Background In the treatment of head and neck cancers (HNCs), radiotherapy (RT) has the advantage of organ preservation compared to surgery. However, treatment toxicities associated with RT can affect important functions for everyday life, both in the acute and late stage. RT to macroscopic tumour in HNC is commonly combined with elective RT to cervical lymph nodes at risk of microscopic involvement. The resulting risk reduction of the elective treatment based on dose-volume parameters is sparsely evaluated. So is the relationship between the elective treatment and treatment toxicity. The present thesis addresses these aspects. A strategy aimed at improving the outcome of RT is accelerated fractionation (AF). AF strives to shorten total treatment time to minimise proliferation of the tumour tissue during the RT period. We have investigated the impact of AF on both disease control and toxicity. Methods In the ARTSCAN study, 750 patients with localised HNC were randomised between AF (68 Gy in 4.5 weeks) and conventional fractionation (CF) (68 Gy in 7 weeks). The elective treatment volume was prescribed 46 Gy with CF in both treatment arms. The thesis is based on four individual papers, investigating the issues above in the whole study population or in sub-populations. Results No difference in disease control or late toxicity between CF and AF was observed at five years. However, there was an increased acute toxicity with AF. Weight loss was associated with treatment volume, independent of tumour stage. The elective treatment volume was found to be an independent risk factor for late aspiration, as well as mean dose to the pharyngeal constrictor muscles, neck dissection, and age at randomisation. There was a significant risk reduction for node relapses in volumes treated to an elective dose. Only a relapse in volumes treated to >60 Gy affected the survival. Conclusion The present thesis questions the benefit of AF in definitive RT as well as extensive elective treatment of the cervical nodes.

La relaxométrie RMN bas champ du proton (20 MHz) a été utilisée au cours de ce travail pour étudier d’une part, les phénomènes mis en jeu dans les composites bois/ciment (CBC) lors de la phase d’hydratation, et d’autre part, le comportement des composites durcis conditionnés en atmosphère humide ou immergés dans l’eau. Dans une première partie, la relaxométrie a permis de suivre simultanément la transformation de l’eau évaporable en hydrates, et l’évolution de la microstructure du ciment, tout au long de la prise. Elle a également permis d’observer les transferts d’eau entre la matière végétale et la matrice de ciment et d’évaluer la compatibilité entre les différentes essences de bois et le ciment. Dans une deuxième partie, l’impact de certaines modifications chimiques du bois ou de la matrice sur l’hydratation du ciment a été étudié. Différents comportements ont été observés en fonction des fonctions chimiques greffées à l’intérieur du bois ou de l’adjuvant chimique incorporé dans la matrice. Enfin, dans une troisième partie, le comportement des composites durcis vis-à-vis de l’eau, ainsi que leurs performances mécaniques, ont été évalués. / In this work, low field proton NMR relaxometry (20 MHz) was applied to study wood-bonded cement composites hydration and the behaviour of hardened composites conditioned in humid atmosphere or immersed in water. In a first part, relaxometry was used to simultaneously follow the transformation of evaporable water into hydrates and the microstructure development during cement setting and hardening. This technique was also used to observe water transfer from wood to the cement matrix and to evaluate wood cement compatibility. In the second part, the influence of wood or matrix chemical modification on cement hydration was studied. Different behaviours were observed depending on the chemical group grafted unto wood or the chemical admixture added to the matrix. In the third part, the effect of moisture and the mechanical properties of hardened composites were evaluated.

Os óleos vegetais são matérias primas de fontes renováveis. São metabolizáveis, biodegradáveis, de fácil disponibilidade e baixo custo. A necessidade de adjuvantes vacinais seguros e que possam modular a resposta imunológica Th1/Th2, favorece a busca por novas substâncias que assim se comportem. Este trabalho tem o objetivo de avaliar e identificar óleo vegetal capaz de mimetizar ação adjuvante dos óleos minerais comercialmente usados e modular a resposta imune. Foram realizados testes de toxicidade aguda in vivo, testes de formação de emulsões, de estabilidade, de qualidade e, por fim, de imunogenicidade com formulações com o vírus rábico. Algumas formulações derivadas dos óleos de girassol, canola e buriti se mostraram não tóxicas, estáveis e de boa qualidade. Os grupos de camundongos inoculados com estas formulações obtiveram respostas imunológicas que apoiam sua capacidade adjuvante, não diferenciando significativamente (p<0,05) dos resultados do óleo mineral comercial. Concluiu-se que é possível elaborar emulsões estáveis não tóxicas a partir de óleos vegetais para sua utilização como veículos e adjuvantes vacinais. Formulações vacinais em forma de emulsões de óleos vegetais, compostas na sua maior parte pelos óleos de girassol e de canola possuem potência e atividade adjuvante semelhantes e tão eficientes quanto aos do óleo mineral. Os óleos vegetais devem estar em seu estado bruto ou semirrefinado, sem a adição de antioxidantes e conservantes. Por último, parece haver uma tendência de equilíbrio de resposta Th1/Th2 para as formulações com óleos vegetais. / Vegetable oils are renewable raw materials. These substances are metabolizable, biodegradable, of easy availability and low cost. The need for safe vaccine adjuvants that can modulate the Th1/Th2 immune response drives the search for new substances with similar behavior. This study aims to evaluate and identify a vegetable oil able to mimic the adjuvant action of the mineral oils used commercially, and modulate the immune response. There were performed tests of in vivo acute toxicity, emulsion formation, stability, quality and immunogenicity with formulations with rabies virus. Some formulations derived from the sunflower, canola and buriti oils proved to be non-toxic, stable and of good quality. Groups of mice inoculated with these formulations had immune responses supporting their adjuvant capacity, not differing significantly (p <0.05) from the results of the commercial mineral oil. It was concluded that it is possible to prepare stable non-toxic emulsions from vegetable oils to be used as vaccine adjuvants and vehicles. Vaccine formulations as emulsions from vegetable oil, composed mostly by the oils of sunflower and canola had adjuvant activity and potency similar to and as effective as the mineral oil. The vegetable oils should be in its raw state or semi refined, without the addition of antioxidants and preservatives. Finally, there seems to be a tendency to balance Th1/Th2 response by formulations with vegetable oils.

Influenza, também conhecida como gripe, é uma doença infecciosa viral que acomete um grande número de indivíduos anualmente, sendo responsável por um elevado número de internações e óbitos. O agente etiológico é o Myxovirus influenzae, vírus envelopado, de RNA de fita simples e polaridade negativa. A vacinação é a forma mais eficaz de se prevenir a infecção pelo vírus, no entanto, a capacidade produtiva dessa vacina não é suficiente para a vacinação da totalidade da população mundial, principalmente em casos de pandemia. Esse projeto teve por objetivo desenvolver uma vacina influenza (fragmentada e inativada) adjuvada, visando aumentar a capacidade produtiva dessa vacina no Instituto Butantan, que hoje é estimada em aproximadamente 40 milhões de doses por campanha. A utilização de adjuvantes na formulação da vacina influenza é capaz de produzir a mesma resposta imunológica protetora contra esse vírus, utilizando uma quantidade menor dos antígenos vacinais, aumentando a capacidade de produção da vacina em até quatro vezes. Foram estudadas 23 formulações adjuvantes utilizando o esqualeno como referência (formulação similar ao MF59®, adjuvante desenvolvido pela Novartis), vitaminas lipossolúveis (vitaminas A, D e E), vitamina B2 (vitamina hidrossolúvel), MPLA (monofosforil lipídio A, produzido pelo Instituto Butantan como subproduto da vacina pertussis low) e gel de hidróxido de alumínio. Para tanto, foram avaliadas a resposta imune conferida a camundongos BALB/c após imunização com diferentes formulações de vacina influenza (fragmentada e inativada) adjuvada e a existência, ou não, de toxicidade induzida pelas formulações vacinais estudadas. As formulações vacinais mais promissoras farão parte das formulações candidatas para realizações de ensaios clínicos. Os animais foram imunizados por via intraperitoneal com as formulações vacinais e foram colhidas amostras de sangue para ensaios sorológicos (inibição de hemaglutinação e ELISA) e células esplênicas para avaliação celular (dosagem de citocinas por citometria de fluxo: IL-2, IL-4, IL-6, IL-10, IL-17 TNF-&#945; e INF-&#947;). Além disso, em um dos experimentos avaliou-se a formação de memória imunológica contra influenza, parâmetro importante em se pensando em uma vacina. Os três primeiros experimentos foram uma triagem a partir da qual selecionaram-se as melhores formulações que foram testadas no último experimento. Nele foram avaliados além da indução de resposta imune a toxicidade e a memória imunológica. Todas as 23 formulações estudadas induziram resposta minimamente protetora nos animais, com exceção da formulação contendo apenas MPLA como adjuvante. As formulações que se mostraram mais promissoras continham além do gel de AI(OH)3 MPLA de B. pertussis ou vitamina B2. Isso sem considerar o tocoferol (vitamina E), que embora tenha apresentado bons resultados acabou preterido em decorrência de sua potencial relação com casos de narcolepsia descritos na literatura. O teste de memória foi capaz de demonstrar que essas formulações produzem resposta de memória imunológica duradoura. Assim, tem-se resultados promissores para novos estudos pré-clínicos e clínicos com a vacina influenza (fragmentada e inativada) sazonal (trivalente). / Influenza, also known as flu, is a viral infectious disease that infects a large number of people annually, being responsible by large morbidity and mortality rates. The etiologic agent is the Myxovirus influenzae, an enveloped virus with single-stranded RNA and negative polarity. Vaccination is the best way to prevent the virus infection; however, the production capacity of this vaccine is not sufficient to vaccinate the entire world population, especially in cases of pandemics. This project aimed to develop an adjuvanted influenza vaccine (split and inactivated), increasing the productive capacity of this vaccine in Instituto Butantan, which is estimated in approximately 40 million of doses by campaign. Influenza vaccines formulated with adjuvants can produce the same protective immunological response against the virus using less amount of antigen increasing the production capacity of this vaccine up to four times. Twenty-three adjuvants containing fat-soluble vitamins (vitamins A, D and E), vitamin B2 (water-soluble vitamin), MPLA (monophosphoryl lipid A, produced by Instituto Butantan as a byproduct of pertussis low vaccine production) and aluminum hydroxide gel were studied. An adjuvant similar to MF59® (Novartis adjuvant) containing squalene was used as control. The immune response elicited in BALB/c mice after immunization with the different formulations of the influenza vaccine and the existence or not of toxicity induced by the vaccines formulations were studied. The most promising formulation will be part of the candidate formulations of clinicai trials. The animais received the vaccine formulations intraperitoneally and at specific days blood samples were taken to serological tests (hemagglutination inhibition and ELISA). At the end, they were euthanized to collect the spleens and splenic cells were cultivated to evaluate cytokines by flow cytometry: IL-2, IL-4, IL-6, IL-10, IL-17 TNF-&#945; and INF-&#947;. Furthermore, in one experiment the immunological memory against influenza was evaluated, an important parameter to vaccines. The most promising formulations contained besides to alum either B. pertussis MPLA or B2 vitamin. Tocopherol (vitamin E) presented good results too, however it has a potential relationship with reported cases of narcolepsy. The memory test was able to demonstrate that these formulations induced long lasting immune memory response. Thus, these are promising results for new pre-clinical and clinical trials with seasonal trivalent influenza vaccine (split and inactivated).

Orientador: Maria Cecília Rui Luvizotto / Resumo: Nas últimas décadas biomarcadores de angiogênese e linfangiogênese tumoral são alvos de estudos e, em humanos, estes aspectos da vasculatura tumoral já são utilizados como fatores prognósticos em neoplasias mamárias. Em cadelas não há estudos suficientes para demonstrar o significado clínico-patológico da linfangiogênese em neoplasias mamárias. De maneira similar, não há estudos que demonstrem efeitos antilinfangiogênicos de terapias como a quimioterapia metronômica (QM), classicamente caracterizada como uma terapia antiangiogênica. Este estudo teve como objetivos estabelecer os efeitos da QM na angiogênese e linfangiogênese tumoral destas neoplasias, assim como obter o significado clínico-patológico da densidade de vasos linfáticos em neoplasias mamárias caninas. Foram utilizadas 40 cadelas portadoras de carcinomas mamários, divididas em dois grupos, sendo um tratado somente com mastectomia e outro tratado com quimioterapia metronômica (QM) seguida de mastectomia. A biópsia do linfonodo sentinela foi preconizada para estabelecimento do “status” do tecido linfoide sentinela. Após, foi realizada a classificação e graduação histopatológica das neoplasias, e procedeu-se a imunomarcação para determinar a densidade de vasos linfáticos (DVL), a densidade microvascular (DMV), índice de proliferação celular (IP), índice apoptótico (IA), escore de COX-2, grau do fator de crescimento endotelial vascular (VEGF) e metástase nodal. As imunomarcações foram comparadas entre os grupos e a DV... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In recent decades biomarkers of tumor angiogenesis and lymphangiogenesis have been studied and, in humans, these aspects of tumor vasculature are already used as prognostic factors in breast cancer. In bitches there are no studies that prove the clinicopathological significance of lymphangiogenesis in mammary neoplasms. Similarly, there are no studies demonstrating anti-lymphangiogenic effects of therapies such as metronomic chemotherapy (MC), classically characterized as anti-angiogenic therapy. This study aimed to establish the effects of MC on tumor angiogenesis and lymphangiogenesis of these neoplasms, as well as to obtain the clinical-pathological significance of lymphatic vessel density in canine mammary tumors. Forty bitches with mammary carcinomas were used, divided into two groups, one treated with mastectomy and the other treated with MC followed by mastectomy. Sentinel lymph node biopsy was recommended for establishing the status of the sentinel lymph node. Afterwards, classification and histopathological graduation of the neoplasms was performed, followed by immunostaining and determination of lymphatic vessel density (LVD), microvascular density (MVD), cell proliferation index (PI), apoptotic index (AI), COX-2 score, and vascular endothelial growth factor (VEGF) grade. Immunostain were compared between the groups and the LVD compared to clinicopathological aspects. Antiangiogenic and pro-apoptotic effects were observed in the group treated with MC, but without an... (Complete abstract click electronic access below) / Doutor

by Paul, Liu Po Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 86-94). / ACKNOWLEDGMENT --- p.i / TABLE OF CONTENT --- p.ii / "LIST OF TABLE, FIGURE & PHOTO" --- p.viii / ABSTRACT --- p.x / Chapter CHAPTER ONE : --- INTRODUCTION --- p.1 / Chapter 1.1 --- ALKALINE PHOSPHATASE / Chapter 1.1.1 --- Alkaline Phosphatase Isoenzyme --- p.2 / Chapter 1.1.2 --- The Properties of Alkaline Phosphatases --- p.4 / Chapter 1.1.3 --- Serum Alkaline Phosphatases --- p.6 / Chapter 1.1.3.1 --- Placental Alkaline Phosphatase --- p.7 / Chapter 1.1.3.2 --- Intestinal Alkaline Phosphatase --- p.7 / Chapter 1.1.3.4 --- Skeletal Alkalne Phosphatase --- p.8 / Chapter 1.1.3.5 --- Hepatic Alkaline Phosphatase --- p.8 / Chapter 1.1.3.3 --- Renal Alkaline Phosphatase --- p.9 / Chapter 1.1.3.6 --- Miscellaneous Alkaline Phosphatase --- p.9 / Chapter 1.1.4 --- Problems in Discriminating the Skeletal and Hepatic Alkaline Phosphatase in Serum --- p.11 / Chapter 1.1.5 --- Quantitative measure of the Bone-Specific Alkaline Phosphatase --- p.12 / Chapter 1.1.6 --- Qualitative Detection of ALP isoenzymes --- p.14 / Chapter 1.2 --- OSTEOSARCOMA --- p.17 / Chapter 1.2.1 --- Definition --- p.17 / Chapter 1.2.2 --- Epidemiology and Statistics --- p.17 / Chapter 1.2.3 --- Clinical Presentation --- p.18 / Chapter 1.2.4 --- Radiographic finding --- p.19 / Chapter 1.2.5 --- Staging of Musculoskeletal Neoplasms --- p.20 / Chapter 1.2.6 --- Treatment of osteosarcoma --- p.21 / Chapter 1.2.6.1. --- Chemotherapy in Prince of Wales Hospital --- p.21 / Chapter 1.3 --- PLASMA AND TISSUE ALKALINE PHOSPHATASE IN NORMAL AND NEOPLASTIC CONDITION --- p.23 / Chapter 1.3.1 --- Normal values of plasma alkaline phosphatase --- p.23 / Chapter 1.3.2 --- Clinical use of elevated plasma & tissue alkaline phosphatase level in neoplastic conditions --- p.25 / Chapter 1.3.2.1 --- Helping the Diagnosis of the Osteosarcoma --- p.25 / Chapter 1.3.2.2 --- Monitoring the effect of chemotherapy --- p.26 / Chapter 1.3.2.3 --- Predicting the clinical course --- p.26 / Chapter 1.3.3 --- Qualitative measurement of ALP in plasma and tissue extract of osteosarcoma patient --- p.29 / Chapter 1.4 --- AIM AND SCOPE OF THE PRESENT DISSERATION --- p.30 / Chapter CHAPTER TWO : --- MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- DIFERENT GROUPS OF PATIENTS --- p.33 / Chapter 2.1.1 --- Monitering the plasma bone specific ALP --- p.33 / Chapter 2.1.1.1 --- Osteosarcoma group --- p.33 / Chapter 2.1.1.2 --- Benign bone tumour group --- p.34 / Chapter 2.1.1.3 --- Metastasis group --- p.34 / Chapter 2.1.2 --- Collection of plasma samples preserve of tumor tissue --- p.34 / Chapter 2.2 --- QUANTITATIVE ANALYSIS OF THE PLASMA AND TISSUE BONE SPECIFIC ALKALINE PHOSPHATASE --- p.36 / Chapter 2.2.1 --- Extraction of tissue ALP --- p.36 / Chapter 2.2.1.1. --- Reagent --- p.36 / Chapter 2.2.1.2. --- Homogenization of the bone tissue --- p.36 / Chapter 2.2.1.3. --- Extraction of ALP --- p.37 / Chapter 2.2.2 --- Assay for Bone-specific ALP --- p.38 / Chapter 2.2.2.1. --- Reagents --- p.38 / Chapter 2.2.2.2. --- Procedures --- p.38 / Chapter 2.3 --- QUALITATIVE MEASUREMENT OF ALP ISOENZYME --- p.40 / Chapter 2.3.1 --- Equipment required --- p.40 / Chapter 2.2.2 --- Practical procedure --- p.40 / Chapter 2.3.3.1 --- Gel casting --- p.40 / Chapter 2.3.3.2 --- Sample preparation and application --- p.42 / Chapter 2.3.3.3 --- Electrofocusing --- p.42 / Chapter 2.3.3.4 --- Western blotting of the protein --- p.43 / Chapter 2.3.3.5 --- Detection methods --- p.45 / Chapter 2.4 --- METHOD OF STATISTICAL ANALYSIS --- p.48 / Chapter CHAPTER THREE : --- RESULTS --- p.49 / Chapter 3.1 --- QUANTITATIVE MEASUREMENT OF PLASMA AND TISSUE BONE SPECIFIC ALKALINE PHOSPHATASE --- p.50 / Chapter 3.1.1 --- General Information of the patients monitoring --- p.50 / Chapter 3.1.2 --- Pretreatment evaluation --- p.52 / Chapter 3.1.3 --- Correlation between the pretreatment plasma ALP levels and prognosis in the osteosarcoma patient group --- p.57 / Chapter 3.1.4 --- "Correlation between the pre-operational, post- operational plasma ALP levels and the prognosis of osteosarcoma" --- p.59 / Chapter 3.1.5 --- Analysis of plasma ALP levels at the time of relapse in osteosarcoma patients --- p.61 / Chapter 3.1.6 --- Usefulness of the plasma ALP levels for monitoring the effectiveness of chemotherapy --- p.62 / Chapter 3.1.7 --- Correlation between the ALP levels in the tumor extract and the prognosis of the osteosarcoma --- p.64 / Chapter 3.2 --- QUALITATIVE ANALYSIS OF THE PLASMA AND TISSUE ALKALINE PHOSPHATASE LEVEL --- p.67 / Chapter 3.2.1 --- Comparison of the result of Isoelectric focusing of the plasma ALP of the osteosarcoma patients and the normal subjects --- p.67 / Chapter 3.2.1 --- Result of Isoelectric focusing of the ALP isoenzymes in the tissue extract of the osteosarcoma and normal bone --- p.70 / Chapter CHAPTER FOUR : --- DISCUSSION --- p.72 / Chapter 4.1 --- USE OF QUANTITATIVE MONITORING OF PLASMA ALP AND MEASURING TISSUE ALP IN OSTEOSARCOMA PATIENTS --- p.73 / Chapter 4.2 --- ISOELECTRIC FORCUSING AS A TECNIQUE FOR QUALITATIVE MEASUREMENT OF PLASMA AND TISSUE ALKALINE PHOSPHATASE --- p.80 / Chapter CHAPTER FIVE : --- CONCLUSION --- p.83 / Chapter CHAPTER SIX : --- BIBILOGRAPHY --- p.85

Influenza, também conhecida como gripe, é uma doença infecciosa viral que acomete um grande número de indivíduos anualmente, sendo responsável por um elevado número de internações e óbitos. O agente etiológico é o Myxovirus influenzae, vírus envelopado, de RNA de fita simples e polaridade negativa. A vacinação é a forma mais eficaz de se prevenir a infecção pelo vírus, no entanto, a capacidade produtiva dessa vacina não é suficiente para a vacinação da totalidade da população mundial, principalmente em casos de pandemia. Esse projeto teve por objetivo desenvolver uma vacina influenza (fragmentada e inativada) adjuvada, visando aumentar a capacidade produtiva dessa vacina no Instituto Butantan, que hoje é estimada em aproximadamente 40 milhões de doses por campanha. A utilização de adjuvantes na formulação da vacina influenza é capaz de produzir a mesma resposta imunológica protetora contra esse vírus, utilizando uma quantidade menor dos antígenos vacinais, aumentando a capacidade de produção da vacina em até quatro vezes. Foram estudadas 23 formulações adjuvantes utilizando o esqualeno como referência (formulação similar ao MF59®, adjuvante desenvolvido pela Novartis), vitaminas lipossolúveis (vitaminas A, D e E), vitamina B2 (vitamina hidrossolúvel), MPLA (monofosforil lipídio A, produzido pelo Instituto Butantan como subproduto da vacina pertussis low) e gel de hidróxido de alumínio. Para tanto, foram avaliadas a resposta imune conferida a camundongos BALB/c após imunização com diferentes formulações de vacina influenza (fragmentada e inativada) adjuvada e a existência, ou não, de toxicidade induzida pelas formulações vacinais estudadas. As formulações vacinais mais promissoras farão parte das formulações candidatas para realizações de ensaios clínicos. Os animais foram imunizados por via intraperitoneal com as formulações vacinais e foram colhidas amostras de sangue para ensaios sorológicos (inibição de hemaglutinação e ELISA) e células esplênicas para avaliação celular (dosagem de citocinas por citometria de fluxo: IL-2, IL-4, IL-6, IL-10, IL-17 TNF-&#945; e INF-&#947;). Além disso, em um dos experimentos avaliou-se a formação de memória imunológica contra influenza, parâmetro importante em se pensando em uma vacina. Os três primeiros experimentos foram uma triagem a partir da qual selecionaram-se as melhores formulações que foram testadas no último experimento. Nele foram avaliados além da indução de resposta imune a toxicidade e a memória imunológica. Todas as 23 formulações estudadas induziram resposta minimamente protetora nos animais, com exceção da formulação contendo apenas MPLA como adjuvante. As formulações que se mostraram mais promissoras continham além do gel de AI(OH)3 MPLA de B. pertussis ou vitamina B2. Isso sem considerar o tocoferol (vitamina E), que embora tenha apresentado bons resultados acabou preterido em decorrência de sua potencial relação com casos de narcolepsia descritos na literatura. O teste de memória foi capaz de demonstrar que essas formulações produzem resposta de memória imunológica duradoura. Assim, tem-se resultados promissores para novos estudos pré-clínicos e clínicos com a vacina influenza (fragmentada e inativada) sazonal (trivalente). / Influenza, also known as flu, is a viral infectious disease that infects a large number of people annually, being responsible by large morbidity and mortality rates. The etiologic agent is the Myxovirus influenzae, an enveloped virus with single-stranded RNA and negative polarity. Vaccination is the best way to prevent the virus infection; however, the production capacity of this vaccine is not sufficient to vaccinate the entire world population, especially in cases of pandemics. This project aimed to develop an adjuvanted influenza vaccine (split and inactivated), increasing the productive capacity of this vaccine in Instituto Butantan, which is estimated in approximately 40 million of doses by campaign. Influenza vaccines formulated with adjuvants can produce the same protective immunological response against the virus using less amount of antigen increasing the production capacity of this vaccine up to four times. Twenty-three adjuvants containing fat-soluble vitamins (vitamins A, D and E), vitamin B2 (water-soluble vitamin), MPLA (monophosphoryl lipid A, produced by Instituto Butantan as a byproduct of pertussis low vaccine production) and aluminum hydroxide gel were studied. An adjuvant similar to MF59® (Novartis adjuvant) containing squalene was used as control. The immune response elicited in BALB/c mice after immunization with the different formulations of the influenza vaccine and the existence or not of toxicity induced by the vaccines formulations were studied. The most promising formulation will be part of the candidate formulations of clinicai trials. The animais received the vaccine formulations intraperitoneally and at specific days blood samples were taken to serological tests (hemagglutination inhibition and ELISA). At the end, they were euthanized to collect the spleens and splenic cells were cultivated to evaluate cytokines by flow cytometry: IL-2, IL-4, IL-6, IL-10, IL-17 TNF-&#945; and INF-&#947;. Furthermore, in one experiment the immunological memory against influenza was evaluated, an important parameter to vaccines. The most promising formulations contained besides to alum either B. pertussis MPLA or B2 vitamin. Tocopherol (vitamin E) presented good results too, however it has a potential relationship with reported cases of narcolepsy. The memory test was able to demonstrate that these formulations induced long lasting immune memory response. Thus, these are promising results for new pre-clinical and clinical trials with seasonal trivalent influenza vaccine (split and inactivated).

Os óleos vegetais são matérias primas de fontes renováveis. São metabolizáveis, biodegradáveis, de fácil disponibilidade e baixo custo. A necessidade de adjuvantes vacinais seguros e que possam modular a resposta imunológica Th1/Th2, favorece a busca por novas substâncias que assim se comportem. Este trabalho tem o objetivo de avaliar e identificar óleo vegetal capaz de mimetizar ação adjuvante dos óleos minerais comercialmente usados e modular a resposta imune. Foram realizados testes de toxicidade aguda in vivo, testes de formação de emulsões, de estabilidade, de qualidade e, por fim, de imunogenicidade com formulações com o vírus rábico. Algumas formulações derivadas dos óleos de girassol, canola e buriti se mostraram não tóxicas, estáveis e de boa qualidade. Os grupos de camundongos inoculados com estas formulações obtiveram respostas imunológicas que apoiam sua capacidade adjuvante, não diferenciando significativamente (p<0,05) dos resultados do óleo mineral comercial. Concluiu-se que é possível elaborar emulsões estáveis não tóxicas a partir de óleos vegetais para sua utilização como veículos e adjuvantes vacinais. Formulações vacinais em forma de emulsões de óleos vegetais, compostas na sua maior parte pelos óleos de girassol e de canola possuem potência e atividade adjuvante semelhantes e tão eficientes quanto aos do óleo mineral. Os óleos vegetais devem estar em seu estado bruto ou semirrefinado, sem a adição de antioxidantes e conservantes. Por último, parece haver uma tendência de equilíbrio de resposta Th1/Th2 para as formulações com óleos vegetais. / Vegetable oils are renewable raw materials. These substances are metabolizable, biodegradable, of easy availability and low cost. The need for safe vaccine adjuvants that can modulate the Th1/Th2 immune response drives the search for new substances with similar behavior. This study aims to evaluate and identify a vegetable oil able to mimic the adjuvant action of the mineral oils used commercially, and modulate the immune response. There were performed tests of in vivo acute toxicity, emulsion formation, stability, quality and immunogenicity with formulations with rabies virus. Some formulations derived from the sunflower, canola and buriti oils proved to be non-toxic, stable and of good quality. Groups of mice inoculated with these formulations had immune responses supporting their adjuvant capacity, not differing significantly (p <0.05) from the results of the commercial mineral oil. It was concluded that it is possible to prepare stable non-toxic emulsions from vegetable oils to be used as vaccine adjuvants and vehicles. Vaccine formulations as emulsions from vegetable oil, composed mostly by the oils of sunflower and canola had adjuvant activity and potency similar to and as effective as the mineral oil. The vegetable oils should be in its raw state or semi refined, without the addition of antioxidants and preservatives. Finally, there seems to be a tendency to balance Th1/Th2 response by formulations with vegetable oils.

In spite of vaccine and treatment strategies, Mycobacterium tuberculosis kills more than 3 people per minute. The emergence of drug-resistant strains makes treating the disease complicated and expensive for government health departments, and unpleasant and laborious for patients. The current vaccine, parenterally administered BCG, is only 50% effective. Oral vaccination has the advantage of targeting the mucosal immune system, which acts at the direct site of initial exposure to the infecting airborne pathogen. In addition, oral vaccines are cheaper and safer to administer than parenteral vaccines. This dissertation provides a conceptual framework for the prevention of the disease by means of oral vaccination and outlines methods that were developed for the production of concentrated purified somatic and extracellular antigens. Immune responses to somatic antigens were also examined in conjunction with established and novel adjuvants. The role of Propionibacterium jensenii 702 as a suitable mucosal adjuvant was supported by the results obtained. / Masters Thesis

Background: The use of chemotherapy and endocrine therapies in the treatment of pre-menopausal women with breast cancer may result in menopausal symptoms, permanent infertility or the need to delay pregnancy. This series of studies investigates the fertility- and menopause-related information needs of pre-menopausal women with a diagnosis of early breast cancer (Studies 1 and 2) and the benefits women need to make undergoing adjuvant endocrine therapies worthwhile (Study 3). Method: Study 1 is a qualitative study of 24 women and Study 2 a survey study amongst 228 women. Study 3 included a subset of 102 women from the sample involved in Study 2 who had been treated with endocrine therapies for a minimum of three months. To be eligible, women had to be aged 40 years or younger (Study 2 and 3) when diagnosed with early stage breast cancer, and be 6-60 months post-diagnosis at the time of participation. For Study 2, participants completed a mailed self-report questionnaire that included a fertility- and menopause-related information needs survey, and standardized measures of distress, quality-of-life, menopausal symptoms and information preferences. For Study 3, participants were asked to complete a face-to-face interview. Results: Study 1 showed that many women thought that the information they had received in the past about fertility and menopausal symptoms was either insufficient or unavailable. Some women felt that, while information on fertility and menopause issues had not been paramount at the time of diagnosis, it became increasingly important after diagnosis. Study 2 showed that 71% of participants discussed fertility-related issues with a health professional as part of their breast cancer treatment and 86% discussed menopause-related issues. Consultation with a fertility or menopause specialist was the most preferred method of obtaining this information. Study 3 demonstrated that the majority of participants considered adjuvant endocrine therapy worthwhile for a 2% absolute gain in survival rates and for a 6-month gain in life expectancy. Conclusions: The results of this series of studies suggest that younger women have unmet needs for fertility- and menopause-related information. Women with early breast cancer who had received adjuvant endocrine therapies judged modest survival gains sufficient to make adjuvant endocrine therapy worthwhile.