas proteínas com as variáveis clínicas, microscópicas, com o índice histopatológico de malignidade e com a sobrevivência livre de doença de 91 pacientes portadores de carcinomas espinocelulares (CEC) de lábio inferior, tratados no Centro de Tratamento e Pesquisa do Hospital do Câncer A.C.Camargo, São Paulo. Os tumores foram corados, separadamente, com os anticorpos anti-podoplanina e anti-CD44v6, sendo avaliada a imunoexpressão destas proteínas pelas células neoplásicas, no front de invasão tumoral, por meio de um método semi-quantitativo de escores. A associação da expressão da podoplanina e do CD44v6 com as variáveis demográficas, clínicas e microscópicas foi feita pelo teste do qui-quadrado ou exato de Fisher. As taxas de sobrevivência livre de doença, acumuladas em cinco e dez anos, foram calculadas pelo teste de Kaplan-Meier e a influência das variáveis clínicas e microscópicas no prognóstico avaliadas pelo modelo de regressão de Cox. A correlação entre a podoplanina e o CD44v6 foi analisada pelo teste de Spearman. Em todos os testes estatísticos utilizou-se um nível de significância de 5%. Os resultados mostraram uma predominância da forte expressão membranosa e citoplasmática da podoplanina pelas células malignas. Verificou-se uma associação significativa da podoplanina citoplasmática com a recidiva locorregional (p=0,028) e da podoplanina membranosa com o índice histopatológico de malignidade tumoral (p=0,026). O CD44v6 foi fortemente expresso pelas células neoplásicas de 95,4% dos CECs e significativamente, associado com o estadiamento clínico T (p=0,034). Não houve correlação entre a podoplanina e o CD44v6 nos CECs de lábio inferior. A forte expressão de podoplanina membranosa (p=0,016) e citoplasmática (p=0,030) pelas células malignas foi fator de prognóstico favorável independente na sobrevivência livre de doença. Concluímos que a podoplanina e o CD44v6 são fortemente expressos pelas células neoplásicas e que a forte imunoexpressão membranosa e citoplasmática da podoplanina pode auxiliar na identificação do risco de recidiva locorregional nos pacientes portadores de carcinoma espinocelular de lábio inferior. / The aim of this study was evalute the podoplanin and CD44v6 immunohistochemical expression by malignant cells and its association with the clinical and microscopic variables, tumor histopathological grading and disease-free survival of 91 patients with lip squamous cell carcinomas (SCC), submitted to surgical treatment at Research and Treatment Center of the Cancer Hospital A.C. Camargo, São Paulo. The tumors were stained separately, with the antibodies anti-podoplanin and anti-CD44v6, and the immunoexpression of these proteins, by the neoplastic cells in the invasion front, was evaluated by a semi-quantitative scores method. Chi-square test or Fishers exact test was used to analyze the association of podoplanin and CD44v6 expression with demographic, clinical, and microscopic variables. Disease-free survival in five and ten years, were calculated by the Kaplan-Meier method and the influence of clinical and microscopic variables on prognosis were evaluated by the Cox regression model. The correlation between podoplanin and CD44v6 expression was analyzed by Spearman\'s test and a significance level of 5% was used in all statistical tests. The results showed a predominance of strong membranous and cytoplasmic podoplanin expression by malignant cells. An association between cytoplasmic podoplanin and locorregional recurrence (p=0,028) and membranous podoplanin with tumor histopathological grading (p=0,026). CD44v6 was strongly expressed in 95.4% of the SCCs neoplastic cells and significantly associated with the clinical staging T (p=0,034). There was no correlation between podoplanin and CD44v6 expression in the lower lip SCC. The strong expression of membranous (p=0.016) and cytoplasmic (p=0.030) podoplanin by malignant cells was a favorable independent prognostic factor in disease-free survival. Concluding, the podoplanin and CD44v6 are strongly expressed by neoplastic cells and the strong membranous and cytoplasmic immunoexpression of podoplanin can help the identification of locoregional recurrence risk in patients with squamous cell carcinoma of the lower lip.

A leptospirose é uma doença endêmica causada por Leptospiras. O genoma da Leptospira interrogans sorovar Copenhageni foi analisado para seleção de potenciais antígenos vacinais. Oito genes foram selecionados e clonados para expressão e purificação dos antígenos. A salmonela SL3261 foi usada como carregadora dos genes de leptospira em vetor pAEsox para expressão das proteínas in vivo. As salmonelas recombinantes induziram resposta imune quando administradas em camundongos por via intraperitoneal. Hamsters foram imunizados com as salmonelas, observando-se que a SLLIC10191 induziu proteção parcial no desafio com L. interrogans sorovar Pomona. Vetores híbridos foram construídos para expressão simultânea de dois antígenos em salmonelas in vivo. Observamos indução de anticorpos específicos, porém, os ensaios de desafio não foram conclusivos. Vários parâmetros do desafio com sorovar Copenhageni foram estudados, como contagem das bactérias e ajuste de dose, variação de virulência por passagens em cultivo e interferência da idade dos animais. / Leptospirosis is an endemic disease caused by Leptospira. The genome of Leptospira interrogans serovar Copenhageni was analyzed for screening potential vaccine antigens. Eight genes were selected and cloned for expression and purification. Salmonella SL3261 was used as carrier of the genes of leptospira in pAEsox vector for in vivo proteins expression of proteins in vivo. Recombinant Salmonella induced immune response when administered intraperitoneally in mice intraperitoneally. Hamsters were immunized with salmonella, resulting we observed that the SLLIC10191 induced partial protection against on challenge with L. interrogans serovar Pomona. Hybrid vectors were constructed for expression of two antigens simultaneously by salmonella in vivo. We observed induction of specific antibodies, however, the challenge tests were not conclusive. Several parameters of the challenge assay with serovar Copenhageni were studied, such as the counting of bacteria count and dose adjustment, changes in virulence by passages in culture and interference backgroundof from the age of animals.

A malária transmitida por transfusão (MTT), embora seja um evento raramente relatado em áreas não endêmicas ou de baixa endemicidade, representa um desafio devido à ocorrência de infecções assintomáticas e à possibilidade de sobrevivência de Plasmodium em hemácias estocadas por até três semanas a temperaturas entre 2 e 6ºC. No Estado de São Paulo, quatro casos de MTT foram relatados, incluindo uma morte. Os doadores infectados foram identificados como portadores assintomáticos que viviam ou haviam se deslocado para o bioma Mata Atlântica do estado. Nesses doadores, geralmente as densidades parasitárias são baixas e indetectáveis pela gota espessa ou testes rápidos. O uso de plataformas incluindo testes sorológicos poderia detectar doadores suspeitos de infecção por Plasmodium, minimizando a possibilidade de MTT. Nesse sentido, o objetivo deste estudo foi padronizar a produção de antígeno recombinante MSP119 de P. vivax, que é a espécie mais prevalente no Brasil, e validar o teste ELISA-PvMSP119, visando à aplicação em serviços hemoterápicos brasileiros de áreas não endêmicas para malária. O antígeno recombinante produzido em condições desnaturantes mostrou-se adequado para a produção em larga escala, pela facilidade de obtenção e purificação. Os resultados de estabilidade obtidos em três lotes-piloto indicaram validade de pelo menos 27 meses sem perda de reatividade. Além disso, o teste apresentou 96,95% de sensibilidade em 197 soros de pacientes com gota espessa positiva para P. vivax e especificidade de 100,00% utilizando soros de 101 indivíduos controles sadios e 99,26% quando considerados também 168 amostras de soro de pacientes com outras doenças. O coeficiente de variação das amostras positivas foi <= 3,8% para a repetitividade e <= 10,6% para a reprodutibilidade. Quanto à reatividade cruzada, obtiveram-se falsos resultados positivos com amostras de doença de Chagas (5,88%) e fator reumatoide (6,67%). Após a validação, avaliou-se a prevalência de anticorpos IgG anti-MSP119 de P. vivax entre doadores de sangue do Sudeste do Brasil, considerados aptos à doação, ensaiando 1.974 amostras de plasma de bancos de sangue, sendo 1.309 do Estado São Paulo (SP) e 665 do Estado do Rio de Janeiro (RJ). A positividade entre as amostras de SP foi 1,15% (N = 15) e entre as do RJ foi 1,65% (N = 11). O índice de reatividade (IR) das amostras positivas variou entre 8,98-1,16 (SP) e 13,03-1,08 (RJ). Em SP e RJ, maior positividade foi encontrada nos municípios que têm contato com o bioma Mata Atlântica e no RJ, também no bairro da Tijuca, onde se encontra a Floresta da Tijuca. A presença de anticorpos IgG anti-P. vivax não é necessariamente um marcador de parasitemia ou doença, porém aponta para o risco de MTT, mesmo em áreas de baixa endemicidade, pois doadores assintomáticos podem ser aceitos com base em triagem clínica. Estes achados constituem-se em alerta que nos impele a rever os critérios adotados para a seleção dos doadores, com o objetivo de reduzir o risco de MTT nessas áreas sem perder doações. / In non-endemic and low endemic areas, transfusion-transmitted malaria (TTM) is a rarely reported event, representing a major challenge, essentially due to the occurrence of asymptomatic infections and to the possibility of Plasmodium survival up to three weeks in stored red blood cells at temperatures between 2 and 6ºC. In São Paulo State, four TTM were detected, including one death. Infected donors were identified as asymptomatic carriers that lived or that had displacements to the Atlantic forest biome in the state. In these donors generally the parasite densities are low and undetectable in the thick blood smear or rapid diagnostic tests. The use of platforms including serological tests might point out donors suspected of harboring Plasmodium, minimizing the possibility of TTM. Accordingly, the aim of this study was to standardize the production of P. vivax MSP119 recombinant antigen, which is the most prevalent species in Brazil, and validate ELISA-PvMSP119, aiming at the application in Brazilian haemotherapic services in malaria non-endemic areas. The recombinant antigen produced under denaturing conditions was suitable for large-scale production, due to the ease of obtaining and purification. The results of stability obtained in three pilot batches indicated that it was valid for at least 27 months without loss of reactivity. Furthermore, the test showed 96.95% sensitivity in sera from 197 patients with positive thick-blood smear for P. vivax and 100.00% specificity in sera from 101 healthy controls, and 99.26% when considered also 168 samples from patients with other diseases. The variation coefficient of positive samples was <= 3.8% for repeatability and <=10.6% for reproducibility. For cross-reactivity, false positive results were obtained with Chagas\' disease (5.88%) and rheumatoid factor (6.67%) samples. After validation, we evaluated the prevalence of IgG anti-MSP119 antibodies to P. vivax among blood donors in Southeastern Brazil, considered suitable for donation, rehearsing 1,974 plasma samples from blood banks, being 1,309 from the State of São Paulo (SP) and 665 from the State of Rio de Janeiro (RJ). The positivity among samples from SP was 1.15% (N = 15) and in RJ was 1.65% (N = 11). The reactivity index (RI) of the positive samples ranged from 8.98 to 1.16 (SP) and from 13.03 to 1.08 (RJ). In SP and RJ, highest positivity was seen in Municipalities in contact to the Atlantic Forest biome, and in RJ, also in the Tijuca neighborhood, where there is the Tijuca Forest. The detection of IgG antibodies is not necessarily a marker of parasitemia or disease, however, points out to the risk of TTM, even in areas of low endemicity, since asymptomatic donors could be accepted based on clinical screening. These findings constitute an alert that impel us to review the adopted criteria for screening of the donors aiming to reduce the risk of TTM in these areas without losing donations.

Malaria affects over half of the world's population and causes half a million deaths annually, especially in Sub-Saharan Africa. Four species of the apicomplexan Plasmodium parasite (P. falciparum, P. ovale, P. malariae and P. vivax) are responsible for malaria in Africa. Both parasite and host factors contribute to heterogeneity in the risk of developing malaria, clinical manifestation of the disease as well as the number of treatments required to clear parasites. The epidemiology of the different species, and the role of exposure to mixed-species Plasmodium co-infections in generating heterogeneity remains poorly studied. Being an obligate intracellular parasite the blood-stage life cycle of the Plasmodium parasite takes place in the erythrocytes of the human host. The surfaces of these erythrocytes are the medically important ABO blood group antigens that have been reported to influence the susceptibility or otherwise of an individual developing severe malaria. In this thesis I have considered the contributions of the species of Plasmodium parasites and the ABO blood group of the host in driving heterogeneity in human malaria. The aims of this thesis were to determine: (i) the seroepidemiology of the different Plasmodium species in two mesoendemic African populations (Zimbabwe and Sudan); (ii) to determine if heterogeneity in clinical presentations of malaria (history of fever, body temperature and parasitaemia) and response to drug treatment is related to exposure to single vs. mixed-Plasmodium species infection; (iii) the spatial and temporal dynamics of malaria prevalence and Plasmodium species distribution in a mesoendemic village in eastern Sudan; (iv) gene expression changes in 3D7 P. falciparum parasites as they infect erythrocytes of different ABO blood group donors. For aims (i to iii) I developed an enzyme-linked immunosorbent assay using antigens derived from Plasmodium merozoite surface protein 1, also known as MSP-119, to detect IgG antibodies to all four malaria parasite species in Zimbabwean and Sudanese populations. In the Zimbabwean study, plasma samples from 100 individuals each (aged 5-18 years) from three villages (Burma Valley, Mutoko and Chiredzi) were screened for exposure to Plasmodium parasites. In Daraweesh, Sudan, plasma samples from 333 individuals (aged 1-74 years) who had experienced a first malaria episode between 1990 and 2000 were recruited into the study. For study aim (iv) I cultured a single clone of 3D7 P. falciparum parasite using erythrocytes of individuals of different ABO blood group types, harvested parasite RNA and sequenced it to determine gene expression changes in the different hosts. I showed that human IgG antibodies to MSP-119 antigens of the four Plasmodium species are species-specific and do not cross-react. In both study populations almost all antibody responses involved P. falciparum, and single-species responses were almost exclusively directed against P. falciparum antigens. Mixed-species responses accounted for more than a third of responses, and were associated with chloroquine treatment failure, with significantly high proportion of individuals with mixed-species infections requiring repeated treatment with chloroquine/sulfadoxine-pyrimethamine for parasite clearance. This finding highlights the need for a sensitive method for detecting mixed-species malaria infections to enable the assessment of the true prevalence and magnitude of the disease burden caused by the non-falciparum species in endemic populations. Drug treatment failures associated with mixed species infections have significant impact on malaria morbidity and mortality. Treatment failure or partial parasite clearance has the potential to allow dormant liver stages of P. vivax and P. ovale to become a source of parasite reservoir for onward transmission. Furthermore, untreated low-grade chronic infections caused by P. malariae have been reported to cause systemic diseases many years after the primary infection. Spatial analysis of malaria epidemiology showed that malaria parasite transmission in Daraweesh was focal, and that infections are not randomly distributed in the village. Two space-time clusters of significantly increased malaria risk were identified (1993- 1999, and 1998-1999) with marked variations between households, but little or no variation in the species of Plasmodium over time. Similarly, multiple significant clusters were identified for the parasite species; three for P. falciparum, two for P. vivax and P. malariae, and one for P. ovale. These clusters had overlapping time frames, with some of the species significantly infecting the same households. This suggests that even in a small geographic area malaria transmission shows heterogeneity, and that such data can provide useful information to guide malaria control efforts. Finally, I demonstrated that 3D7 P. falciparum parasite growth was similar in the erythrocytes of different blood group donors, and provide preliminary data to show that the non-coding RNA gene, PF3D7_1370800, is differentially expressed in blood group A donors relative to blood groups B and O donors. Further research is needed to better understand the role of this gene in malaria pathology. All together, these findings will aid malaria researchers and other stakeholders in making informed choices about tools for diagnosing Plasmodium species, and control programmes targeting eradication of malaria caused by all Plasmodium species, as is the case of incorporating these findings into current malaria research in Sudan.

INTRODUÇÃO: O desenvolvimento das glândulas salivares envolve um processo coordenado de interações moleculares complexas, nas quais as integrinas têm papel fundamental. As integrinas são uma família de receptores transmembrânicos, heterodímeros, compostos por duas subunidades: alfa e beta que estão ligadas de forma não covalente e dependentes de cations bivalentes. Estes heterodímeros medeiam sinais intra e extracelulares envolvidos na organização das células em tecidos e órgãos durante seu desenvolvimento. Em particular a integrina beta1 está envolvida na proliferação e diferenciação de células no desenvolvimento dos tecidos epiteliais. OBJETIVOS: Para compreender o papel da integrina beta1 no desenvolvimento das glândulas salivares humanas, estudamos a sua expressão por meio da hibridização in situ em tecido fetal humano em desenvolvimento. MATERIAL E MÉTODOS: A hibridização in situ com o método chromogenic in situ hibridization (CISH) foi utilizada para investigar a expressão genética da integrina beta1 em espécimes de glândulas salivares em desenvolvimento derivados de 30 fetos humanos em vários estágios gestacionais (de 825 semanas de vida intrauterina). Os resultados obtidos com a hibridização in situ foram relacionados com os aspectos morfológicos das glândulas salivares em desenvolvimento em cada fase da evolução da morfogênese glandular, por meio de análise qualitativa. RESULTADOS: Expressão da integrina beta1 foi detectada em raras células na fase prebotão. A expressão dessa molécula foi detectada em mais estruturas com a evolução do desenvolvimento morfogenético das glândulas salivares. CONCLUSÕES: A expressão a integrina beta1 parece ser regulada de forma temporoespacial e é associada ao estabelecimento do fenótipo maduro das glândulas salivares / INTRODUCTION: Salivary gland development entails coordinated processes involving complex molecular interactions in which integrins have a fundamental role. The integrins are a family of heterodimeric transmembrane receptors comprising alpha and beta subunits that mediate intercellular and extracellular signals involved in the organisation of cells in tissues and organs during development. The beta1 integrin in particular have been implicated in proliferation and differentiation of cells involved in the development of epithelial tissues. OBJECTIVE: To understand the role of beta1 integrin in salivary gland development we have studied its expression in human foetal tissues. MATERIAL AND METHODS: In situ hybridisation CISH technique was used to compare the expression and localisation of integrin beta1 with differentiation markers in developing human salivary glands obtained from foetuses of 825 gestational weeks. RESULTS: Integrin beta1 first appeared during bud stage in a few cells and its distribution increased as salivary gland morphogenesis progressed. CONCLUSIONS: The developmentally regulated expression of integrin beta1 in association with the establishment of a mature phenotype of salivary glands is suggestive of its role in salivary gland morphogenesis

O objetivo deste estudo consistiu em avaliar a expressão imuno-histoquímica da podoplanina e do CD44v6 pelas células malignas, verificando a associação destas proteínas com as variáveis clínicas, microscópicas, com o índice histopatológico de malignidade e com a sobrevivência livre de doença de 91 pacientes portadores de carcinomas espinocelulares (CEC) de lábio inferior, tratados no Centro de Tratamento e Pesquisa do Hospital do Câncer A.C.Camargo, São Paulo. Os tumores foram corados, separadamente, com os anticorpos anti-podoplanina e anti-CD44v6, sendo avaliada a imunoexpressão destas proteínas pelas células neoplásicas, no front de invasão tumoral, por meio de um método semi-quantitativo de escores. A associação da expressão da podoplanina e do CD44v6 com as variáveis demográficas, clínicas e microscópicas foi feita pelo teste do qui-quadrado ou exato de Fisher. As taxas de sobrevivência livre de doença, acumuladas em cinco e dez anos, foram calculadas pelo teste de Kaplan-Meier e a influência das variáveis clínicas e microscópicas no prognóstico avaliadas pelo modelo de regressão de Cox. A correlação entre a podoplanina e o CD44v6 foi analisada pelo teste de Spearman. Em todos os testes estatísticos utilizou-se um nível de significância de 5%. Os resultados mostraram uma predominância da forte expressão membranosa e citoplasmática da podoplanina pelas células malignas. Verificou-se uma associação significativa da podoplanina citoplasmática com a recidiva locorregional (p=0,028) e da podoplanina membranosa com o índice histopatológico de malignidade tumoral (p=0,026). O CD44v6 foi fortemente expresso pelas células neoplásicas de 95,4% dos CECs e significativamente, associado com o estadiamento clínico T (p=0,034). Não houve correlação entre a podoplanina e o CD44v6 nos CECs de lábio inferior. A forte expressão de podoplanina membranosa (p=0,016) e citoplasmática (p=0,030) pelas células malignas foi fator de prognóstico favorável independente na sobrevivência livre de doença. Concluímos que a podoplanina e o CD44v6 são fortemente expressos pelas células neoplásicas e que a forte imunoexpressão membranosa e citoplasmática da podoplanina pode auxiliar na identificação do risco de recidiva locorregional nos pacientes portadores de carcinoma espinocelular de lábio inferior. / The aim of this study was evalute the podoplanin and CD44v6 immunohistochemical expression by malignant cells and its association with the clinical and microscopic variables, tumor histopathological grading and disease-free survival of 91 patients with lip squamous cell carcinomas (SCC), submitted to surgical treatment at Research and Treatment Center of the Cancer Hospital A.C. Camargo, São Paulo. The tumors were stained separately, with the antibodies anti-podoplanin and anti-CD44v6, and the immunoexpression of these proteins, by the neoplastic cells in the invasion front, was evaluated by a semi-quantitative scores method. Chi-square test or Fishers exact test was used to analyze the association of podoplanin and CD44v6 expression with demographic, clinical, and microscopic variables. Disease-free survival in five and ten years, were calculated by the Kaplan-Meier method and the influence of clinical and microscopic variables on prognosis were evaluated by the Cox regression model. The correlation between podoplanin and CD44v6 expression was analyzed by Spearman\'s test and a significance level of 5% was used in all statistical tests. The results showed a predominance of strong membranous and cytoplasmic podoplanin expression by malignant cells. An association between cytoplasmic podoplanin and locorregional recurrence (p=0,028) and membranous podoplanin with tumor histopathological grading (p=0,026). CD44v6 was strongly expressed in 95.4% of the SCCs neoplastic cells and significantly associated with the clinical staging T (p=0,034). There was no correlation between podoplanin and CD44v6 expression in the lower lip SCC. The strong expression of membranous (p=0.016) and cytoplasmic (p=0.030) podoplanin by malignant cells was a favorable independent prognostic factor in disease-free survival. Concluding, the podoplanin and CD44v6 are strongly expressed by neoplastic cells and the strong membranous and cytoplasmic immunoexpression of podoplanin can help the identification of locoregional recurrence risk in patients with squamous cell carcinoma of the lower lip.