Les thérapies anticancéreuses ont apporté un gain largement reconnu en matière de réduction de la charge tumorale, de survie des patients et d’amélioration de leur qualité de vie, dans un certain nombre de cancers. Hélas, ces thérapies exercent un effet immunosuppresseur en détruisant les effecteurs ou en bloquant l’activité de certains facteurs biologiques impliqués dans le recrutement des acteurs du système immunitaire. D’autre part, plusieurs travaux ont permis de démontrer que ces traitements pouvaient avoir un effet contraire en générant ou en favorisant l’induction d’une réponse immunitaire anti-tumorale, soit par effet direct sur le recrutement et l’activation des effecteurs de l’immunité, soit en potentialisant les interactions cellulaires par des mécanismes biologiques. Ces derniers faisant intervenir les cytokines, la stimulation des TLR, l’augmentation des interactions entre cellules du SI; ce qui permet de passer d’une anergie immunologique vers un véritable système d’éradication des cellules cancéreuses.Dans notre laboratoire, nous avons essayé d’évaluer l’implication du système immunitaire dans la réponse thérapeutique induite par des agents cytotoxiques conventionnels. Ici, nous décrivons les effets d’un inhibiteur de cyclines kinases multi-cibles « CDKi PHA-793 887 » testé dans un essai de phase I mené sur deux sites en Europe. C’est le constat inattendu que 6 des 15 patients, traités par ce médicament (PHA-793887) ont développé de graves infections bactériennes et virales et que 6 d’entre eux ont présenté la réactivation du virus de l’herpès qui nous a conduit à étudier ces effets sur le système immunitaire et en particulier sur le dialogue entre cellules dendritiques (CD) et cellules natural killer (NK). Ce travail met en évidence que ce médicament inhibe le signalling des récepteurs toll-like (TLR) réduisant par conséquent l’interaction CD/NK in vitro. Enfin la stimulation des cellules des patients sous traitement démontre une réduction importante de ce signalling ex-vivo. Ainsi, cet effet immunosuppresseur inattendu a permis une réactivation virale chez 40% des patients. La deuxième partie de ce travail, concerne les effets du cyclophosphamide (CTX) utilisé à faible dose. L’injection d'une faible dose chez la souris ou d’un dosage métronomique chez l'homme, promeut la différenciation des cellules lymphocytaires vers Th17 (sécrétant de l’interleukine-17 (IL-17)) et Th1 (sécrétant de l’interféron-γ (IFN)). Ceux-ci ont été retrouvés dans le sang et dans des ascites carcinomateuses de patients. Ainsi, le CTX pourrait participer à la génération de réponses anti-tumorale via la différenciation Th 17 comme cela fut suggéré par de récentes études précliniques montrant l’existence d’une corrélation étroite entre le taux des lymphocytes Th17 infiltrant la tumeur et la destruction tumorale. / Cancer therapies have made a gain widespread recognition in the reduction of tumor burden, patient survival and improved quality of life in a number of cancers. Unfortunately, these therapies exert an immunosuppressive effect by killing effectors or blocking the activity of certain biological factors involved in recruiting of the immune system. On the other hand, several studies have shown that these treatments could have the opposite effect by generating or promoting the induction of antitumor immune response, either by direct effect on the recruitment and activation of effectors immunity, either by potentiating cellular interactions by biological mechanisms. The latter involving cytokines, TLR stimulation, increased interactions between cells of the IS; which toggles between immunological anergy to a real system to eradicate cancer cells. In our laboratory, we tried to evaluate the involvement of the immune system in the therapeutic response induced by conventional cytotoxic agents. Here, we describe the effects of an inhibitor of cyclin kinases multi-target "CDKIs PHA-793887" tested in a phase I trial conducted at two sites in Europe. This unexpected finding is that 6 of 15 patients treated with this drug (PHA-793887) developed severe bacterial and viral infections and six of them showed reactivation of the herpes virus that has led us to study these effects on the immune system and in particular on the dialogue between dendritic (DCs) and natural killer (NK) cells. This work shows that this drug inhibits the signaling of toll-like receptor (TLR) thereby reducing the interaction DC / NK in vitro. Finally, stimulation of the cells of treated patients demonstrated a significant reduction of this signaling ex vivo. Thus, this immunosuppressive effect has an unexpected viral reactivation in 40% of patients. The second part of this work concerns the effects of metronomic dose of cyclophosphamide (CTX). The injection of a low dose in mice or metronomic dosing in humans, markedly promotes the differentiation of CD4+ T helper 17 (Th17) cells that can be recovered in both blood and tumor beds. However, CTX does not convert regulatory T cells into Th17 cells and promotes cell differentiation into Th17 lymphocytes (secreting interleukin-17 (IL-17)) and Th1 (secreting interferon-γ (IFN)). These were found in blood and in ascites carcinoma patients. Thus, CTX may participate in the generation of antitumor responses through Th 17 differentiation as was suggested by recent preclinical studies showing the existence of a correlation between the rate of Th17 lymphocytes infiltrating the tumor and tumor destruction.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-18T17:36:59Z No. of bitstreams: 1 MARIANE MAGALHÃES ZANCHI.pdf: 1185149 bytes, checksum: 5b02afd42e868819a353f05a3e1fb99a (MD5) / Made available in DSpace on 2016-04-18T17:36:59Z (GMT). No. of bitstreams: 1 MARIANE MAGALHÃES ZANCHI.pdf: 1185149 bytes, checksum: 5b02afd42e868819a353f05a3e1fb99a (MD5) Previous issue date: 2014-07-26 / A ciclofosfamida (CF) é um agente antineoplásico e imunossupressor, usada no tratamento de diversos tipos de tumores e em algumas doenças auto-imunes. A CF é considerada um pró-fármaco, portanto, precisa sofrer biotransformação hepática para formar seus metabólitos ativos, como a mostarda fosforamida e a acroleína. Seu metabólito terapêutico, a mostarda fosforamida, é responsável pelo efeito citotóxico na célula tumoral, enquanto a acroleína é conhecida por apresentar um efeito tóxico secundário, por aumentar a produção de espécies reativas ao oxigênio, causando estresse oxidativo. Esse, por sua vez, poderia estar diretamente relacionado com a redução da fertilidade causada por esta droga. Diante disso, compostos naturais com atividade antioxidante poderiam ser uma alternativa ao dano causado pela CF. O chá verde, obtido da planta Camellia sinensis, tem ação antioxidante e scavenger de radicais livres, devido ao alto conteúdo de polifenóis. O presente estudo avalia o papel protetor da infusão do chá verde sobre o dano oxidativo no sistema reprodutor de camundongos machos, e a sua relação com a fertilidade, após uma administração aguda de CF. Os camundongos foram pré-tratados por gavagem com veículo ou chá verde diariamente, por 14 dias, na dose de 250 mg/kg. A CF foi administrada via intraperitoneal no 14o dia, 1 hora após a administração do chá, na dose de 100 mg/kg. Os camundongos foram eutanasiados 24 horas após a administração da CF, e os testículos e epidídimos foram retirados para posteriores análises bioquímicas e avaliação espermática. O conteúdo de catequinas, principais constituintes do chá verde foi avaliada por HPLC. Epigalocatequina galato (EGCG) está presente em alta concentração na nossa infusão (1340,2 μg/mL), seguido por epicatequina (EC-500,95 μg/mL) e epicatequina galato (ECG-302,84 μg/mL). CF causou estresse oxidativo no sistema reprodutor dos animais, evidenciado neste trabalho por danificar tanto lipídios, como proteínas e DNA. A CF causou um aumento na peroxidação lipídica (níveis de MDA), dano de DNA (ensaio cometa) e atividade da enzima superóxido dismutase (SOD), enquanto diminuiu a atividade da glutationa peroxidase (GPx), glutationa-S-transferase (GST) e 17β–hidroxiesteróide desidrogenase (17β–HSD) em ambos os tecido testados. A atividade da enzima catalase (CAT) e a quantificação de proteína carbonil foram alteradas somente em testículo, mas não em epidídimo. Em relação à avaliação espermática, CF reduziu significativamente apenas a concentração de espermatozóides, comparado ao controle. O pré-tratamento com a infusão do chá verde reduziu significativamente a produção de MDA, carbonilação de proteína, dano de DNA e restaurou a atividade da GPx e GST. Em epidídimo, o chá verde também aumentou a concentração espermática e restaurou a atividade da enzima 17β–HSD. Pode-se concluir que a infusão do chá verde melhora o dano induzido pela CF, no sistema reprodutor de camundongos, e este efeito poderia ser atribuído ao alto conteúdo de catequinas presentes no chá e à sua atividade antioxidante. / Cyclophosphamide (CP) is an antineoplastic and immunosuppressive agent used to treat various types of tumors and in some autoimmune diseases. CP is considered a prodrug, therefore, must undergo hepatic biotransformation to form its active metabolites, such as phosphoramide mustard and acrolein. Its therapeutic metabolite, phosphoramide mustard, is responsible for the cytotoxic effect on tumor cells, while acrolein is known to provide a secondary toxic effect by increasing the reactive oxygen species production, causing oxidative stress. This might be directly related to the fertility reduction caused by this drug. Therefore, natural compounds with antioxidant activity could be an alternative to the damage caused by CP. Green tea, obtained from the plant Camellia sinensis, has antioxidant and free radical scavenging effect, due to the high content of polyphenols. The present study evaluates the protective role of the green tea infusion on oxidative damage on male mice reproductive system, and their relationship to fertility after an acute administration of CP. The mice were pre-treated by gavage with vehicle or green tea daily for 14 days at dose of 250 mg/kg. CP was administered intraperitoneally on day 14, 1 hour after administration of tea at dose of 100 mg/kg. Mice were euthanized 24 hours after the administration of the CP and testes and epididymis were removed for further biochemical analysis and sperm assessment. The content of catechins, the main constituents of green tea were evaluated by HPLC. Epigallocatechin gallate (EGCG) is present in high concentrations in our infusion (1340.2 μg/ml), followed by epicatechin (EC-500.95 μg/ml) and epicatechin gallate (ECG-302.84 μg/mL). CP caused oxidative stress in the reproductive system of animals, evidenced in this work by damaging not only lipids, but also proteins and DNA. CP increased lipid peroxidation (MDA), DNA damage (comet assay) and superoxide dismutase (SOD) activity, while decreased glutathione peroxidase (GPx), glutathione-S-transferase (GST) and 17β-hydroxysteroid dehydrogenase (17β-HSD) activity in both tissues tested. Catalase (CAT) activity and quantification of protein carbonyl were altered only in testes but not in the epididymis. Regarding sperm evaluation, CF significantly decreased only sperm concentration, compared to the control group. Pre-treatment with green tea infusion significantly reduced MDA production, protein carbonylation, DNA damage and restored GPx and GST activity. In epididymis, green tea also increased sperm concentration and restored 17 β-HSD activity. Green tea infusion improves the damage induced by CP in the reproductive system of mice, and this effect is attributed to the high content of catechins in tea and its antioxidant activity.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-05-02T13:45:06Z No. of bitstreams: 1 Natasha Pavin.pdf: 478428 bytes, checksum: 248a3efadd02d2695494d5e6ef077e2b (MD5) / Made available in DSpace on 2016-05-02T13:45:07Z (GMT). No. of bitstreams: 1 Natasha Pavin.pdf: 478428 bytes, checksum: 248a3efadd02d2695494d5e6ef077e2b (MD5) Previous issue date: 2016-04-02 / A ciclofosfamida (CP) é um agente antineoplásico e imunossupressor. É uma droga utilizada no tratamento de diversos tipos de tumores, usada para evitar a rejeição em transplante de órgãos e em doenças autoimunes, como artrite reumática e lúpus eritematoso. A CP é um pró-fármaco que sofre biotransformação no fígado pelo citocromo P450 para gerar metabólitos ativos como a mostarda fosfaramida e a acroleína. Seus metabólitos podem ocasionar um quadro de estresse oxidativo causando danos a diversos órgãos, entre eles o sistema reprodutor. A Tribulus terrestris (TT) faz parte da família das Zygophyllaceae, é uma erva rasteira perene com uma distribuição generalizada no Mediterrâneo, climas subtropicais e no deserto de todo o mundo. Na medicina popular tradicional, tem sido utilizada desde a antiguidade como um afrodisíaco, para energizar, vitalizar e melhorar a função sexual e desempenho físico em homens, bem como para tratar infecções urinárias, inflamações, edemas e outras doenças. Apesar de estudos experimentais e clínicos confirmarem parcialmente o fato de alguns efeitos da TT sobre a libido e a produção de esperma estarem envolvidos com o componente majoritário desta planta, a protodioscina, ainda há muito debate a respeito dos possíveis mecanismos de ação, bem como da sua aplicação terapêutica. Considerando o potencial antioxidante e esteroidogênico da TT, este estudo possui como objetivo investigar o potencial protetor do extrato seco da TT contra o dano testicular induzido pela ciclofosfamida em camundongos machos. Os camundongos Swiss machos receberam o extrato seco de TT como pré-tratamento por gavagem, durante 14 dias, na dose de 11 mg/Kg. No 14 ° dia, receberam uma única injeção intraperitoneal de ciclofosfamida na dose de 100 mg/Kg. Após 24 horas, esses animais foram anestesiados e eutanasiados, e o sangue, os testículos e os epidídimos foram retirados para posteriores análises bioquímicas, avaliação espermática e histopatológica. A presença e quantificação de protodioscina no vii extrato de TT foram avaliadas por HPLC (Cromatografia Líquida de Alta Eficiência), onde foi verificada uma concentração de 1,48% no extrato seco. A CP causou um desequilíbrio no sistema reprodutor destes animais, através do aumento das espécies reativas (ER), peroxidação lipídica (TBARS) e carbonilação de proteínas, bem como uma alteração nas enzimas antioxidantes: superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa S-transferase (GST) e glutationa redutase (GR). O pré tratamento com TT foi eficaz em proteger contra esses danos. Além disso, foi verificado uma redução na atividade da enzima 17β-hidroxiesteróide desidrogenase (17β-HSD), corroborando com a redução do nível de testosterona sérica verificado neste trabalho. Ao mesmo tempo, ao analisarmos a histopatologia dos testículos destes animais, verificamos uma moderada desorganização do epitélio espermatogenico, bem como, uma congestão dos vasos sanguíneos intersticiais. Estes achados corroboraram com os resultados encontrados na avaliação seminal dos animais tratados com CP, onde foi possível verificar uma diminuição da motilidade (41,07%), do vigor (23,52%), da integridade de membrana (43,44%) e da velocidade curvo-retilinea (25,43%), e o pré tratamento com TT foi eficaz em proteger contra esses danos. Em conclusão, este trabalho visou demonstrar pela primeira vez o potencial protetor do extrato seco de TT contra os danos ao sistema reprodutor masculino de camundongos expostos ao quimioterápico CP. À melhora dos parâmetros avaliados pode estar relacionada ao potencial antioxidante e esteroidogênico, bem como pela quantidade de protodioscina observada neste extrato. / Cyclophosphamide (CP) is an antineoplastic and immunosuppressive agent. It is a drug used to treat various tumours types, and also to prevent rejection in organ transplantation and autoimmune diseases such as rheumatoid arthritis and lupus erythematosus. The CP is a prodrug which is biotransformed in the liver by cytochrome P450 enzymes to generate active metabolites phosphoramide mustard and acrolein. Its metabolites can cause oxidative stress state, leading to damage in various organs, including the reproductive system. Tribulus terrestris (TT) is part of the family of Zygophyllaceae, it is a perennial creeping herb with a widespread distribution in the Mediterranean, subtropical climates and desert around the world. In traditional folk medicine, it has been used since ancient times as an aphrodisiac, to energize, vitalize and improve sexual function and physical performance in men and to treat urinary infections, inflammation, edema and other diseases. Although experimental and clinical studies partly confirm that some effects of TT on libido and sperm production are involved with the major component of this plant, protodioscin, there is still much debate about the possible mechanisms of action, as well as their therapeutic application. Considering the antioxidant and steroidogenic potential of TT, this study has the objective of investigating the protective potential of TT dry extract against testicular damage induced by cyclophosphamide in male mice. The male Swiss mice received the TT dry extract as pretreatment by gavage for 14 days at a dose of 11 mg kg-1. On day 14, animals received a single intraperitoneally injection of cyclophosphamide at a dose of 100 mg kg-1. After 24 hours, the animals were anesthetized and euthanized, and blood, testes and epididymis were removed for further biochemical analyzes, sperm and histological evaluation. The presence and quantification of protodioscin in TT extract were analyzed by HPLC (High Performance Liquid Chromatography), where a concentration of 1.48% of dry extract was verified. CP caused an imbalance in the reproductive system of these animals by increasing the reactive species (RS), ix lipid peroxidation (TBARS) and protein carbonylation, as well as change in antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR). The pre-treatment with TT was effective in protecting against this damage. Furthermore, a reduction in the activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) was found, confirming the reduction of serum testosterone levels observed in this study. Additionally, when analyzing the histopathology of the animals’ testes, we perceived a moderate disruption of spermatogenesis epithelium, as well as a congestion of interstitial blood vessels. These findings corroborate the results found in seminal evaluation of the animals treated with CP, where we observed a decrease in motility (41.07%), vigor (23.52%), membrane integrity (43,44%) and curved-straight speed (25.43%), and the pre-treatment with TT was effective in protecting against this damage. In conclusion, this study aimed to demonstrate for the first time the protective potential of TT dry extract against damage to the male reproductive system of mice exposed to CP chemotherapy. Improvement of these parameters may be related to antioxidant and steroidogenic potential as well as the amount of protodioscin observed in this extract.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-06T09:39:55Z No. of bitstreams: 2 Dissertação - Rangel Moreira Silva - 2016.pdf: 3905320 bytes, checksum: 6b2881f7623815dc402d04f906727e47 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-06T09:40:57Z (GMT) No. of bitstreams: 2 Dissertação - Rangel Moreira Silva - 2016.pdf: 3905320 bytes, checksum: 6b2881f7623815dc402d04f906727e47 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-06T09:40:57Z (GMT). No. of bitstreams: 2 Dissertação - Rangel Moreira Silva - 2016.pdf: 3905320 bytes, checksum: 6b2881f7623815dc402d04f906727e47 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-06-30 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Myrciaria cauliflora (Mart), commonly known as “Jabuticaba” (Brazilian Grape) occurs in all of South America, especially in Brazil. This species presents medicinal properties and it is popularly used to treat diarrhea and respiratory diseases. It has already been proven several biological activities such as antioxidant, antimutagenic and anti-proliferative. The phytochemical analysis of M. cauliflora detected the presence of several compounds, including the ellagitannin pedunculagin. Due to the widespread use of this species for therapeutic and food purposes, this study aimed to evaluate the genotoxic, antigenotoxic, cytotoxic and anticytotoxic actions of the aqueous fraction (AF) and pedunculagin from M. cauliflora seed, by micronucleus (MN) test and the comet assay in mice bone marrow cells. In order to analyze AF and pedunculagin activities prior to CP’s toxic effects, the animals were co-, pre- and post-treated with these substances. The results showed that AF at 24 h treatment did not show genotoxic or cytotoxic effects in the MN test and comet assay. However, AF showed cytotoxic action, but did not show genotoxic effect at 120 h treatment in the MN test. In the comet assay it was able to significantly reduce the frequency of DNA breaks when compared to the negative control. In the evaluation of the antigenotoxic and anticytotoxic effects, it was observed that in all treatments, in both assays, AF and pedunculagin showed antigenotoxic and anticytotoxic activities, but in the post-treatment pedunculagin increased the cytotoxic effect of CP. In general, our results indicated that AF and pedunculagin were able to protect the mice bone marrow cells against DNA damage induced by CP in the MN test and comet assay. These findings indicated that AF and pedunculagin could be probable candidates for the development of new drugs. / Myrciaria cauliflora (Mart), comumente conhecida como jabuticaba, ocorre em toda América do Sul e especialmente no Brasil. Esta espécie apresenta propriedades medicinais e é popularmente usada para tratar diarreia e doenças respiratórias. A essa espécie já foram atribuídas várias atividades biológicas, tais como: antioxidante, antimutagênica e antiproliferativa. A análise fitoquímica de M. cauliflora detectou a presença de vários compostos, incluindo o elagitanino pedunculagina. Devido ao grande uso desta espécie para fins terapêuticos e alimentícios, este estudo teve como objetivo avaliar as ações genotóxica, antigenotóxica, citotóxica e anticitotóxica da fração aquosa (FA) e do composto pedunculagina da semente de M. cauliflora pelo teste do micronúcleo e ensaio do cometa em células da medula óssea de camundongos. A fim de avaliar a atividade da FA e da pedunculagina frente aos efeitos genotóxicos da CP, os animais foram co, pré e pós-tratados com essas substâncias. Os resultados mostraram que a FA no tempo de 24 horas não apresentou efeitos genotóxico e citotóxico no teste do micronúcleo e no ensaio do cometa. No tempo de 120 horas, pelo teste do micronúcleo, a FA não mostrou ação genotóxica, porém exibiu efeito citotóxico e pelo ensaio do cometa, a frequência de quebras de DNA foi menor, quando comparada ao controle negativo. Na avaliação dos efeitos da antigenotoxicidade e anticitotoxicidade, em ambos os ensaios, a FA e a pedunculagina mostraram atividades antigenotóxica e anticitotóxica, porém, no pós-tratamento, o composto pedunculagina potencializou o efeito citotóxico da CP. De modo geral, nossos resultados indicaram que a FA e a pedunculagina foram capazes de proteger as células da medula óssea de camundongos contra os danos no DNA induzidos pela CP no teste do micronúcleo e no ensaio do cometa. Esses resultados indicaram que a FA e a pedunculagina podem ser prováveis candidatos para o desenvolvimento de novas drogas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cyclophosphamide (CP) is an alkylating agent widely used in anticancer and immunosuppression therapy. Like other chemotherapies, this drug can induce damage in healthy tissues, generating multiple organ toxicity. The study of natural substances able to prevent or reduce the toxicity induced by the chemotherapy is very important. Pecan nut shell (Carya illinoensis) is a vegetal by-product with low cost and elevated antioxidant potential and therefore can be considered a promissory substance. The aim of this study was to verify whether the cited antioxidant protects against multiple tissue damage induced by CP. Wistar rats received water or pecan shell aqueous extract (AE - 5%) ad libitum in place of drinking water up to the end of the experiment. On day 30, half of each group received a single administration of vehicle or CP 200 mg/kg-ip. After 7 days, the animals were sacrificed by exsanguination. The blood was collected to obtain plasma and erythrocytes. Vital tissues as heart, liver, kidney, testis and bladder were removed. The occurrence of oxidative stress (OS) was observed through the determination of lipid peroxidation (LP), measured by the thiobarbituric acid reactive substances (TBARS) formation and reduced glutathione (GSH) in all removed tissues, the enzyme catalase (CAT) in the heart, liver, kidney and testis, the enzyme superoxide dismutase (SOD) in the testis and the plasmatic vitamin C (VIT C) levels. Furthermore, the testicular function was evaluated by the enzyme lactate deydrogenase (LDH) and the macroscopic and histopathological analysis of the bladder was carried out. Results demonstrated that the CP treatment increased TBARS and decreased GSH levels in all studied tissues. Moreover, the activities of SOD in the testis and CAT in the heart were increased. However, in the other organs, the activity of the latter was diminished. The testicular LDH and plasmatic VIT C levels were also decreased. Finally, the bladder of the CP treated rats showed dark color and various histological alterations, such as thickening of the lines, hemorrhage, edema, leukocyte infiltration and vascular proliferation. The co-treatment with AE was able to prevent the TBARS increase and GSH depletion in all tissues, except heart and plasma, respectively. The extract also prevented changes in the testicular SOD and CAT activity in all organs, except the kidney. Besides, the extract prevented alteration in plasmatic VIT C and testicular LDH as well as various visual and histological changes in the rat bladder. These findings provided important evidences of the protective effect of pecan shell AE against the general organic toxicity induced by CP. Therefore, this natural substance may be considered to minimize adverse effects related with this chemotherapy, improving the life quality of patients who need to use this type of drug. / A ciclofosfamida (CP) é um agente alquilante muito usado na terapia anticâncer e imunossupressora. Assim como outros quimioterápicos, este fármaco pode causar danos aos tecidos saudáveis, gerando toxicidade em múltiplos órgãos. Desta forma, o estudo de substâncias naturais capazes de prevenir ou minimizar a toxicidade decorrente do tratamento quimioterápico vem ganhando sua importância. A casca da noz pecã (Carya illinoensis), é um sub-produto de origem vegetal de baixo custo e com elevado potencial antioxidante, constituindo uma substância promissora. O objetivo deste estudo foi verificar se o extrato aquoso bruto do vegetal citado apresenta efeito protetor contra os danos teciduais múltiplos induzidos pela CP. Ratos Wistar receberam água potável ou extrato aquoso bruto (EAB 5%) da casca da noz pecã, ad libitum, no lugar da água de beber até o final do experimento. No trigésimo dia, a metade de cada grupo recebeu uma administração única de veículo ou CP 200 mg/kg-ip. Após 7 dias, os animais foram sacrificados por exsanguinação, para obtenção de plasma e eritrócitos, enquanto alguns tecidos como coração, fígado, rim, testículos e bexiga foram removidos. Parâmetros de estresse oxidativo (EO) foram avaliados através da peroxidação lipídica (LPO), medida por meio da formação de espécies reativas ao ácido tiobarbitúrico (TBARS) e glutationa reduzida (GSH) em todos os tecidos avaliados, bem como a enzima catalase (CAT) no coração, fígado, rim e testículos. A enzima superóxido dismutase (SOD) testicular e os níveis plasmáticos de vitamina C (VIT C) também foram determinados. A função testicular foi determinada através dos níveis da enzima lactato desidrogenase (LDH). A bexiga foi submetida à avaliação macroscópica e histopatológica, já que é um tecido intensamente danificado pela CP. Os resultados obtidos demonstraram que o tratamento com CP aumentou os níveis de TBARS e diminuiu os níveis de GSH em todos os tecidos avaliados. A atividade da SOD testicular apresentou-se aumentada, bem como a atividade da CAT no coração. Nos demais órgãos, a atividade desta última enzima encontrou-se diminuída. Os níveis da enzima LDH testicular e os níveis plasmáticos de VIT C também se apresentaram reduzidos. Por último, as bexigas dos ratos tratados com CP mostraram coloração escura e diversas alterações histológicas, tais como: espessamento das camadas, hemorragia, edema, infiltração leucocitária e proliferação vascular. O co-tratamento com EAB foi capaz de prevenir o aumento do TBARS e diminuição do GSH em todos os tecidos, exceto coração e plasma, respectivamente. O extrato também preveniu modificações na atividade da SOD testicular e da CAT em todos os órgãos, exceto no rim. Além disso, o extrato também preveniu as alterações nos níveis de VIT C plasmática e LDH testicular, bem como as modificações macroscópicas e histopatológicas na bexiga dos ratos tratados com CP. Os resultados apresentados aqui evidenciam os efeitos protetores do EAB das casca da noz pecã sobre a toxicidade generalizada da CP. Deste modo, este sub-produto da indústria da noz pode ser considerado para minimizar os efeitos indesejáveis relacionados ao tratamento com CP, melhorando a qualidade de vida dos pacientes que necessitam deste quimioterápico.

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Le souhait de réduire des effets secondaires associés aux anticancéreux a mené à considérer l'utilisation de prodrogues activables au site d'action, comme la cyclophosphamide (CPA). La CPA est activée majoritairement par le CYP2B6 humain avec une faible efficacité, obligeant à l'utilisation de concentrations importantes de prodrogue. Celles-ci peuvent être réduites par transfection au niveau de la tumeur d'un gène codant pour un P450 optimisé, possédant une efficacité catalytique élevée vis-à-vis de la CPA tout en étant le moins immunogène possible. Pour ce faire, en partant de la modélisation du CYP2B6 et du CYP2B11, forme canine à bas Km pour l'activation de la CPA, un gène synthétique du CYP2B11 pour l'expression dans la levure a été dessiné et divisé en 15 "modules" structuraux. Quinze chimères à points de jonction définis entre les deux CYP2Bs ont ensuite été construites par échange de ces modules, via une méthode originale de génération de banques ordonnées de chimères, SIGNAL, indépendante de l'homologie de séquence des enzymes parentaux. SIGNAL, à mi-chemin entre les processus classiques d'évolution dirigée et de mutagenèse dirigée, nous a permis, après analyse fonctionnelle des chimères, de mieux comprendre le mécanisme de métabolisation de la CPA par les deux enzymes et d'identifier des modules structuraux jouant potentiellement un rôle important dans la haute affinité du CYP2B11. En parallèle, la mise au point d'un système de sélection à haut débit des variants les plus efficaces pour l'activation de la CPA dans la levure, basé sur le principe du gène rapporteur, a été débutée, afin de pouvoir raffiner l'optimisation du P450 par un processus de mutagenèse aléatoire.

Trabalhadores da área da saúde, em laboratórios, hospitais e/ou outros locais, estão potencialmente expostos a numerosos riscos ocupacionais. A ampla e crescente utilização de fármacos antineoplásicos, na quimioterapia, é considerada um importante risco químico para o pessoal envolvido no preparo e na administração destas substâncias. O manuseio destes fármacos sem cuidados pode levar a inúmeros efeitos tóxicos. Alguns destes fármacos foram classificados pela IARC como carcinógenos e provavelmente carcinógenos para humanos. Para tais fármacos é difícil atingir uma dose de não efeito observado, pois a patologia provocada por eles é considerada multifatorial. Este estudo objetivou validar método para a determinação de ciclofosfamida, um dos fármacos mais utilizados e, classificado como carcinógeno para humanos, para posterior aplicação em amostras coletadas em situação de real exposição. O analito foi identificado e quantificado por CG-EM após extração do analito em fase sólida e derivação com anidrido trifluoroacético. A ifosfamida foi utilizada como padrão interno e o fármaco foi determinado em amostras provenientes de wipe test e luvas, coletadas em quatro hospitais italianos, dentro de um intervalo de calibração de 1 a 100 ng/mL. Os intervalos de coeficiente de variação obtidos, no ensaio da precisão do método, foram entre 0,5 e 10% (intra-ensaio) e O e 19% (interensaio). Amostras contendo ciclofosfamida foram analisadas durante um mês, sem perda significativa do analito. A porcentagem de recuperação foi 98,9%. Os valores de ciclofosfamida nas 176 amostras coletadas variaram de inferior ao limite de quantificação (1,0 ng/mL) a 141.186 ng. Os resultados, bem correlacionados com aqueles relatados na literatura, sugerem que o método pode ser utilizado na identificação da ciclofosfamida em diversas superfícies e materiais e pode ser considerado ferramenta útil na monitorização da exposição ocupacional à esta substância. / Workers in laboratories and hospitals have potential exposure to numerous occupational hazards. The widespread use of antineoplastic drugs in chemotherapy is considered an important risk to staff involved in the preparation and administration to these drugs. Careless handling may lead toxic effects among the occupational exposure subjects. Some antineoplastic drugs were classified by IARC as carcinogenic and probably carcinogenic for humans. For carcinogenic agents the absence of a no-adverse-effect-Ievel is supposed. In this study cyclophosphamide was quantified adapting a previous analytical method by gas chromatography coupled mass spectrometry (GC-MS) after solid phase extraction and derivatization with trifluoroacetic anhydride. This drug in fact is one of the most frequently used alkylating antineoplastic agent for different types of tumors and classified as human carcinogen. The ifosfamide was used as internal standard and the drug was measured by analysis in wipe samples and gloves, collected from four different hospitals, within a range from 1 to 100 ng/mL. The values of variation coefficient varied from 0.5 to 10% (intra-assay) and from 0 to 19% (interassay). Frozen reference wipe samples containing cyclophosphamide were analysed over one month and no significant loss was observed. The range obtained for bias assay was 83-116% and the recovery was 98.9%. The cyclophosphamide was measured in 176 samples with values ranging from below limit of quantification (1.0 ng/mL) to 141186 ng. The results, well related with those reported in literature, suggest that this method can be used to identify the cyclophosphamide from surfaces and materials samples and can be considered useful in exposure assessment to this drug.

Trabalhadores da área da saúde, em laboratórios, hospitais e/ou outros locais, estão potencialmente expostos a numerosos riscos ocupacionais. A ampla e crescente utilização de fármacos antineoplásicos, na quimioterapia, é considerada um importante risco químico para o pessoal envolvido no preparo e na administração destas substâncias. O manuseio destes fármacos sem cuidados pode levar a inúmeros efeitos tóxicos. Alguns destes fármacos foram classificados pela IARC como carcinógenos e provavelmente carcinógenos para humanos. Para tais fármacos é difícil atingir uma dose de não efeito observado, pois a patologia provocada por eles é considerada multifatorial. Este estudo objetivou validar método para a determinação de ciclofosfamida, um dos fármacos mais utilizados e, classificado como carcinógeno para humanos, para posterior aplicação em amostras coletadas em situação de real exposição. O analito foi identificado e quantificado por CG-EM após extração do analito em fase sólida e derivação com anidrido trifluoroacético. A ifosfamida foi utilizada como padrão interno e o fármaco foi determinado em amostras provenientes de wipe test e luvas, coletadas em quatro hospitais italianos, dentro de um intervalo de calibração de 1 a 100 ng/mL. Os intervalos de coeficiente de variação obtidos, no ensaio da precisão do método, foram entre 0,5 e 10% (intra-ensaio) e O e 19% (interensaio). Amostras contendo ciclofosfamida foram analisadas durante um mês, sem perda significativa do analito. A porcentagem de recuperação foi 98,9%. Os valores de ciclofosfamida nas 176 amostras coletadas variaram de inferior ao limite de quantificação (1,0 ng/mL) a 141.186 ng. Os resultados, bem correlacionados com aqueles relatados na literatura, sugerem que o método pode ser utilizado na identificação da ciclofosfamida em diversas superfícies e materiais e pode ser considerado ferramenta útil na monitorização da exposição ocupacional à esta substância. / Workers in laboratories and hospitals have potential exposure to numerous occupational hazards. The widespread use of antineoplastic drugs in chemotherapy is considered an important risk to staff involved in the preparation and administration to these drugs. Careless handling may lead toxic effects among the occupational exposure subjects. Some antineoplastic drugs were classified by IARC as carcinogenic and probably carcinogenic for humans. For carcinogenic agents the absence of a no-adverse-effect-Ievel is supposed. In this study cyclophosphamide was quantified adapting a previous analytical method by gas chromatography coupled mass spectrometry (GC-MS) after solid phase extraction and derivatization with trifluoroacetic anhydride. This drug in fact is one of the most frequently used alkylating antineoplastic agent for different types of tumors and classified as human carcinogen. The ifosfamide was used as internal standard and the drug was measured by analysis in wipe samples and gloves, collected from four different hospitals, within a range from 1 to 100 ng/mL. The values of variation coefficient varied from 0.5 to 10% (intra-assay) and from 0 to 19% (interassay). Frozen reference wipe samples containing cyclophosphamide were analysed over one month and no significant loss was observed. The range obtained for bias assay was 83-116% and the recovery was 98.9%. The cyclophosphamide was measured in 176 samples with values ranging from below limit of quantification (1.0 ng/mL) to 141186 ng. The results, well related with those reported in literature, suggest that this method can be used to identify the cyclophosphamide from surfaces and materials samples and can be considered useful in exposure assessment to this drug.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.