Submitted by MARCELA PETROLINI CAPOBIANCO (mpcapobianco@yahoo.com.br) on 2017-04-24T11:43:11Z No. of bitstreams: 1 TESE MARCELA PETROLINI CAPOBIANCO.pdf: 3177156 bytes, checksum: 6b6af4a4ea15e5048723eabdb0659191 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-25T20:25:33Z (GMT) No. of bitstreams: 1 capobianco_mp_dr_sjrp.pdf: 3177156 bytes, checksum: 6b6af4a4ea15e5048723eabdb0659191 (MD5) / Made available in DSpace on 2017-04-25T20:25:33Z (GMT). No. of bitstreams: 1 capobianco_mp_dr_sjrp.pdf: 3177156 bytes, checksum: 6b6af4a4ea15e5048723eabdb0659191 (MD5) Previous issue date: 2017-04-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No Brasil o Plasmodium vivax é a espécie mais prevalente, responsável por aproximadamente 85% dos casos de malária. Ademais as variantes da proteína circumesporozoíta (CSP - VK210, VK247 e P. vivax-like) já foram identificadas em várias áreas endêmicas no país. Diversos estudos analisando a influência da variabilidade genética de receptores celulares e moléculas envolvidas na resposta imune, com diferentes peptídeos do Plasmodium, têm obtido resultados variáveis de acordo com o antígeno utilizado e a população analisada. Este trabalho apresenta resultados sobre o estudo de polimorfismos genéticos no TCR (T cell Receptor) e nas Interleucinas 1 e 2 em pacientes infectados por P. vivax provenientes do município de Goianésia do Pará, no Estado do Pará. Avaliou-se estes polimorfismos com a parasitemia do indivíduo, com os genótipos da CSP e com a resposta sorológica contra os peptídeos da CSP. Foram relacionados também estes polimorfismos com os níveis de citocinas. Os polimorfismos foram analisados por técnicas de PCR-RFLP e PCR alelo específico. As análises sorológicas da CSP foram realizadas por ELISA. Foram comparadas as frequências genotípicas observadas segundo o teorema de Hardy-Weinberg (HW). Os níveis de IL1 e IL2 foram avaliados por citometria de fluxo, seguindo protocolo descrito pelo fabricante. Associação dos polimorfismos com os níveis de interleucinas foi avaliada por Análise de variância. As frequências genotípicas e alélicas foram obtidas no programa R v 2.11.1. A parasitemia variou de 15 a 70.000, com mediana de 1.500 parasitos/mm3. Os SNPS investigados mostraram frequências variadas na amostra estudada. Todo o polimorfismo avaliado encontra-se em Equilíbrio de Hard Wenberg. Não houve diferença significativa da parasitemia em relação aos SNPs investigados. Infecções contendo apenas a variante VK247 foram as mais comuns e também não foi observado diferença significativa na resposta de anticorpos de acordo com a variante da CSP presente no momento da infecção. Correlações significativas entre os níveis destas interleucinas com a parasitemia e os níveis de anticorpos contra as variantes da CSP não foram observadas. Além disso, as variantes da CSP não influenciaram os níveis plasmáticos das citocinas e não houve associações positivas entre os SNPs das IL1 e IL2 e seus níveis plasmáticos. Os resultados poderão contribuir na identificação e participação efetiva de genes humanos na modulação da resposta imune, essenciais no estabelecimento de estratégias de imunização contra a doença, em área de transmissão ativa no Estado do Pará. / In Brazil, Plasmodium vivax is the most prevalent species responsible for approximately 85% of malaria cases. In addition, variants of the circosporozoite protein (CSP - VK210, VK247 and P. vivax - like) have already been identified in several endemic areas in the country. Several studies analyzing the influence of the genetic variability of cellular receptors and molecules involved in the immune response with different Plasmodium peptides have obtained variable results according to the antigen used and the analyzed population. This work presents results on the study of genetic polymorphisms in TCR (T cell Receptor) and in Interleukins 1 and 2 in patients infected by P. vivax from the city of Goianésia do Pará, in the State of Pará. These polymorphisms were evaluated with Parasitemia, CSP genotypes and serological response to CSP peptides. These polymorphisms were also related to cytokine levels. Polymorphisms were analyzed by PCR-RFLP and allele-specific PCR techniques. Serological tests of CSP were performed by ELISA. The genotypic frequencies observed according to the Hardy-Weinberg (HW) theorem were compared. Levels of IL1 and IL2 were evaluated by flow cytometry following the protocol described by the manufacturer. Association of polymorphisms with interleukin levels was evaluated by analysis of variance. The genotypic and allelic frequencies were obtained in program R v 2.11.1. The parasitemia ranged from 15 to 70,000, with a median of 1,500 parasites / mm3. The SNPS investigated showed varied frequencies in the sample studied. All polymorphism evaluated is in Hard Wenberg Equilibrium. There was no significant difference in parasitemia in relation to the investigated SNPs. Infections containing only the VK247 variant were the most common and also no significant difference in antibody response was observed according to the CSP variant present at the time of infection. Significant correlations between the levels of these interleukins with parasitemia and levels of antibodies against variants of CSP were not observed. In addition, the CSP variants did not influence plasma cytokine levels and there were no positive associations between IL1 and IL2 SNPs and their plasma levels. The results may contribute to the identification and effective participation of human genes in the modulation of the immune response, essential in the establishment of immunization strategies against the disease, in an active transmission area in the State of Pará.

A desnutrição é capaz de induzir diversas alterações metabólicas afetando marcadamente a composição corporal e o sistema imunológico. O exercício físico, por sua vez, produz alterações no organismo para uma melhor capacidade de adaptação a situações de estresse. O desvio da situação de homeostase produzida pelo exercício físico induz uma reorganização de seus mecanismos funcionais, principalmente dos mecanismos endócrinos e imunológicos. Ainda é pouco conhecida a influência do exercício sobre a desnutrição e também as conseqüências sobre o sistema imunológico quando as duas variáveis são combinadas. Assim, esse trabalho teve como objetivo avaliar os efeitos do exercício físico de endurance sobre ratos submetidos a um protocolo de desnutrição crônica. Avaliamos ratos Wistar machos, desnutridos por 16 semanas, divididos em 4 grupos: eutrófico sedentário (ES), eutrófico treinado (ET), desnutrido sedentário (DS), desnutrido treinado (DT). O treinamento físico foi realizado em esteira, por 10 semanas, 5 vezes por semana, com intensidade aproximada de 60- 65% do consumo máximo de oxigênio. Avaliou-se a composição corporal, através da aferição do peso corporal, peso dos tecidos muscular esquelético e adiposo, do fígado, do conteúdo de gordura e proteína na carcaça, e a concentração de leptina, ACTH, glicose, insulina, e glutamina no plasma. Avaliamos também, através de citometria de fluxo, os marcadores de superfície celular CD3 e CD4, bem como a celularidade no timo. O consumo máximo de oxigênio e o desempenho através de um teste até a exaustão também foram analisados. A análise estatística utilizada foi o teste de variância ANOVA two-way com pós teste de Bonferroni e, nível de significância adotado de p<0,05. Os resultados encontrados demonstraram que o treinamento de endurance em ratos submetidos à desnutrição crônica promoveu uma acentuada redução do peso e da adiposidade corporal; um aumento da massa muscular relativa ao peso corporal; um restabelecimento da glicemia aos valores normais; uma melhor relação da concentração insulina/glicose, sugerindo uma sensibilidade à insulina aumentada; um aumento dos estoques de glicogênio muscular; um maior consumo máximo de oxigênio; e uma recuperação na morfologia e fisiologia tímica, uma maior resposta proliferativa do baço e linfonodos estimulados com IL2. Concluímos desta forma que o exercício foi capaz de recuperar a morfologia, como também a maturação timócitos CD3 e CD4 e sua celuraridade em ratos desnutridos. A resposta proliferativa à estimulação da IL2 também foi recuperada. / Malnutrition is capable of inducing diverse metabolic alterations, markedly affecting body composition and the immune system. Physical exercise, on the other hand, induces a renders the organism more capable of adaptation to stress. Still, little is known about the influence of exercise training upon malnutrition-related alterations and its consequences on the immune system. Our aim was to evaluate the effect of moderate intensity exercise training in rats submitted to a protocol (16wk) of chronic malnutrition. Male Wistar rats were divided in to 4 groups: sedentary, fed ad libitum (SF); trained fed ad libitum (TF); sedentary energy restricted (RES); and trained energy restricted (TER). Training was carried out on a treadmill for 10 weeks, 5 time wk, under an intensity of 60-65% of the maximal oxygen consumption. We evaluated the Corporal composition, the variation of body weight, and the weight of the skeletal muscle, adipose tissues, and liver; as well as fat and protein content in the carcass; and also plasma leptin, ACTH, glucose, insulin and glutamine concentration. We also examined through flow cytometry CD3 and CD4, as well as the celularity in the thymus. The maximum consumption of oxygen and the performance were also assessed. The results demonstrate that endurance training in rats submitted to the chronic malnutrition protocol promoted reduction of body weight and of corporal adiposity; an increase in the relative contribution of muscle to body weight; the reestablishment of glicemia; improval of insulin/glucose reason, suggesting increased sensitivy to insulin; an increase of muscle glycogen content; enhanced oxygen consumption; are recovery of the morphology and physiology of the thymus, together with a proliferative response of the spleen and lymph nodes stimulated with IL2. We conclude in such a way that moderate intensity training restored thymus morphology and the capacity of maturation of CD3 and CD4 and also timocyte number and the of proliferative response to IL2 stimulation.

A desnutrição é capaz de induzir diversas alterações metabólicas afetando marcadamente a composição corporal e o sistema imunológico. O exercício físico, por sua vez, produz alterações no organismo para uma melhor capacidade de adaptação a situações de estresse. O desvio da situação de homeostase produzida pelo exercício físico induz uma reorganização de seus mecanismos funcionais, principalmente dos mecanismos endócrinos e imunológicos. Ainda é pouco conhecida a influência do exercício sobre a desnutrição e também as conseqüências sobre o sistema imunológico quando as duas variáveis são combinadas. Assim, esse trabalho teve como objetivo avaliar os efeitos do exercício físico de endurance sobre ratos submetidos a um protocolo de desnutrição crônica. Avaliamos ratos Wistar machos, desnutridos por 16 semanas, divididos em 4 grupos: eutrófico sedentário (ES), eutrófico treinado (ET), desnutrido sedentário (DS), desnutrido treinado (DT). O treinamento físico foi realizado em esteira, por 10 semanas, 5 vezes por semana, com intensidade aproximada de 60- 65% do consumo máximo de oxigênio. Avaliou-se a composição corporal, através da aferição do peso corporal, peso dos tecidos muscular esquelético e adiposo, do fígado, do conteúdo de gordura e proteína na carcaça, e a concentração de leptina, ACTH, glicose, insulina, e glutamina no plasma. Avaliamos também, através de citometria de fluxo, os marcadores de superfície celular CD3 e CD4, bem como a celularidade no timo. O consumo máximo de oxigênio e o desempenho através de um teste até a exaustão também foram analisados. A análise estatística utilizada foi o teste de variância ANOVA two-way com pós teste de Bonferroni e, nível de significância adotado de p<0,05. Os resultados encontrados demonstraram que o treinamento de endurance em ratos submetidos à desnutrição crônica promoveu uma acentuada redução do peso e da adiposidade corporal; um aumento da massa muscular relativa ao peso corporal; um restabelecimento da glicemia aos valores normais; uma melhor relação da concentração insulina/glicose, sugerindo uma sensibilidade à insulina aumentada; um aumento dos estoques de glicogênio muscular; um maior consumo máximo de oxigênio; e uma recuperação na morfologia e fisiologia tímica, uma maior resposta proliferativa do baço e linfonodos estimulados com IL2. Concluímos desta forma que o exercício foi capaz de recuperar a morfologia, como também a maturação timócitos CD3 e CD4 e sua celuraridade em ratos desnutridos. A resposta proliferativa à estimulação da IL2 também foi recuperada. / Malnutrition is capable of inducing diverse metabolic alterations, markedly affecting body composition and the immune system. Physical exercise, on the other hand, induces a renders the organism more capable of adaptation to stress. Still, little is known about the influence of exercise training upon malnutrition-related alterations and its consequences on the immune system. Our aim was to evaluate the effect of moderate intensity exercise training in rats submitted to a protocol (16wk) of chronic malnutrition. Male Wistar rats were divided in to 4 groups: sedentary, fed ad libitum (SF); trained fed ad libitum (TF); sedentary energy restricted (RES); and trained energy restricted (TER). Training was carried out on a treadmill for 10 weeks, 5 time wk, under an intensity of 60-65% of the maximal oxygen consumption. We evaluated the Corporal composition, the variation of body weight, and the weight of the skeletal muscle, adipose tissues, and liver; as well as fat and protein content in the carcass; and also plasma leptin, ACTH, glucose, insulin and glutamine concentration. We also examined through flow cytometry CD3 and CD4, as well as the celularity in the thymus. The maximum consumption of oxygen and the performance were also assessed. The results demonstrate that endurance training in rats submitted to the chronic malnutrition protocol promoted reduction of body weight and of corporal adiposity; an increase in the relative contribution of muscle to body weight; the reestablishment of glicemia; improval of insulin/glucose reason, suggesting increased sensitivy to insulin; an increase of muscle glycogen content; enhanced oxygen consumption; are recovery of the morphology and physiology of the thymus, together with a proliferative response of the spleen and lymph nodes stimulated with IL2. We conclude in such a way that moderate intensity training restored thymus morphology and the capacity of maturation of CD3 and CD4 and also timocyte number and the of proliferative response to IL2 stimulation.

<p>Bacillus Calmette Guérin (BCG), or attenuated Mycobacterium bovis, is the gold standard of immunotherapy in the clinic to treat superficial bladder cancer. However, setbacks remain due to a high recurrence rate, side effects, and BCG-refractory disease. In this thesis, we explored the use of novel immunotherapeutic agents such as CpG oligodeoxynucleotides (CpG ODNs) or synthetic ODNs containing unmethylated CpG dinucleotides. Since unmethylated CpG motifs are predominant in bacterial but not vertebrate DNA, they function as a “danger signal” leading to a potent immune response.</p><p>To be able to test various immunotherapeutic agents, we optimized subcutaneous (s.c.), metastatic, and orthotopic models using the murine bladder-49 (MB49) cancer cell line. In the orthotopic model, we show that poly-L-lysine promotes MB49 attachment to the bladder leading to 100% tumor take. In addition, Clorpactin (sodium oxychlorosene) potently enhances adenoviral transduction in the bladder.</p><p>Utilizing the MB49 model, we compare CpG ODNs with BCG and demonstrate the increased efficacy of CpG ODNs which could cure both s.c. and aggressive orthotopic bladder cancer. In our model, type B ODNs were most optimal and the antitumor response required T cells in order to induce regression and tumor-specific immunity. We also combined CpG ODNs with adenoviral vectors (Ad) expressing the immunostimulatory molecules CD40L, TRANCE, lymphotactin, IL2 or IL15. However, we show that CpG ODNs are effective as a monotherapy and adenoviral vectors did not enhance the effect.</p><p>AdCD40L was also used to genetically modify human dendritic cells (DCs). AdCD40L-transduced DCs not only had a higher and prolonged expression of the Th1 cytokine IL12 compared to TNFα-matured DCs, but CD40L-activated DCs could also resist the suppressive effects of IL10 and TGFβ. Since TNFα is commonly used in clinical DC vaccination protocols and because tumors often secrete immunosuppressive cytokines, these data have important implications for optimizing cancer immunotherapy.</p>

Bacillus Calmette Guérin (BCG), or attenuated Mycobacterium bovis, is the gold standard of immunotherapy in the clinic to treat superficial bladder cancer. However, setbacks remain due to a high recurrence rate, side effects, and BCG-refractory disease. In this thesis, we explored the use of novel immunotherapeutic agents such as CpG oligodeoxynucleotides (CpG ODNs) or synthetic ODNs containing unmethylated CpG dinucleotides. Since unmethylated CpG motifs are predominant in bacterial but not vertebrate DNA, they function as a “danger signal” leading to a potent immune response. To be able to test various immunotherapeutic agents, we optimized subcutaneous (s.c.), metastatic, and orthotopic models using the murine bladder-49 (MB49) cancer cell line. In the orthotopic model, we show that poly-L-lysine promotes MB49 attachment to the bladder leading to 100% tumor take. In addition, Clorpactin (sodium oxychlorosene) potently enhances adenoviral transduction in the bladder. Utilizing the MB49 model, we compare CpG ODNs with BCG and demonstrate the increased efficacy of CpG ODNs which could cure both s.c. and aggressive orthotopic bladder cancer. In our model, type B ODNs were most optimal and the antitumor response required T cells in order to induce regression and tumor-specific immunity. We also combined CpG ODNs with adenoviral vectors (Ad) expressing the immunostimulatory molecules CD40L, TRANCE, lymphotactin, IL2 or IL15. However, we show that CpG ODNs are effective as a monotherapy and adenoviral vectors did not enhance the effect. AdCD40L was also used to genetically modify human dendritic cells (DCs). AdCD40L-transduced DCs not only had a higher and prolonged expression of the Th1 cytokine IL12 compared to TNFα-matured DCs, but CD40L-activated DCs could also resist the suppressive effects of IL10 and TGFβ. Since TNFα is commonly used in clinical DC vaccination protocols and because tumors often secrete immunosuppressive cytokines, these data have important implications for optimizing cancer immunotherapy.

Over the past several decades, there have been numerous attempts to utilize synthetic dsRNA to control tumor growth in animal models and clinical trials. Recently, it has become clear that intracellular dsRNA is more effective than extracellular dsRNA in promoting apoptosis and orchestrating adaptive immune response. To overcome the difficulty in delivering a large dose of synthetic dsRNA into tumors, while avoiding systemic toxicity we propose to deliver a RNA replicase-based plasmid DNA, hypothesizing that the dsRNA generated by the replicase-based plasmid in tumor cells will inhibit tumor growth. We evaluated the anti-tumor activity of a plasmid (pSIN-beta) that encodes the sindbis RNA replicase genes in mice with model tumors (TC-1 lung cancer cells or B16 melanoma cells) and compared it to a traditional pCMV-beta plasmid. In cell culture, transfection of tumor cells with pSIN-beta generated dsRNA. In mice with model tumors, pSIN-beta more effectively inhibited tumor growth than pCMV-beta, and in some cases, eradicated the tumors. RNA replicase-based plasmid may be exploited to generate intracellular dsRNA to control tumor growth. The feasibility of further improving the antitumor activity of the RNA replicase-based plasmid by targeting it into tumors cells was also evaluated. An epidermal growth factor (EGF)-conjugated, PEGylated cationic liposome was developed to deliver the RNA replicase-based plasmid, pSIN-beta, into EGFR-over-expressing human breast cancer cells (MDA-MB-468) in vitro and in vivo. Delivery of the pSIN-beta using the EGF receptor-targeted liposome more effectively controlled the growth of MDA-MB-468 tumors in mice than using un-targeted liposome. Finally the potential of further improving the antitumor activity of the pSIN-beta plasmid by incorporating interleukin-2 (IL2) gene into the plasmid was investigated. The resultant pSIN-IL2 plasmid was delivered to mouse melanoma cells that over-express the sigma receptor. The pSIN-IL2 plasmid was more effective at controlling the growth of B16 melanoma in mice when complexed with sigma receptor targeted AA-PEG-liposomes than with the untargeted liposomes. Importantly, the pSIN-IL2 plasmid was more effective than pSIN-beta plasmid at controlling the growth of B16 melanoma in mice, and B16-bearing mice that were treated with pSIN-IL2 had an elevated number of activated CD4+, CD8+, and natural killer cells, compared to those treated with pSIN-beta. / text