The Western Fence Lizard (Sceloporus occidentalis) is a major host of juvenile stages of the Western Black-legged Tick (Ixodes pacificus), which is the vector for the Lyme disease causative spirochete bacterium Borrelia burgdorferi in the western United States. Because S. occidentalis is reservoir incompetent and capable of eliminating spirochetes from infected ticks, it has been implicated as a major factor in the ecology of Lyme disease in the West. Although complement proteins in lizard blood have been established as the borreliacidal factor, no studies have examined intraspecific variability in host lizard borreliacidal capacity. In Chapter 1 of this thesis, we introduce the complexity of the Borrelia burgdorferi transmission cycle and it’s implications for transmission risk. In Chapter 2 we tested the hypothesis that host lizard physiological condition impacts their borreliacidal capacity. Blood plasma of lizards in varying physiological conditions was challenged against cultured B. burgdorferi, and the complement-mediated inactivation of spirochetes was quantified. Adult lizards had higher bactericidal activity than first-year juveniles, suggesting that complement-mediated inactivation develops with maturity and/or exposure to spirochete antigens. Also, bactericidal activity was positively associated with lizard tick load and body condition. Adult lizard sex did not significantly affect spirochete mortality. Lizards from an inland site with little exposure to ticks had higher bactericidal activity than lizards from a coastal population that is heavily parasitized by ticks.

A Senna occidentalis (So), Fabaceae-Caesalpinoideae (Leguminosae), conhecida como fedegoso, entre outros nomes populares causa, em animais de criação, intoxicação pelo consumo, tanto de suas folhas a campo, como pela ingestão de suas sementes quando presentes em rações. Estas intoxicações podem ser de caráter crônico ou agudo, em ruminantes ou monogástricos, tendo sido já reproduzida em animais de laboratório. Os principais achados histológicos são degenerações em músculos esquelético e cardíaco, fígado, rim e, recentemente publicado, lesões no sistema nervoso central. Porém, pouco se sabe sobre os efeitos do princípio ativo desta planta, a diantrona, na toxicologia perinatal. O objetivo do presente trabalho foi estudar os possíveis efeitos teratogênicos desta planta em caprinos, utilizando um protocolo de avaliação de toxicologia do desenvolvimento. Para tanto, foi somado a um protocolo pré-existente de acompanhamento da gestação de cabras, por meio de ultra-sonografia e dados clínicos, como pesagem semanal e coleta quinzenal de sangue para realização de bioquímica sérica, a avaliação de filhotes por morfometria corporal esde o nascimento ao 4º mês de idade, além de avaliação neurocomportamental até a 10ª semana de idade. Assim, trinta e oito fêmeas nulíparas, receberam rações contendo 0% (controle), 1% (So1), 2% (So2), 3% (So3) e 4% (So4) de sementes da planta, desde a confirmação da gestação, no 27º dia pós-coito, até o parto. Outro grupo de cabras (LAC) consumiu ração contendo 4% de sementes da planta durante a lactação. Amostras de leite das cabras foram colhidas para a tentativa de detecção da diantrona. Foi observado queda no ganho de peso nas fêmeas pertencentes ao grupo So3 durante a gestação. No grupo So4 ocorreu a morte de uma fêmea, duas mortes fetais, e morte pós-natal de um filhote. À análise histológica da cabra que morreu, observou-se severas lesões em fígado, rim, músculo esquelético e cardíaco. Foi observado, pela primeira vez na literatura, lesões em nervo ciático. Nas cabras do grupo LAC foram observadas lesões teciduais brandas, exceto em nervo ciático. Não se observaram malformações físicas nos filhotes. Somente pequenas alterações bioquímicas foram observadas nas mães e na prole. A morfometria evidenciou poucas alterações nos filhotes do grupo LAC. Histologicamente, estes filhotes LAC tiveram lesões focais brandas em musculatura esquelética e cardíaca, fígado e rim, revelando que o princípio ativo da planta pode ser transferido para o leite. Os testes neurocomportamentais revelaram déficit de aprendizado dos filhotes do grupo So3. Não foi possível a detecção da diantrona no leite avaliado. Este estudo permitiu verificar que a So, embora não seja um potente teratógeno, quando consumida durante a gestação, pode causar efeitos tóxicos no feto, como evidenciado pela avaliação neurocomportamental. O protocolo sugerido poderá ter grande valia para estudar o potencial teratogênico de diversas substâncias as quais os ruminantes possam estar expostos. / Senna occidentalis (So - formerly Cassia occidentalis), Fabaceae-Caesalpinoideae (Leguminosae) is a weed growing in pastures and in cultivated fields such as corn and soybeans in many tropical and subtropical areas worldwide. Many reports have shown that So is toxic to different animal species. Histologic lesions plant-related are degeneration skeltal and cardiac muscles, liver and kidney. Recently was related leions in the central nervous system. Even so, very little is known about the possible effects of prolonged exposure at low doses of So on developmental toxicology. Thus, the present study proposes an approach to evaluate the perinatal toxicity from gestational intoxication of So seeds in goats. Third-eight pregnant goats were fed rations containing 0% (control group), 1% (So1 group), 2% (So2 group), 3% (So3) and 4% (So4 group) mature So seeds from pregnancy detection on day 27 after mating until parturition. Another group (LAC) was fed with ration containing 4 % of So seeds only in the lactation period; clinic observations such weight gains, general body status and serum biochemistry were evaluated. Fetuses were evaluated during the pregnancy using ultrasonographic measurements; neonates were evaluated after birth using body morphometry, weight gains, corporal status and serum biochemistry. Neurobehavioral tests was performed on kids from So3 group. In the So4 group was observed fetal dead from 2 dams, dead of one newborn and dead of one pregnant dam. None malformations was observed. Only a few minor alterations in serum biochemistry were observed in dams and kids; even so one So4 group dam had several and diffuse tissue lesions as vacuolations in hepatocytes; vacuolations in the endothelium of the convoluted tubules in kidneys, necrosis in skeletal and cardiac muscles, and for the first time vacuolation was observed in sciatic nerve. Alterations in body morphometry were observed only in kids from LAC group during first month of lactation. Also, tissue lesions was observed in these kids, showing the capacity of the diantrona (active principle of the palnt) be carried to milk. Neurobehavioral tests revealed deficit on learning hability in the So3 kids. Diantrone didn\'t was detected in milk samples. This study shows that, despite S. occidentalis isn\'t a powerfull teratogen when ate during gestation, can cause toxic effects on the fetus, as neurobehavioral test revealed. Protocol utilized here is usefull to study the potential teratogenic effects of drugs and other substancies whose an animal could be exposed.

A Senna occidentalis (So), Fabaceae-Caesalpinoideae (Leguminosae), conhecida como fedegoso, entre outros nomes populares causa, em animais de criação, intoxicação pelo consumo, tanto de suas folhas a campo, como pela ingestão de suas sementes quando presentes em rações. Estas intoxicações podem ser de caráter crônico ou agudo, em ruminantes ou monogástricos, tendo sido já reproduzida em animais de laboratório. Os principais achados histológicos são degenerações em músculos esquelético e cardíaco, fígado, rim e, recentemente publicado, lesões no sistema nervoso central. Porém, pouco se sabe sobre os efeitos do princípio ativo desta planta, a diantrona, na toxicologia perinatal. O objetivo do presente trabalho foi estudar os possíveis efeitos teratogênicos desta planta em caprinos, utilizando um protocolo de avaliação de toxicologia do desenvolvimento. Para tanto, foi somado a um protocolo pré-existente de acompanhamento da gestação de cabras, por meio de ultra-sonografia e dados clínicos, como pesagem semanal e coleta quinzenal de sangue para realização de bioquímica sérica, a avaliação de filhotes por morfometria corporal esde o nascimento ao 4º mês de idade, além de avaliação neurocomportamental até a 10ª semana de idade. Assim, trinta e oito fêmeas nulíparas, receberam rações contendo 0% (controle), 1% (So1), 2% (So2), 3% (So3) e 4% (So4) de sementes da planta, desde a confirmação da gestação, no 27º dia pós-coito, até o parto. Outro grupo de cabras (LAC) consumiu ração contendo 4% de sementes da planta durante a lactação. Amostras de leite das cabras foram colhidas para a tentativa de detecção da diantrona. Foi observado queda no ganho de peso nas fêmeas pertencentes ao grupo So3 durante a gestação. No grupo So4 ocorreu a morte de uma fêmea, duas mortes fetais, e morte pós-natal de um filhote. À análise histológica da cabra que morreu, observou-se severas lesões em fígado, rim, músculo esquelético e cardíaco. Foi observado, pela primeira vez na literatura, lesões em nervo ciático. Nas cabras do grupo LAC foram observadas lesões teciduais brandas, exceto em nervo ciático. Não se observaram malformações físicas nos filhotes. Somente pequenas alterações bioquímicas foram observadas nas mães e na prole. A morfometria evidenciou poucas alterações nos filhotes do grupo LAC. Histologicamente, estes filhotes LAC tiveram lesões focais brandas em musculatura esquelética e cardíaca, fígado e rim, revelando que o princípio ativo da planta pode ser transferido para o leite. Os testes neurocomportamentais revelaram déficit de aprendizado dos filhotes do grupo So3. Não foi possível a detecção da diantrona no leite avaliado. Este estudo permitiu verificar que a So, embora não seja um potente teratógeno, quando consumida durante a gestação, pode causar efeitos tóxicos no feto, como evidenciado pela avaliação neurocomportamental. O protocolo sugerido poderá ter grande valia para estudar o potencial teratogênico de diversas substâncias as quais os ruminantes possam estar expostos. / Senna occidentalis (So - formerly Cassia occidentalis), Fabaceae-Caesalpinoideae (Leguminosae) is a weed growing in pastures and in cultivated fields such as corn and soybeans in many tropical and subtropical areas worldwide. Many reports have shown that So is toxic to different animal species. Histologic lesions plant-related are degeneration skeltal and cardiac muscles, liver and kidney. Recently was related leions in the central nervous system. Even so, very little is known about the possible effects of prolonged exposure at low doses of So on developmental toxicology. Thus, the present study proposes an approach to evaluate the perinatal toxicity from gestational intoxication of So seeds in goats. Third-eight pregnant goats were fed rations containing 0% (control group), 1% (So1 group), 2% (So2 group), 3% (So3) and 4% (So4 group) mature So seeds from pregnancy detection on day 27 after mating until parturition. Another group (LAC) was fed with ration containing 4 % of So seeds only in the lactation period; clinic observations such weight gains, general body status and serum biochemistry were evaluated. Fetuses were evaluated during the pregnancy using ultrasonographic measurements; neonates were evaluated after birth using body morphometry, weight gains, corporal status and serum biochemistry. Neurobehavioral tests was performed on kids from So3 group. In the So4 group was observed fetal dead from 2 dams, dead of one newborn and dead of one pregnant dam. None malformations was observed. Only a few minor alterations in serum biochemistry were observed in dams and kids; even so one So4 group dam had several and diffuse tissue lesions as vacuolations in hepatocytes; vacuolations in the endothelium of the convoluted tubules in kidneys, necrosis in skeletal and cardiac muscles, and for the first time vacuolation was observed in sciatic nerve. Alterations in body morphometry were observed only in kids from LAC group during first month of lactation. Also, tissue lesions was observed in these kids, showing the capacity of the diantrona (active principle of the palnt) be carried to milk. Neurobehavioral tests revealed deficit on learning hability in the So3 kids. Diantrone didn\'t was detected in milk samples. This study shows that, despite S. occidentalis isn\'t a powerfull teratogen when ate during gestation, can cause toxic effects on the fetus, as neurobehavioral test revealed. Protocol utilized here is usefull to study the potential teratogenic effects of drugs and other substancies whose an animal could be exposed.

O câncer de próstata é a segunda causa de morte entre homens no Brasil. É tipo um câncer de crescimento lento, podendo levar anos para o tumor atingir 1 cm3, porém, em alguns casos ele pode se espalhar pelo corpo, sendo o osso o principal sítio de metástase. No estágio de desenvolvimento do câncer conhecido como metástase, o principal tratamento consiste em terapia de restrição andrógena, levando as células prostáticas a pararem de proliferar, uma vez que elas crescem em resposta a presença de hormônios andrógenos, como a diidrotestosterona e testosterona. Porém, em alguns casos, as células proliferam mesmo na ausência de andrógenos e isto se deve a diversos fatores que, em geral, estão associados a mutações no receptor andrógeno e/ou alterações no metabolismo andrógeno. Quando isto acontece, os tratamentos disponíveis são menos efetivos e costumam falhar. Porém, estudos sugerem que o γ-orizanol, um fitoesterol extraído do óleo do farelo do arroz; e extratos amplamente utilizados na medicina popular, como o extrato hidroalcólico de Thuya occidentalis, poderiam atuar inibindo o desenvolvimento e progressão do câncer de próstata. Neste estudo, com o uso de abordagens bioquímicas e de biologia molecular, foi demonstrado que o tratamento com γ-orizanol diminui a viabilidade e biomassa celular em cultura, associado ao aumento da morte celular por apoptose e/ou necrose, em linhagens celulares responsivas (LNCaP) e não-responsivas a andrógenos (PC3 e DU145), além de aumentar a pERK1/2 em células LNCaP e DU145. O γ-orizanol também foi capaz de bloquear o ciclo celular em G2/M nas células PC3 e LNCaP e em G0/G1 nas células DU145. Estes efeitos foram ainda acompanhados por uma redução da expressão do gene e proteína caveolina-1- uma importante molécula envolvida no aumento da agressividade do câncer de próstata, e também, na progressão da doença para o fenótipo andrógeno resistente - nas células não-responsivas a andrógenos, e do gene PCGEM1 - gene específico da próstata regulado por andrógeno - nas células LNCaP e DU145. Ainda, γ-orizanol também mostrou capacidade de regular vários miRNAs - pequenas moléculas de RNA não codificantes de proteínas - envolvidos no controle de funções associadas ao desenvolvimento, progressão e invasão no câncer de próstata, como o miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 e miR222. O extrato hidroalcólico de Thuya occidentalis reduziu a viabilidade e biomassa celular nas linhagens responsiva (LNCaP) e não-responsivas (DU145 e PC3) a andrógenos, além de induzir parada do ciclo celular na fase G0/G1 nas células DU145 e aumentar a morte celular por apoptose e/ou necrose em todas as linhagens. Da mesma forma que o γ-orizanol, este extrato reduziu a expressão da caveolina-1 nas linhagens não-responsivas a andrógenos. Trabalhos anteriores mostram que o monoterpeno α-tujona é o principal composto ativo do extrato de Thuya occidentalis. Por cromatografia gasosa acoplada a detector de massas foi mostrada a existência de 0,0016 μg de α-tujona na dose de extrato usada neste estudo. No entanto, o tratamento com 0,0016μg de α-tujona foi efetivo somente sobre linhagem LNCaP, não tendo efeito sobre as outras linhagens estudadas, reforçando a hipótese da diferença de sensibilidade entre as linhagens responsivas e não responsivas a andrógeno e mostrando a contribuição de outros componentes do extrato nos efeitos observados neste estudo. Concluindo, estes resultados demonstram que tanto γ-orizanol como o extrato de Thuya occidentalis podem vir a ser agentes terapêuticos promissores no tratamento de câncer de próstata, não só por inibirem o crescimento celular, mas também e principalmente pela possibilidade de induzirem a recuperação da sensibilidade a andrógenos, aumentando as possibilidades de tratamento da doença. / Prostate cancer is the second cause of death among men in Brazil. It is a slowgrowing cancer and it may take years for tumor to reach 1 cm3, but in some cases it can spread throughout the body and the bone is the main site of metastasis. At this cancer stage known as metastasis, the principal treatment involves antiandrogen therapy, leading to prostate cells stop proliferating, because they grow in response to presence of androgens such as testosterone and dihydrotestosterone. However, in some cases, the cells can proliferate even in the absence of androgens and this fact occurs due to many factors and they are generally associated with mutations in the androgen receptor and/or alterations in androgen metabolism. In this stage, the treatments available are less effective and usually fail. However, studies suggest that γ-oryzanol, a phytosterol extracted of rice bran oil; and extracts widely used in folk medicine, as Thuya occidentalis hidroalcolic extract, could act inhibiting the development and progression of prostate cancer. In this study, using molecular biology and biochemical approaches we showed that γ- oryzanol treatment was able to decrease cell viability and biomass in culture, and this fact was linked to increased cell death by apoptosis and/or necrosis in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) prostate cancer cell lines, besides increasing pERK1/2 in LNCaP and DU145 cells. γ- oryzanol was also able to cause cell cycle arrest at G2/M phase in LNCaP and PC3 cells and at G0/G1 phase in DU145 cells. These effects were also accompanied by a reduction in caveolin-1 gene and protein expression - an important molecule related to high aggressiveness in prostate cancer and also in the progression of the disease to androgen resistant phenotype - in androgen unresponsive cells, and also PCGEM1 gene - a prostate specific gene regulated by androgens - in LNCaP and DU145 cells. γ-oryzanol also showed ability to regulate several miRNAs - small non-coding RNA molecules - involved in the control of many functions associated with the development, progression and invasion of prostate cancer, such as miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 and miR222. Thuya occidentalis hidroalcolic extract also reduce cell viability and biomass in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) cells, in addition to inducing cell cycle arrest at G0/G1 phase in DU145 cells and to increase apoptosis and/or necrosis cell death in all cell lines. The same way that γ-oryzanol, this extract reduced the caveolin-1 expression in androgen unresponsive prostate cancer cells. Prior studies showed that the monoterpene α-thujone is the main active compound in the T. occidentalis extract. By gas chromatography coupled to mass detector it was showed the existence of 0.0016 μg of α-thujone in extract dose used in this study. However, the treatment with 0.0016 μg of α-thujone was effective only on LNCaP cell line, having no effect on the other studied lines, supporting the hypothesis of difference in sensitivity between responsive and unresponsive cell lines and showing the contribution of other components in the effects caused by the extract, observed it this study. In conclusion, these results demonstrate that both γ-oryzanol as T. occidentalis extract may become promising therapeutic agents in treatment of prostate cancer, not only inhibit cell growth but also and manly by the possibility of inducing the recovery of androgen sensitivity, increasing the treatment chances of treatment this disease.

O câncer de próstata é a segunda causa de morte entre homens no Brasil. É tipo um câncer de crescimento lento, podendo levar anos para o tumor atingir 1 cm3, porém, em alguns casos ele pode se espalhar pelo corpo, sendo o osso o principal sítio de metástase. No estágio de desenvolvimento do câncer conhecido como metástase, o principal tratamento consiste em terapia de restrição andrógena, levando as células prostáticas a pararem de proliferar, uma vez que elas crescem em resposta a presença de hormônios andrógenos, como a diidrotestosterona e testosterona. Porém, em alguns casos, as células proliferam mesmo na ausência de andrógenos e isto se deve a diversos fatores que, em geral, estão associados a mutações no receptor andrógeno e/ou alterações no metabolismo andrógeno. Quando isto acontece, os tratamentos disponíveis são menos efetivos e costumam falhar. Porém, estudos sugerem que o γ-orizanol, um fitoesterol extraído do óleo do farelo do arroz; e extratos amplamente utilizados na medicina popular, como o extrato hidroalcólico de Thuya occidentalis, poderiam atuar inibindo o desenvolvimento e progressão do câncer de próstata. Neste estudo, com o uso de abordagens bioquímicas e de biologia molecular, foi demonstrado que o tratamento com γ-orizanol diminui a viabilidade e biomassa celular em cultura, associado ao aumento da morte celular por apoptose e/ou necrose, em linhagens celulares responsivas (LNCaP) e não-responsivas a andrógenos (PC3 e DU145), além de aumentar a pERK1/2 em células LNCaP e DU145. O γ-orizanol também foi capaz de bloquear o ciclo celular em G2/M nas células PC3 e LNCaP e em G0/G1 nas células DU145. Estes efeitos foram ainda acompanhados por uma redução da expressão do gene e proteína caveolina-1- uma importante molécula envolvida no aumento da agressividade do câncer de próstata, e também, na progressão da doença para o fenótipo andrógeno resistente - nas células não-responsivas a andrógenos, e do gene PCGEM1 - gene específico da próstata regulado por andrógeno - nas células LNCaP e DU145. Ainda, γ-orizanol também mostrou capacidade de regular vários miRNAs - pequenas moléculas de RNA não codificantes de proteínas - envolvidos no controle de funções associadas ao desenvolvimento, progressão e invasão no câncer de próstata, como o miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 e miR222. O extrato hidroalcólico de Thuya occidentalis reduziu a viabilidade e biomassa celular nas linhagens responsiva (LNCaP) e não-responsivas (DU145 e PC3) a andrógenos, além de induzir parada do ciclo celular na fase G0/G1 nas células DU145 e aumentar a morte celular por apoptose e/ou necrose em todas as linhagens. Da mesma forma que o γ-orizanol, este extrato reduziu a expressão da caveolina-1 nas linhagens não-responsivas a andrógenos. Trabalhos anteriores mostram que o monoterpeno α-tujona é o principal composto ativo do extrato de Thuya occidentalis. Por cromatografia gasosa acoplada a detector de massas foi mostrada a existência de 0,0016 μg de α-tujona na dose de extrato usada neste estudo. No entanto, o tratamento com 0,0016μg de α-tujona foi efetivo somente sobre linhagem LNCaP, não tendo efeito sobre as outras linhagens estudadas, reforçando a hipótese da diferença de sensibilidade entre as linhagens responsivas e não responsivas a andrógeno e mostrando a contribuição de outros componentes do extrato nos efeitos observados neste estudo. Concluindo, estes resultados demonstram que tanto γ-orizanol como o extrato de Thuya occidentalis podem vir a ser agentes terapêuticos promissores no tratamento de câncer de próstata, não só por inibirem o crescimento celular, mas também e principalmente pela possibilidade de induzirem a recuperação da sensibilidade a andrógenos, aumentando as possibilidades de tratamento da doença. / Prostate cancer is the second cause of death among men in Brazil. It is a slowgrowing cancer and it may take years for tumor to reach 1 cm3, but in some cases it can spread throughout the body and the bone is the main site of metastasis. At this cancer stage known as metastasis, the principal treatment involves antiandrogen therapy, leading to prostate cells stop proliferating, because they grow in response to presence of androgens such as testosterone and dihydrotestosterone. However, in some cases, the cells can proliferate even in the absence of androgens and this fact occurs due to many factors and they are generally associated with mutations in the androgen receptor and/or alterations in androgen metabolism. In this stage, the treatments available are less effective and usually fail. However, studies suggest that γ-oryzanol, a phytosterol extracted of rice bran oil; and extracts widely used in folk medicine, as Thuya occidentalis hidroalcolic extract, could act inhibiting the development and progression of prostate cancer. In this study, using molecular biology and biochemical approaches we showed that γ- oryzanol treatment was able to decrease cell viability and biomass in culture, and this fact was linked to increased cell death by apoptosis and/or necrosis in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) prostate cancer cell lines, besides increasing pERK1/2 in LNCaP and DU145 cells. γ- oryzanol was also able to cause cell cycle arrest at G2/M phase in LNCaP and PC3 cells and at G0/G1 phase in DU145 cells. These effects were also accompanied by a reduction in caveolin-1 gene and protein expression - an important molecule related to high aggressiveness in prostate cancer and also in the progression of the disease to androgen resistant phenotype - in androgen unresponsive cells, and also PCGEM1 gene - a prostate specific gene regulated by androgens - in LNCaP and DU145 cells. γ-oryzanol also showed ability to regulate several miRNAs - small non-coding RNA molecules - involved in the control of many functions associated with the development, progression and invasion of prostate cancer, such as miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 and miR222. Thuya occidentalis hidroalcolic extract also reduce cell viability and biomass in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) cells, in addition to inducing cell cycle arrest at G0/G1 phase in DU145 cells and to increase apoptosis and/or necrosis cell death in all cell lines. The same way that γ-oryzanol, this extract reduced the caveolin-1 expression in androgen unresponsive prostate cancer cells. Prior studies showed that the monoterpene α-thujone is the main active compound in the T. occidentalis extract. By gas chromatography coupled to mass detector it was showed the existence of 0.0016 μg of α-thujone in extract dose used in this study. However, the treatment with 0.0016 μg of α-thujone was effective only on LNCaP cell line, having no effect on the other studied lines, supporting the hypothesis of difference in sensitivity between responsive and unresponsive cell lines and showing the contribution of other components in the effects caused by the extract, observed it this study. In conclusion, these results demonstrate that both γ-oryzanol as T. occidentalis extract may become promising therapeutic agents in treatment of prostate cancer, not only inhibit cell growth but also and manly by the possibility of inducing the recovery of androgen sensitivity, increasing the treatment chances of treatment this disease.

O câncer de próstata é a segunda causa de morte entre homens no Brasil. É tipo um câncer de crescimento lento, podendo levar anos para o tumor atingir 1 cm3, porém, em alguns casos ele pode se espalhar pelo corpo, sendo o osso o principal sítio de metástase. No estágio de desenvolvimento do câncer conhecido como metástase, o principal tratamento consiste em terapia de restrição andrógena, levando as células prostáticas a pararem de proliferar, uma vez que elas crescem em resposta a presença de hormônios andrógenos, como a diidrotestosterona e testosterona. Porém, em alguns casos, as células proliferam mesmo na ausência de andrógenos e isto se deve a diversos fatores que, em geral, estão associados a mutações no receptor andrógeno e/ou alterações no metabolismo andrógeno. Quando isto acontece, os tratamentos disponíveis são menos efetivos e costumam falhar. Porém, estudos sugerem que o γ-orizanol, um fitoesterol extraído do óleo do farelo do arroz; e extratos amplamente utilizados na medicina popular, como o extrato hidroalcólico de Thuya occidentalis, poderiam atuar inibindo o desenvolvimento e progressão do câncer de próstata. Neste estudo, com o uso de abordagens bioquímicas e de biologia molecular, foi demonstrado que o tratamento com γ-orizanol diminui a viabilidade e biomassa celular em cultura, associado ao aumento da morte celular por apoptose e/ou necrose, em linhagens celulares responsivas (LNCaP) e não-responsivas a andrógenos (PC3 e DU145), além de aumentar a pERK1/2 em células LNCaP e DU145. O γ-orizanol também foi capaz de bloquear o ciclo celular em G2/M nas células PC3 e LNCaP e em G0/G1 nas células DU145. Estes efeitos foram ainda acompanhados por uma redução da expressão do gene e proteína caveolina-1- uma importante molécula envolvida no aumento da agressividade do câncer de próstata, e também, na progressão da doença para o fenótipo andrógeno resistente - nas células não-responsivas a andrógenos, e do gene PCGEM1 - gene específico da próstata regulado por andrógeno - nas células LNCaP e DU145. Ainda, γ-orizanol também mostrou capacidade de regular vários miRNAs - pequenas moléculas de RNA não codificantes de proteínas - envolvidos no controle de funções associadas ao desenvolvimento, progressão e invasão no câncer de próstata, como o miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 e miR222. O extrato hidroalcólico de Thuya occidentalis reduziu a viabilidade e biomassa celular nas linhagens responsiva (LNCaP) e não-responsivas (DU145 e PC3) a andrógenos, além de induzir parada do ciclo celular na fase G0/G1 nas células DU145 e aumentar a morte celular por apoptose e/ou necrose em todas as linhagens. Da mesma forma que o γ-orizanol, este extrato reduziu a expressão da caveolina-1 nas linhagens não-responsivas a andrógenos. Trabalhos anteriores mostram que o monoterpeno α-tujona é o principal composto ativo do extrato de Thuya occidentalis. Por cromatografia gasosa acoplada a detector de massas foi mostrada a existência de 0,0016 μg de α-tujona na dose de extrato usada neste estudo. No entanto, o tratamento com 0,0016μg de α-tujona foi efetivo somente sobre linhagem LNCaP, não tendo efeito sobre as outras linhagens estudadas, reforçando a hipótese da diferença de sensibilidade entre as linhagens responsivas e não responsivas a andrógeno e mostrando a contribuição de outros componentes do extrato nos efeitos observados neste estudo. Concluindo, estes resultados demonstram que tanto γ-orizanol como o extrato de Thuya occidentalis podem vir a ser agentes terapêuticos promissores no tratamento de câncer de próstata, não só por inibirem o crescimento celular, mas também e principalmente pela possibilidade de induzirem a recuperação da sensibilidade a andrógenos, aumentando as possibilidades de tratamento da doença. / Prostate cancer is the second cause of death among men in Brazil. It is a slowgrowing cancer and it may take years for tumor to reach 1 cm3, but in some cases it can spread throughout the body and the bone is the main site of metastasis. At this cancer stage known as metastasis, the principal treatment involves antiandrogen therapy, leading to prostate cells stop proliferating, because they grow in response to presence of androgens such as testosterone and dihydrotestosterone. However, in some cases, the cells can proliferate even in the absence of androgens and this fact occurs due to many factors and they are generally associated with mutations in the androgen receptor and/or alterations in androgen metabolism. In this stage, the treatments available are less effective and usually fail. However, studies suggest that γ-oryzanol, a phytosterol extracted of rice bran oil; and extracts widely used in folk medicine, as Thuya occidentalis hidroalcolic extract, could act inhibiting the development and progression of prostate cancer. In this study, using molecular biology and biochemical approaches we showed that γ- oryzanol treatment was able to decrease cell viability and biomass in culture, and this fact was linked to increased cell death by apoptosis and/or necrosis in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) prostate cancer cell lines, besides increasing pERK1/2 in LNCaP and DU145 cells. γ- oryzanol was also able to cause cell cycle arrest at G2/M phase in LNCaP and PC3 cells and at G0/G1 phase in DU145 cells. These effects were also accompanied by a reduction in caveolin-1 gene and protein expression - an important molecule related to high aggressiveness in prostate cancer and also in the progression of the disease to androgen resistant phenotype - in androgen unresponsive cells, and also PCGEM1 gene - a prostate specific gene regulated by androgens - in LNCaP and DU145 cells. γ-oryzanol also showed ability to regulate several miRNAs - small non-coding RNA molecules - involved in the control of many functions associated with the development, progression and invasion of prostate cancer, such as miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 and miR222. Thuya occidentalis hidroalcolic extract also reduce cell viability and biomass in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) cells, in addition to inducing cell cycle arrest at G0/G1 phase in DU145 cells and to increase apoptosis and/or necrosis cell death in all cell lines. The same way that γ-oryzanol, this extract reduced the caveolin-1 expression in androgen unresponsive prostate cancer cells. Prior studies showed that the monoterpene α-thujone is the main active compound in the T. occidentalis extract. By gas chromatography coupled to mass detector it was showed the existence of 0.0016 μg of α-thujone in extract dose used in this study. However, the treatment with 0.0016 μg of α-thujone was effective only on LNCaP cell line, having no effect on the other studied lines, supporting the hypothesis of difference in sensitivity between responsive and unresponsive cell lines and showing the contribution of other components in the effects caused by the extract, observed it this study. In conclusion, these results demonstrate that both γ-oryzanol as T. occidentalis extract may become promising therapeutic agents in treatment of prostate cancer, not only inhibit cell growth but also and manly by the possibility of inducing the recovery of androgen sensitivity, increasing the treatment chances of treatment this disease.

Herbivory by Western Flower Thrips (Frankliniella occidentalis Pergande) (WFT) and drought-stress due to limited water availability are currently two major factors that can severely impact cotton (Gossypium hirsutum L.) production. This dissertation examines the effects of drought-stress on cotton and host-plant resistance (HPR) to WFT in laboratory conditions, and seeks to identify the physiological and morphological mechanisms that underlie drought-tolerance and HPR. A life-history systems-approach was developed that provides a new level of detail for understanding how environmental variation impacts adult female WFT. The approach was illustrated by investigating the combined effects of cotton genotype, periodic drought-stress, and prey availability on the adult female omnivorous thrips using a factorial design. Three treatment conditions were significantly different, none of which were predicted based on prevailing ecological-hypotheses. At the same time, the approach produced three novel insights about WFT life-history and reproductive strategy. The roles of negative photo-taxis and leaf biomechanical properties were investigated as potential mechanisms that influence WFT foraging-decisions on individual cotyledons. Results showed that WFT foraging-decisions could be considered adaptive, but there was limited support for either of the mechanisms investigated. The physiological responses to drought stress and drought recovery were investigated for three transgenic cotton cultivars and an untransformed wild-type (WT). At peak drought, ABA levels, stomatal area, and stomatal apertures in the transgenic isolone, AtRAV1-1 were 48% lower, 27.7%, and 16.3% smaller than WT. These results suggest that AtRAV1-1 was the most drought-tolerant and support the hypothesis that changes in stomatal morphology may have functionally contributed to drought-tolerance. Lastly, I investigated whether changes in phytohormone concentrations associated with periodic-drought stress in four cotton cultivars (three transgenic and WT) were correlated with WFT feeding, fitness and state-dependent reproductive responses (i.e., the relationship between initial weight and reproduction). Results showed that JA-Ile and JA were positively correlated with state-dependent egg viability and fecundity, respectively, and negatively correlated with total egg viability and fecundity, respectively, supporting the hypothesis that JA and JA-Ile underlie the negative effects on WFT reproduction and the associated shift to state-dependent reproduction.

Ring-width chronologies from Pinus resinosa Ait., Pinus strobus L., and Thuja occidentalis L. were developed in two areas of the Boundary Waters Canoe Area Wilderness to assess their growth climate response and their potential for developing reconstructions of climate. New red pine chronologies were combined with existing chronologies to extend the ring-width record both into the past and into the present. Ring-width response to climate, assessed using correlation analysis and response functions, was broadly similar among all three species with relatively significant positive relationships with June–July precipitation and significant negative (but less consistent) associations with June–July temperatures (p < 0.05). White-cedar appeared to have a broader phenological window of response with a stronger spring influence when compared to other species included in this study. Comparisons with other nearby proxies showed relatively strong coherence overall but with some important regional differences. Overall, these species may be useful for placing current climatic patterns in the Boundary Waters within a longer term perspective but care should be taken with respect to identifying appropriate climatic records for calibration.

I studied the habitat characteristics of Mexican Spotted Owls (Strix occidentalis lucida), a federally threatened species, in the canyonlands region of southern Utah. Vegetative and geologic features were measured within 10m wide belt plots at each current or historic nest/roost site. Based on our findings, past research, and species life history characteristics, I constructed a species distribution model (SDM) predicting Mexican Spotted Owl distribution in Utah for the Colorado Plateau region. The SDM was generated using the following inputs as important habitat variables: elevation, aspect, surface ratio, curvature, slope, geology, and vegetation. Program R was used for model development and generation. The SDM was generated using an ensemble model approach by combining three modeling techniques: random forest, logistic regression, and maximum entropy. This study combines measured habitat characteristics, with sophisticated geographic information system (GIS) tools and SDMs to provide managers with an informative and useful toolkit for Mexican Spotted Owl conservation. Chapter 2 discusses modeling techniques and SDM development. I detail how individual models were constructed using random forest, logistic regression, and maximum entropy and how these were combined into an ensemble model. Final models indicated that several vegetative and geologic characteristics were considered important habitat characteristics for predicting Mexican Spotted Owl presence within the Colorado Plateau. The SDMs produced eight distribution maps predicting Mexican Spotted Owl presence and probability of occurrence in Utah for the Colorado Plateau region. Chapter 3 explains the use of SDMs by managers and synthesizes findings of measured habitat characteristics for southern Utah. For habitat characteristics I measured a combination of vegetative and geologic features within 10m wide belts at current and historic Mexican Spotted Owl sites. Vegetative features measured included: height and species of all trees and shrubs, position of tree or shrub within plot, presence of canopy cover, and tree diameter at breast height (DBH). Geologic features measured included: geologic formation type, wall height, structure type, number of caves, and number of solution cavities. I found that canyon width and density of vegetation > 2.5 m tall were significantly correlated with Mexican Spotted Owl presence.

[Truncated abstract] Eucalyptus occidentalis, a timber species from south Western Australia, is highly salt and waterlogging tolerant. Screening identified genotypes tolerant of high salt concentrations and waterlogging. Tolerance at provenance, family and individual level, and how phenotypic performance under salt and waterlogging was inherited was explored to provide a breeding population. Salt and/or waterlogged screening was carried out under controlled conditions up to extreme salt levels to determine tolerance between genotypes. This tank method was shown to produce repeatable results. Seedlings of 30 families from 9 provenances were used for screening. At low salt concentration (up to 300 mM NaCl), differentiation occurred for some traits but in general there was only a slight reduction in growth under salt, and waterlogging alone was not detrimental. At high salt concentration (550 mM) differentiation occurred among genotypes for all traits. Equivalent genotypes were also planted in field trials at three sites, two with medium (583 - 847 mm) and one with low rainfall (372 - 469 mm), in southern Western Australia. Survival was low (<53%) after 9 months due to an exceptional dry season followed by 3 months waterlogging in Kirkwood (38 - 1360 mSm-1), but was high >89% after 33 months in saline fields in Sandalwindy (96 - 976 mSm-1) and Roberts (88 - 1424 mSm-1). Some families were similarly in high rank for height under saline conditions in controlled and field trials. Height had the highest narrow-sense heritability value, especially under controlled saltwaterlogging (0.85) treatment and 20% selection enabled a gain of 8-14% under controlled conditions and in the field. Leaf production under salt was not an inherited trait. Systems were developed to hasten deployment of selected material. Extended daylength (16 h) and paclobutrazol (1 mg a.i/mm stem circumference) stimulated flowering in 2 year-old plants. Clonal propagation was possible. Grafting success varied from 0-100% depending on scion/rootstock provenances. ... There was only a slight reduction in heterozygosity from species level to provenance and family levels, and two superior genotypes maintained high diversity. v Crossing was possible using one stop pollination of cut immature styles and capsule retention varied from 0-34% and germination rate from 2-96%. Genetic distance between parents was correlated with seed set and offspring fitness. Wider genetic distances increased capsule retention, seed germination and seedling survival. Under 500 mM salt-waterlogging, offspring heights were similar when parental genetic distances were similar. High heritability value for height from ANOVA-REML parental screening was confirmed using parent-offspring regression. Screened superior genotypes, which withstood very high salt concentration, provide a breeding population for further breeding and for plantations under saline regions in low-medium rainfall areas in Western Australia and other parts of the world. These trees provide an economic return in areas where no other plants may survive and an environmental service in potentially reducing waterlogging, salinity and its spread.