Investigations of structural and functional relationships of rat transient receptor potential cation channel, subfamily V, member 1 (TRPV1), also known as the capsaicin receptor, and human transient receptor potential cation channel, subfamily A, member 1, also known as TRPA1, are presented. Capsaicin induced Ca2+ -dependent desensitization of rat TRPV1 channel is studied and lead to the identification of key amino acid residues in the C- terminal domain of TRPV1 interacting with the membrane phospholipid PIP2 and an intradomain interaction that controls the open and desensitized state of the TRPV1 channel. Further the molecular basis of agonist AITC- and voltage-dependent gating on TRPA1 is explained. Hereby, residue P949 located near the center of the sixth transmembrane spanning helix (S6) is structurally required for normal functioning of the receptor and the distal bi-glycine G958XXXG962 motif controls its activation/deactivation properties. Furthermore, the gating region is extended towards the cytoplasmic part of the channel, putatively located near the inner mouth of the channel pore. A following series of experiments lead to the identification of a limited number of residues that appear important for allosteric regulation of the channel by chemical and voltage stimuli (K969, R975, K989, K1009, K1046, K1071, K1092 and K1099). In addition, three charge-neutralizing `gain-of- function{\crq} mutants (R975A, K988A, and K989A) which exhibited higher sensitivity to depolarizing voltages were characterized, indicating that these residues are directly involved in voltage-dependent modulation of TRPA1.

Os receptores acoplados à proteína G (GPCRs) são proteínas integrais de membrana caracterizados por possuírem sete alfa-hélices transmembranares. Esses receptores são importantes alvos de estudos biomédicos e aproximadamente 40% dos medicamentos atualmente comercializados agem sobre estes receptores. O receptor de Angiotensina II do tipo 1 (AT1) é um GPCR e o principal mediador do Sistema Renina-Angiotensina que tem como principal efetor o octopeptídeo Angiotensina II (AngII). Recentemente foi descrito que as mutações A244S e I103T-A244S no receptor AT1 podem estar relacionadas com a predisposição à fibrilação atrial. Neste trabalho foi realizada a construção, caracterização bioquímica e farmacológica destes mutantes, bem como do mutante I103T, com o objetivo de compreender como a funcionalidade desses receptores mutantes poderiam contribuir para a predisposição à fibrilação atrial. Os mutantes I103T e I103T-A244S revelaram ser mais eficientes e potentes que o receptor selvagem em aumentar os níveis de cálcio intracelular em resposta à AngII. Todos os mutantes estudados apresentaram baixa eficiência quanto à ativação da via das MAPKs e apresentaram comportamento diferente do receptor selvagem quando bloqueados com o antagonista Losartan, seletivo para o receptor AT1 e muito usado na clínica como medicamento anti-hipertensivo. Esses dados ressaltam a relevância do estudo tanto em termos de melhor compreender as bases moleculares da relação entre as mutações e a doença, bem como possível prevenção ao uso de medicamentos que possam interagir e agir diferentemente em receptores com essas mutações. / G-protein coupled receptors (GPCRs) are integral membrane proteins characterized by having seven transmembrane alpha-helices. These receptors are important targets of biomedical studies and approximately 40% of currently marketed drugs act on such receptors. The angiotensin II type 1 receptor (AT1) is a GPCR and the main mediator of the Renin-Angiotensin System whose main effector is the octapeptide Angiotensin II (Ang II). It was recently described that I103T and A244S mutations in the AT1 receptor may be related to the susceptibility to atrial fibrillation. In this study we carried out the construction of these mutants and their biochemical and functional characterization. The I103T and I103T/A244S mutants were shown to be more efficient and potent than the wild-type receptor on the increase of intracellular calcium levels. All mutants showed lower efficcacy for MAPK pathway activation and showed different behavior when compared to the wild-type receptor after antagonism with Losartan. These data highlight the relevance of the present study concerning a better understanding of the molecular basis of cardiovascular diseases and showing that conventional therapies for certain diseases may lead to adverse effects on patients carrying point mutations on the receptor sequence.

Ischemic heart disease represents a leading cause of death worldwide. Ischemia denotes an insufficient supply of oxygenated blood to the heart due to occlusion of the coronary vessels. Timely reperfusion, i.e., restoring blood flow to the ischemic part of the heart, limits ischemic damage. However, reperfusion itself induces injury to the heart. This phenomenon is referred as ischemia/reperfusion (I/R) injury. Osteopontin (OPN), also known as cytokine Eta-1, is a cell-secreted extracellular matrix protein. Expression of OPN increases in the heart in response to a variety of pathological conditions. Mice lacking OPN exhibit exaggerated left ventricular dilation in non-reperfused model of myocardial remodeling. Cardioprotective role of OPN has also been demonstrated in a mouse model of repetitive I/R injury for 7 days. The objective of this study was to examine the role of OPN in modulation of systolic and diastolic parameters of the heart following I/R injury in a time-dependent manner. For this study, wild type (WT) and OPN knockout (KO) mice aged ~4 months were subjected to cardiac ischemia by the ligation of left anterior descending coronary artery (LAD). Following 45 min of ischemia, the LAD was reperfused by snipping the ligature. Heart function was measured using echocardiography at baseline, 3, 7, 14, and 27 days following I/R injury. M-mode echocardiographic images were used to calculate the systolic parameters (% fractional shortening [%FS], % ejection fraction [%EF], and end-systolic volume [ESV]), while pulse wave Doppler images were used to calculate diastolic parameter (aortic ejection time; [AET]). Global cardiac function was evaluated using myocardial performance index (MPI; a Doppler-derived index which combines systolic and diastolic functions). At basal levels, most of the systolic and diastolic parameters remained unchanged between the two groups. I/R injury decreased %FS and EF in both groups vs the baseline values at 3, 7, 14 and 27 days post-I/R. However, the decrease in %FS and EF was significantly greater in KO-I/R vs WT-I/R group. ESV was significantly higher in WT mice 7 days post-I/R, and stayed higher 14 and 27 days post-I/R vs baseline. However, the increase in ESV was significantly greater in KO mice 3 day post-I/R, and remained higher vs WT-I/R during the time course. AET was lower in WT group 14 days post-I/R vs baseline. On the other hand, AET was significantly lower in KO group 3, 7, 14 and 27 days post-I/R vs WT-I/R. MPI was higher in WT group 7 days post-IR vs baseline. MPI decreased significantly in WT group 27 days vs 7 days post-I/R. In KO group, MPI was significantly higher than WT mice at baseline, and remained higher 3 and 27 day post-I/R vs WT-I/R. Thus, lack of OPN decreases systolic and diastolic functional parameters of the heart following I/R injury, suggesting a cardioprotective role of OPN in myocardial remodeling post-IR.

<p dir="ltr">Bat population numbers are declining in the Midwestern United States. Reasons for decline are multifaceted (habitat degradation, fatalities at wind turbines, White Nose Syndrome, and declining insect populations), and many species are listed as endangered (Myotis sodalis, Myotis septentrionalis, Perimyotis subflavus). Other species in the Midwestern United States have no conservation status (Eptesicus fuscus), or are only listed as a species of concern (Lasiurus cinereus, Lasiurus borealis, Lasionycteris noctivagans). Bats play a crucial role in our ecosystems, providing both ecological and economic benefit as pollinators and insect population regulators. Thus, conserving these species is vital. To gain better insight into the ecology of Midwestern bat species I studied five species in two respects. First, I investigated the availability of roosts for a colony of endangered Myotis sodalis near Indianapolis. This colony has withstood high levels of urbanization and habitat degradation. Therefore, understanding what aspects of the roosting area has allowed for continued use by the colony is crucial for future conservation efforts. I used an Akaike’s Information Criteria approach to rank models that best differentiate between the current roosting area and surrounding landscape. I identified that the roosting area contained a greater number of large standing dead trees (Snags >42.6 cm) that are able to serve as primary roosts for the colony. These results demonstrate that a colony of Indiana bats may be able to withstand urbanization if they have enough large DBH snags available in the area. Future conservation efforts in a heavily urbanized environment should focus on the maintaining large snags as primary roosts. Second, I studied the morphological variation of four species (Lasiurus cinereus, Lasiurus borealis, Lasionycteris noctivagans, and Eptesicus fuscus) to parse out differences that may lead to niche specialization. These four species share habitat and foraging range and therefore may directly compete for resources. However, despite declining insect populations these four species have moderately stable populations. I collected approximately 30 craniums and mandibles for each species and compared the linear size differences between landmarks and the overall shape variation from these landmarks. Due to different phylogenies and body sizes, the four species were different from one another in all 24 linear measurements. In regards to shape variation, the Eptesicini bat craniums had characteristics of more gracile species. In addition, the mandibles of Eptesicini were highly distinct. Lasionycteris noctivagans was the least durable and Eptesicus fuscus had 10 specialization for hard bodied prey consumption. These results suggest potential niche specialization due variation in morphology.</p>

Ketohexokinase (KHK), in an adenosine triphosphate (ATP) dependent reaction, catalyzes the first reaction in fructose metabolism, which converts the furanose form of D-fructose into fructose-1-phosphate. This enzyme has become a target for pharmacological development against fatty liver and metabolic syndrome. KHK exists in two isoforms, A and C, which differs by alternative splicing of exon 3 which encodes 45 out of 298 amino acids. Normally KHK exists as a homodimer and is comprised of an alpha/beta domain interlocking with a β-clasp domain. For KHK-C, there appear to be at least two conformations of the β-clasp domain. Previous work on KHK-A reveals it does not adopt the same conformations. A structure of the mouse KHK-A in its unliganded form is solved and shows that these two conformations also exist for KHK-A. Furthermore, this property is conserved across species. While crystals of human KHK-A in its unliganded form were grown, a structure was not achieved. However, unpublished structures of human KHK-A in its unliganded form also shows different conformations in β-clasp domain when in juxtaposition with the same enzyme complex with ligands. Defining the role of conformational changes in KHK-A is important, because this isozyme has been reported to have a role in cancer metastasis.

No description available.

Streams and terrestrial ecosystems are linked through allochthonous organic matter inputs from streamside vegetation. This allochthonous material makes up the energy base for forested aquatic food webs. Therefore, removal of riparian vegetation associated with agricultural land use affects stream ecosystem structure and function. The objectives of this study were to measure and compare allochthonous input and leaf breakdown rates along a gradient of agricultural land use in southern Appalachian streams. Study streams were placed into the following land use categories: forest and light, moderate, and heavy agriculture. Several physical, chemical, and biological parameters also were measured including discharge, temperature, nutrient concentrations, macroinvertebrate abundance and density, periphyton biomass, and chlorophyll a concentration. In forested, light agricultural, and moderate agricultural streams, the quantity and quality of allochthonous input were not significantly different. However, the timing and composition of allochthonous materials were related to land use. Chlorophyll a and periphyton biomass did not vary among land use types. Leaf breakdown rates were significantly faster in light and moderate agricultural streams in comparison to forested and heavy agricultural streams. Slow breakdown rates in forested streams resulted from low nutrient concentration and cool stream temperature. The scarcity of shredding macroinvertebrates and sedimentation probably limited leaf breakdown in heavy agricultural streams. Though limited riparian vegetation along agricultural streams resulted in an energy supply equivalent to forested streams, agricultural land use may still have long term impacts on stream structure including nutrient concentrations, temperature, macroinvertebrate community, and sedimentation thus affecting stream ecosystem function. / Master of Science

A importância das matas ciliares para a manutenção de diversas funções ecológicas e hidrológicas, reconhecida a ponto de tais formações receberem proteção legal específica, não impediu que estas fossem destruídas indiscriminadamente durante o processo de ocupação do território brasileiro. Estima-se que, só no Estado de São Paulo, cerca de 600 mil hectares de áreas marginais aos corpos d\'água encontram-se ocupados com outros usos que não a vegetação natural, necessitando, portanto, de ações de recuperação. Embora exista um número significativo de trabalhos de revegetação de áreas degradadas, principalmente em matas ciliares, poucos são os resultados de pesquisas sobre o desenvolvimento a longo-prazo de plantios de recomposição de mata ciliar. Nem os plantios mais antigos, aleatórios, e nem os mais recentes, baseados nos processos sucessionais, têm sido submetidos a análises que possibilitem avaliar a eficácia destes métodos de plantio na restauração da mata ciliar com sua forma e funções originais. O objetivo geral desta pesquisa foi o de avaliar a auto-sustentabilidade e as tendências sucessionais de uma mata ciliar implantada, com base em aspectos da estrutura e funcionamento da floresta em trechos de diferentes idades. Neste sentido avaliou-se a floresta existente atualmente, do ponto de vista da composição e estrutura da comunidade; caracterizou-se e quantificou-se o estrato regenerativo da floresta, visando compreender os processos de regeneração natural;caracterizou-se a sazonalidade da queda de folhedo através de sua quantificação periódica e sua contribuição para o retorno de nutrientes ao solo; caracterizou-se o solo sob a floresta, e comparou-se os resultados com área não reflorestada; comparou-se a floresta implantada com florestas naturais através dos parâmetros analisados: riqueza e diversidade florística do estrato arbóreo, densidade absoluta, densidade relativa, área basal, estratificação da floresta, similaridade florística, cobertura das copas, regeneração natural, características químicas do solo e produção e conteúdo de nutrientes no folhedo. Analisando-se as áreas reflorestadas como um todo, constata-se a existência de uma mata ciliar exuberante e fisionomicamente bem estruturada, cujo papel de proteção aos recursos hídricos e ao solo tem sido desempenhado de forma evidente. Alguns resultados obtidos são de extrema importância: no setor de 28 anos, 69% das árvores amostradas surgiram espontaneamente e apenas 31% são indivíduos plantados, demonstrando que está havendo um aumento natural da riqueza florística, o que prova que a natureza encarrega-se de perpetuar a floresta fazendo os \"ajustes\" necessários. Processos ecológicos como a dispersão de sementes e a regeneração natural estão incorporados na dinâmica florestal do local, podendo-se afirmar que a auto-sustentabilidade florestal da comunidade que se formou a partir do plantio de mata ciliar já está garantida. A hipótese deste trabalho foi confirmada pelos resultados obtidos: com o decorrer do tempo a mata ciliar implantada tende a se tornar cada vez mais semelhante à mata ciliar natural, em estrutura e função. / The importance of riparian forests to maintain the many hydrologic and ecological functions, acknowledged to the point of receiving specific legal protection, did not prevent their indiscriminate destruction during the process of land settlement in Brazil. It is estimated that in São paulo state alone about 600 thousands hectares of stream-side areas have been occupied by other uses rather than natural vegetation, therefore, requiring actions of restoration. Although there is a significant number of studies in restoration of degraded areas mainly in riparian forests, the results in research on development of restoration planting of riparian forests in the long term are few. Neither older random plantings nor more recent ones based on sucession processes have been subjected to analysis that would make it possible to evaluate the efficacy of these methods of planting for restoration of riparian forest with its original form and functions. The general aim of this study was to evaluate self-sustainability and sucession tendencies of a planted riparian forest, based on aspects of structure and functioning of the forest in patches of different ages. Thus, the current existing forest was evaluated regarding composition and structure of the community; the regenerative stratum of the forest was characterized and quantified with the purpose of understanding natural regeneration processes; the seasonability of fall foliage through its periodic quantification and itscontribution to the return of nutrients to the soil was characterized; the soil underneath the forest was characterized, and the results in areas that were not restored were compared; planted forests and natural forests were compared through parameters analyzed: richness and floristic diversity of the tree stratum, absolute density, relative density, basal area, forest stratification, floristic similarity, canopy cover, natural regeneration, chemical characteristics of the soil and production and nutrients content in the foliage. By analyzing restored areas as a whole, we can confirm an exuberant and physiognomically well structured riparian forest, whose role of protecting hydric resources and the soil has been easily noticed. Some ecological indicators assessed such as the abundant natural regeneration existing under the planted forest and the spontaneous development of new species show that there has been a natural increase in floristic richness, which proves that nature manages to perpetuate forests by making the necessary adjustments. The hypothesis of this work was confirmed by the results obtained: with time the planted forest tends to become more and more similar to the natural forest, both in structure and function.

Esta pesquisa tem como enfoque as personagens femininas de Graciliano Ramos em suas obras Caetés, São Bernardo, Angústia, Vidas Secas e Infância. Partindo dos conceitos de Antonio Candido sobre estrutura e função, busca-se analisar como a construção literária se relaciona com os dados sociais e históricos da época em que os livros foram escritos e/ou publicados, a década de 1930. A partir das análises feitas, desenvolve-se a ideia da importância dessas personagens que, mesmo deixadas em segundo plano, são essenciais para a construção dos textos literários. / The following research focuses on the female characters of Graciliano Ramos in his works Caetés, São Bernardo, Angústia, Vidas Secas and Infância. Thinking about the concepts of Antonio Candido on structure and function, we analyze how the literary construction relates to the social and historical life in the moment that the books were written, the decade of 1930. From the analyzes made, we realized the importance of these female characters, even when left in the background.

Pentameric ligand-gated ion channels (pLGICs) are membrane bound receptors found in the nervous system. They are responsible for detecting neurotransmitters released from neurons and subsequently mediating responses of the cells on which they are found. Thus, pLGICs play an invaluable role in communication between cells of the nervous system and understanding their function is pivotal to understanding how the nervous system works in general. One factor which is known to mediate pLGIC function is lipids found in the membrane environment in which pLGICs are embedded. This dissertation explores the various ways in which lipids interact with and modulate the function of pLGIC. Potential mechanisms and biological consequences of this modulation will be presented and discussed within the context of our current state of knowledge of pLGIC and nervous system function.