Triptofano (TRP), um amino ácido essencial, é metabolizado por duas vias principais, a via das quinureninas e a via serotonérgica. Em ambas as vias há a possibilidade de formação de compostos ativos no sistema imune que se caracterizam pelas ações imunossupressoras e indutoras de tolerância. Na via serotonérgica há a formação de serotonina (5-HT) e em alguns tecidos de melatonina (MEL). Este composto pode ainda ser oxidado por ação de peroxidases aos seus produtos de abertura de anel indólico o AFMK (N1-acetil-n2-formil-5-metoxiquinuramina) e AMK (N1-acetil-5-metoxiquinuramina). Já na via das quinureninas, o TRP é diretamente metabolizado à N-formilquinurenina (NFK) e este é rapidamente deformilado a quinurenina (QUIN). Neste projeto avaliamos qual o efeito do choque endotóxico induzido por injeção endovenosa de LPS (1 mg/kg) sobre a biodisponibilidade de TRP e formação de seu metabólito QUIN. Este estudo foi realizado em condições controle e na vigência de sobrecarga de TRP (administração subcutânea de 0,8 mg/kg). Utilizamos ratos machos Wistar com 30 dias separados em quatro grupos: GI (controle), GII (LPS), GIII (TRP) e GIV (TRP+LPS). TRP (0,8 mg/Kg) foi injetado por via subcutânea nos tempos 0 e 2 horas. Quando injetado, LPS (1 mg/kg) foi administrado por via intravenosa no tempo 2 horas. Após 1 hora da última administração, sangue e cérebro foram coletados. O cérebro foi seccionado em três regiões: cerebelo, córtex e mesencéfalo, os quais foram processados para obtenção de homogenatos. Tanto os homogenatos quanto o soro foram tratados com acetona para extração de TRP e seus metabólitos. A análise destes compostos foi realizada por cromatografia líquida de alta eficiência (HPLC). A administração de TRP elevou significativamente a sua concentração no soro e no SNC. Quando da administração de LPS no grupo que já havia recebido sobrecarga de TRP (GIV) houve uma marcada elevação de TRP e de QUIN séricos e das regiões do SNC, especialmente na região do córtex. Concluímos que na vigência de choque endotóxico há um aumento da biodisponibilidade de TRP, tanto no soro como no SNC e que há um aumento da metabolização deste pela rota das quinureninas, possivelmente via IDO. Estes resultados contribuem para a compreensão da toxicidade de TRP, especialmente relevante no caso em que haja um choque endotóxico concomitante e evidencia o córtex como uma região mais susceptível para os efeitos tóxicos do TRP. / Tryptophan (TRP) is an essential amino acid, metabolized by two main paths; the kynurenine and the serotonergic pathways. In both, there is the possibility of generation of biologic active compounds, especially on the immune system leading to immunosuppression and tolerance. In the serotonergic path there is the formation of serotonine (5-HT) and in some tissues of melatonine (MEL). The latter can be oxidized by the action of peroxidases to its indole ring opening product AFMK (N1-acetil-n2-formil-5-methoxikynuramine) and AMK (N1-acethyl-5-methoxykynuramine). In the kynurenine path, TRP is metabolized to N-formylkynurenine (NFK) that is deformilated to kynurenine (KYN). In this study we evaluated the effect of a endotoxic skock induced by an intravenous injection of LPS (1 mg/kg) on the bioavailability of TRP and formation of KYN. This study was carried out in control conditions and on TRP overload (subcutaneous administration of 0,8 mg/Kg). One month old male Wistar rats were divide in four groups: GI(control), GII(LPS), GIII(TRP) and GIV (TRP+LPS). TRP (0,8 mg/kg) was subcutaneously injected at zero and 2h times. When injected, LPS (1mg/kg) was intravenously administered at 2 h. After one hour from the last administration, blood and brain were collected. Brain is separated in cerebellum, midbrain and cortex and was lysed for the preparation of homogenates. Both, serum and homogenates were extracted in acetone; TRP and KYN were analyzed by HPLC. TRP overload caused a significant increase in its concentration in serum and brain. When LPS was administered in conjunction with TRP overload (GIV) there was a remarkable increase in TRP and KYN in serum and brain, especially in cortex. Our conclusion is that in the bioavailability of TRP, in serum and in brain, and its metabolization to kynurenine is increased by inflammation. IDO is probably involved in this condition. Our results contribute to the knowledge of TRP toxicity, particularly with a concomitant inflammation and demonstrate the cortex as a region of more susceptibility to TRP toxicity.

Štěpána Boukalová Mechanisms of activation and modulation of vanilloid TRP channels TRPV1 and TRPV3 are thermosensitive ion channels from the vanilloid subfamily of TRP receptors. TRPV1, which is primarily expressed in nociceptive sensory neurons, is an important transducer of painful stimuli and is also involved in the detection of noxious heat. TRPV3 is expressed mainly in the skin where it regulates proliferation and differentiation of keratinocytes. Similarly to voltage-dependent potassium (Kv) channels, TRP receptors are comprised of four subunits, each with six transmembrane segments (S1-S6). Using mutational approach, we tried to elucidate the role of S1 in TRPV1 functioning. Our results indicate that the extracellular portion of S1 plays a crucial role in TRPV1 gating. TRPV1 channels with a conservative mutation of positively charged residue in this region (R455K substitution) were overactive. However, they were neither activated nor potentiated by low pH; on the contrary, protons stabilized the closed conformation of this mutant channel. Very similar phenotypic properties were found in other TRPV1 mutants with substitution in S4/S5-S5 region and in the pore helix. In Kv channels, extracelular portion of S1 forms a small contact surface with the pore helix, which allows efficient transmission of...

O surgimento e a evolução da vida na terra foram possíveis graças ao desenvolvimento de mecanismos temporais precisos capazes de ajustar os processos fisiológicos que ocorriam no interior do organismo com os ciclos ambientais, promovendo assim, ganhos na capacidade adaptativa e reprodutiva dos indivíduos. Neste contexto, luz e temperatura são as duas pistas temporais mais relevantes para resetar o relógio endógeno e, aparentemente, esses dois zeitgebers trabalham juntos para manter os ritmos circadianos. Uma ampla gama de fotorreceptores e fotopigmentos evoluiu no sentido de perceber com alta sensibilidade a informação fótica fornecida pelo ambiente e, recentemente, foi demonstrado que a detecção de temperatura também pode ser exercida pelos fotopigmentos rodopsina e melanopsina, sendo mediada por canais TRP (Shen et al., 2011). Consideramos as células B16-F10 Per1::Luc como um modelo promissor para o estudo de luz e temperatura em relógios periféricos, uma vez que essa linhagem expressa os dois fotopigmentos apontados com função de termorreceptores em Drosophila. Nossos estudos nos permitiram verificar que a luz não atua como um agente sincronizador nessas células, que se mantiveram em livre curso mesmo após um pulso de 10 min de luz azul (650 lux). Por outro lado, um pulso de temperatura de 2,5º C acima da temperatura de manutenção por 1h atuou ajustando a expressão do gene Per1, imprimindo um ritmo circadiano, diferentemente do observado no controle. Com base nessas informações, hipotetizamos que a informação de luz, percebida via melanopsina na retina de mamíferos, levaria a regulação da temperatura circadiana pelo NSQ, e a temperatura corporal, por sua vez, poderia atuar como uma pista interna para a sincronização dos tecidos periféricos, tendo os canais TRP como mediadores. Para responder esta questão, utilizamos camundongos WT e TrpV1 KO submetidos a diferentes protocolos de luz e avaliamos a expressão de genes do relógio Per1, Per2, Clock e Bmal1 e dos canais TrpV1 e TrpA1 em tecidos periféricos. Identificamos que a glândula suprarrenal, fígado e tecido adiposo marrom possuem uma maquinaria do relógio tipicamente ativa e acreditamos que a oscilação dos genes de relógio observada nesses tecidos é expressiva. Interessantemente, vimos também que o TrpV1, além de ser expresso nos tecidos analisados em animais WT, apresenta uma transcrição rítmica no fígado e tecido adiposo marrom de animais em LD, corroborando nossa hipótese de que canais TRP atuam como mediadores da informação de luz aos tecidos periféricos. Dadas as diferenças encontradas entre os animais WT e TrpV1 KO, sugerimos que a presença do canal TRPV1 pode ser essencial, embora seu grau de envolvimento varie de acordo com o tecido. No que diz respeito ao canal TRPA1, encontramos dois resultados que merecem ser destacados. Primeiramente, identificamos no fígado de camundongos TrpV1 KO mantidos em LD uma provável compensação da expressão de TrpA1 na ausência de TrpV1 e, curiosamente, que o tecido adiposo marrom não expressa o canal TrpA1. Considerando os resultados deste trabalho sobre o envolvimento dos canais TRP em resposta à luz e temperatura, acreditamos ter fortalecido nossa hipótese inicial, principalmente após demonstrarmos o papel do canal TRPV1 e que tecidos periféricos são sincronizados por alterações de temperatura. / The life emergence and evolution on Earth were made possible by the development of precise temporal mechanisms able to adjust the physiological processes within an organism with environmental cycles, thus promoting gains in the adaptive and reproductive capacity of the individuals. In this context, light and temperature are the two most relevant time cues to reset the endogenous clock; apparently these two zeitgebers work together to keep the circadian rhythms. A wide variety of photoreceptors and photopigments evolved in order to precisely perceive the photic information provided by the environment, and recently it has been shown that the temperature detection can also be exerted by the photopigments rhodopsin and melanopsin, being mediated by TRP channels (Shen et al., 2011). We have identified B16-F10 Per1::Luc cells as a promising model for the study of light and temperature effects on peripheral clocks, since this cell line expresses both photopigments pointed as thermoreceptors in Drosophila. Our studies allowed us to demonstrate that light does not act as a synchronizing agent on those cells, which remained in free running after a 10 min pulse of blue light (650 lux). On the other hand, a temperature pulse of 2.5º C above the maintenance temperature, for 1h, adjusted Per1 gene expression, imprinting a circadian rhythm, which was not observed in the control. Based on this information, we hypothesized that the light perceived via melanopsin by the mammalian retina would lead to the regulation of the circadian temperature by the SCN, and the body temperature, in turn, could act as an inner cue for the synchronization of the peripheral tissues, having the TRP channels as mediators. To answer this question, we have used WT and TrpV1 KO mice under different light protocols and evaluated the expression of clock genes Per1, Per2, Clock and Bmal1 and TrpV1 and TrpA1 channels in peripheral tissues. We found that the adrenal gland, liver and brown adipose tissue have a typically active clock machinery, and the oscillation of clock genes observed in these tissues is significant. Interestingly, we observed that TrpV1 is expressed in those tissues, and presents a rhythmic transcription in the liver and brown adipose tissue of LD maintained animals, confirming our hypothesis that TRP channels act as mediators of light information to peripheral tissues. In face of the differences between WT and trpV1 KO animals, we suggest that the presence of the TRPV1 channel may be essential, although its degree of involvement may vary according to the tissue. In terms of TRPA1 channel, we found two results that deserve to be highlighted. Firstly, we identified in the liver of TrpV1 KO mice maintained in LD a presumable compensation of TrpA1 expression in the absence of TrpV1 and, interestingly, the brown adipose tissue does not express TrpA1 channel. Considering the findings of this study on the participation of TRP channels in responses to light and temperature, we believe we have strengthened our initial hypothesis, especially after we have demonstrated the role of TRPV1 channel, and that peripheral tissues may be synchronized by temperature changes.

O surgimento e a evolução da vida na terra foram possíveis graças ao desenvolvimento de mecanismos temporais precisos capazes de ajustar os processos fisiológicos que ocorriam no interior do organismo com os ciclos ambientais, promovendo assim, ganhos na capacidade adaptativa e reprodutiva dos indivíduos. Neste contexto, luz e temperatura são as duas pistas temporais mais relevantes para resetar o relógio endógeno e, aparentemente, esses dois zeitgebers trabalham juntos para manter os ritmos circadianos. Uma ampla gama de fotorreceptores e fotopigmentos evoluiu no sentido de perceber com alta sensibilidade a informação fótica fornecida pelo ambiente e, recentemente, foi demonstrado que a detecção de temperatura também pode ser exercida pelos fotopigmentos rodopsina e melanopsina, sendo mediada por canais TRP (Shen et al., 2011). Consideramos as células B16-F10 Per1::Luc como um modelo promissor para o estudo de luz e temperatura em relógios periféricos, uma vez que essa linhagem expressa os dois fotopigmentos apontados com função de termorreceptores em Drosophila. Nossos estudos nos permitiram verificar que a luz não atua como um agente sincronizador nessas células, que se mantiveram em livre curso mesmo após um pulso de 10 min de luz azul (650 lux). Por outro lado, um pulso de temperatura de 2,5º C acima da temperatura de manutenção por 1h atuou ajustando a expressão do gene Per1, imprimindo um ritmo circadiano, diferentemente do observado no controle. Com base nessas informações, hipotetizamos que a informação de luz, percebida via melanopsina na retina de mamíferos, levaria a regulação da temperatura circadiana pelo NSQ, e a temperatura corporal, por sua vez, poderia atuar como uma pista interna para a sincronização dos tecidos periféricos, tendo os canais TRP como mediadores. Para responder esta questão, utilizamos camundongos WT e TrpV1 KO submetidos a diferentes protocolos de luz e avaliamos a expressão de genes do relógio Per1, Per2, Clock e Bmal1 e dos canais TrpV1 e TrpA1 em tecidos periféricos. Identificamos que a glândula suprarrenal, fígado e tecido adiposo marrom possuem uma maquinaria do relógio tipicamente ativa e acreditamos que a oscilação dos genes de relógio observada nesses tecidos é expressiva. Interessantemente, vimos também que o TrpV1, além de ser expresso nos tecidos analisados em animais WT, apresenta uma transcrição rítmica no fígado e tecido adiposo marrom de animais em LD, corroborando nossa hipótese de que canais TRP atuam como mediadores da informação de luz aos tecidos periféricos. Dadas as diferenças encontradas entre os animais WT e TrpV1 KO, sugerimos que a presença do canal TRPV1 pode ser essencial, embora seu grau de envolvimento varie de acordo com o tecido. No que diz respeito ao canal TRPA1, encontramos dois resultados que merecem ser destacados. Primeiramente, identificamos no fígado de camundongos TrpV1 KO mantidos em LD uma provável compensação da expressão de TrpA1 na ausência de TrpV1 e, curiosamente, que o tecido adiposo marrom não expressa o canal TrpA1. Considerando os resultados deste trabalho sobre o envolvimento dos canais TRP em resposta à luz e temperatura, acreditamos ter fortalecido nossa hipótese inicial, principalmente após demonstrarmos o papel do canal TRPV1 e que tecidos periféricos são sincronizados por alterações de temperatura. / The life emergence and evolution on Earth were made possible by the development of precise temporal mechanisms able to adjust the physiological processes within an organism with environmental cycles, thus promoting gains in the adaptive and reproductive capacity of the individuals. In this context, light and temperature are the two most relevant time cues to reset the endogenous clock; apparently these two zeitgebers work together to keep the circadian rhythms. A wide variety of photoreceptors and photopigments evolved in order to precisely perceive the photic information provided by the environment, and recently it has been shown that the temperature detection can also be exerted by the photopigments rhodopsin and melanopsin, being mediated by TRP channels (Shen et al., 2011). We have identified B16-F10 Per1::Luc cells as a promising model for the study of light and temperature effects on peripheral clocks, since this cell line expresses both photopigments pointed as thermoreceptors in Drosophila. Our studies allowed us to demonstrate that light does not act as a synchronizing agent on those cells, which remained in free running after a 10 min pulse of blue light (650 lux). On the other hand, a temperature pulse of 2.5º C above the maintenance temperature, for 1h, adjusted Per1 gene expression, imprinting a circadian rhythm, which was not observed in the control. Based on this information, we hypothesized that the light perceived via melanopsin by the mammalian retina would lead to the regulation of the circadian temperature by the SCN, and the body temperature, in turn, could act as an inner cue for the synchronization of the peripheral tissues, having the TRP channels as mediators. To answer this question, we have used WT and TrpV1 KO mice under different light protocols and evaluated the expression of clock genes Per1, Per2, Clock and Bmal1 and TrpV1 and TrpA1 channels in peripheral tissues. We found that the adrenal gland, liver and brown adipose tissue have a typically active clock machinery, and the oscillation of clock genes observed in these tissues is significant. Interestingly, we observed that TrpV1 is expressed in those tissues, and presents a rhythmic transcription in the liver and brown adipose tissue of LD maintained animals, confirming our hypothesis that TRP channels act as mediators of light information to peripheral tissues. In face of the differences between WT and trpV1 KO animals, we suggest that the presence of the TRPV1 channel may be essential, although its degree of involvement may vary according to the tissue. In terms of TRPA1 channel, we found two results that deserve to be highlighted. Firstly, we identified in the liver of TrpV1 KO mice maintained in LD a presumable compensation of TrpA1 expression in the absence of TrpV1 and, interestingly, the brown adipose tissue does not express TrpA1 channel. Considering the findings of this study on the participation of TRP channels in responses to light and temperature, we believe we have strengthened our initial hypothesis, especially after we have demonstrated the role of TRPV1 channel, and that peripheral tissues may be synchronized by temperature changes.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-07T11:31:21Z No. of bitstreams: 1 brunoguilhermedafonseca.pdf: 3400228 bytes, checksum: 5e2e24048845084805ad960c6e33ecac (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-02T13:29:49Z (GMT) No. of bitstreams: 1 brunoguilhermedafonseca.pdf: 3400228 bytes, checksum: 5e2e24048845084805ad960c6e33ecac (MD5) / Made available in DSpace on 2016-07-02T13:29:49Z (GMT). No. of bitstreams: 1 brunoguilhermedafonseca.pdf: 3400228 bytes, checksum: 5e2e24048845084805ad960c6e33ecac (MD5) Previous issue date: 2016-02-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho investigou-se a adsorção do triptofano (Trp), seus derivados e do 2-mercaptoetanol (MET) na superfície de nanopartículas metálica de prata ou ouro. Utilizou-se como ferramentas para o estudo, as técnicas espectroscópicas de absorção no ultravioleta e visível (UV-VIS), espalhamento Raman normal e intensificado pela superfície (surface-enhanced Raman scattering – SERS) e cálculos teóricos baseados na teoria do funcional da densidade (density functional theory – DFT). A banda da ressonância do plásmon de superfície localizado (localized surface plasmon resonance – LSPR) foi monitorada nos espectros UV-VIS para a investigação das propriedades eletrônicas das nanopartículas metálicas, envolvidas nas interações com os adsorbatos e nas distribuições de tamanhos. Foram sintetizados nanoprismas de prata estabilizados por citrato de sódio em diferentes condições de temperatura, envolvendo múltiplas etapas de crescimento e usando cobre como dopante indutor de geometria. A inclusão de pequena quantidade de cobre, em relação a prata, levou à maior formação de nanoprismas triangulares que o método original. Estas superfícies foram utilizadas nos estudos da adsorção do MET. Os nanoprismas, dopados ou não, mostraram-se inadequados para uso como substrato SERS de outros adsorbatos que não fossem mercaptanas. Sintetizou-se também nanoesferas de prata ou ouro para o estudo da adsorção do Trp e seus derivados em diferentes ambientes químicos. Os nanoprismas foram utilizados para estudos LSPR na presença do MET em diferentes concentrações, além da obtenção do espectro SERS da molécula. Observou-se que a concentração possui papel determinante na auto-organização de uma monocamada molecular, fazendo com que a conformação predominante se altere na superfície metálica. Os cálculos por DFT permitiram a simulação da adsorção do MET em superfície (111) de prata e obtenção de resultados relacionados às estruturas e estabilidades dos confôrmeros. Teoria e experimento mostraram-se em acordo dentro das condições estudadas e permitindo inferir que a ligação de hidrogênio possui um papel chave nas propriedades da monocamada. Estudou-se a adsorção dos isômeros L-Trp e D-Trp e dos peptídeos Ala-Trp, Trp-Gly, pGlu-Lys-Trp-Ala-Pro e Trp-His-Trp-Leu-Gln-Leu através das espectroscopias SERS e UV-VIS. Em prata, os espectros mostraram que os aminoácidos e os dipeptídeos adsorvem preferencialmente através dos grupos carboxilato e amina. Em ouro, a análise espectral permitiu identificar a adsorção das moléculas de Trp via nitrogênio do anel indólico. Os espectros foram obtidos em concentração de 10-3 mol L-1 nos diferentes metais, contudo a adição de HCl permitiu a obtenção de espectros em 10-5 mol L-1. As diferenças entre os espectros em ouro permitiram concluir que o Trp interage mais intensamente com a superfície na presença de HCl. A partir de todos os resultados obtidos, pode-se sugerir que as nanopartículas de ouro mostram-se úteis para o estudo de estruturas mais complexas por diferencia o triptofano adsorvido à superfície de outros presentes na estrutura de interesse. / The adsorption of tryptophan (Trp), his derivatives and 2-mercaptoethanol (MET) on metallic nanoparticle surface had been studied on this thesis. For such investigations it was employed the following tools: ultraviolet and visible absorption spectroscopy (UV-VIS), Raman scattering spectroscopy, surface enhanced Raman spectroscopy (SERS) and density functional theory. The localized surface plasmon resonance (LSPR) band was monitored on UV-VIS spectra to study the electronic properties of metallic nanoparticles involved in the interactions with the adsorbates and the size distributions. Silver nanoplates were synthesized in different conditions of temperature, multiple growing steps and using copper as a stabilizing doping. A small addition of copper, compared to silver, increased the triangular nanoprism yield of the original synthesis. These nanoparticles were used for adsorption study of MET. It was synthesized gold and silver nanospheres for studying Trp adsorption and other peptides on different chemical surroundings. The nanoprisms were utilized for LSPR study in the presence of MET at different concentrations, in addition to SERS spectra of this molecule. It has been observed the molecule concentration has a key role for self-assembled monolayer formation, which changes the most common conformer on the metallic surface. DFT calculation allowed us to simulate MET adsorption on silver surface (111) obtaining results related to the structures and stabilities of the conformers. Theory and experiment showed in agreement in the conditions studied and conclude the hydrogen bond is a key player in the monolayer properties. It was studied the adsorption of the isomers, L-Trp and D-Trp, and the peptides, Ala-Trp, Trp-Gly, pGlu-Lys-Trp-Ala-Pro and Trp-His-Trp-Leu-Gln-Leu, through SERS spectroscopy. In silver, the spectra showed amino acids and dipeptides tend to adsorb by the acid and amine group. In gold, an enhancement and shifting of the bands allowed us to identify the adsorption by the indole ring, specifically by the nitrogen atom. The spectra were obtained at concentration of 10-3mol.L-1 on different metals, however in presence of HCl the molecular concentration was 10-5mol.L-1. When HCl was added, the spectra showed a slightly different pattern, which suggest us that, in this condition, Trp interacts stronger to the surface.

Vascular resistance is mainly determined by the contraction of vascular smooth muscle (VSM), which is regulated by the phosphorylation of myosin light chain (MLC). VSM contraction is initiated by calcium influx into the VSM cells, which is mediated by transient receptor potential (TRP) channels and L-type voltage- dependent calcium channels (L-VDCC). On the other hand, calcium sensitization is a mechanism enhancing vascular contractile response at a given level of intracellular calcium by RhoA/Rho kinase pathway-mediated inhibition of myosin light chain phosphatase. In this thesis I present the data about i) the role of TRP channels in the mechanisms of vascular smooth muscle contraction, ii) enhanced contractility of arteries from spontaneously hypertensive rats (SHR), and iii) the differences in contraction of arteries from normotensive and hypertensive rats related to the role of RhoA/Rho kinase pathway in three types of experimental hypertension (SHR, Ren-2 transgenic rats and salt-sensitive Dahl rats). In the study concerning TRP channels, I compared the effects of three commonly used non-selective TRP channels inhibitors (2-APB, SKF-96365, FFA) on isolated arteries. Among them 2-APB was the most interesting because the observed inhibitory effects of 2-APB were dependent on the type of...

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Mit dem Tryptophandepletionstest kann kurzfristig die zentralnervöse Serotoninsynthese reduziert werden. Das TRP-LNAA-Ratio dient dabei zur Abschätzung der zentralnervösen Verfügbarkeit von TRP. In der Literatur besteht jedoch weder über die zu verwendenden LNAAs im Nenner des Ratios noch über die genaue Berechnungsmethode Einigkeit. Ziel dieser Arbeit war es, mithilfe von statistischen Methoden die verschiedenen LNAA-Kombinationen sowie die reguläre und die Km-normalisierte Berechnungsmethode zu vergleichen. Dafür wurden die LNAA-Konzentrationen verwendet, die in der dieser Arbeit zugrundeliegenden Studie erhoben wurden. Das Herausarbeiten von Unterschieden und Übereinstimmung, diente dem Zweck, eine Empfehlung für die Verwendung einer bestimmten LNAA-Kombination und einer Berechnungsmethode geben zu können. Die Ergebnisse dieser Arbeit zeigen, dass die Frage, welche Berechnungsmethode zur Bestimmung des TRP-LNAA-Ratios angewandt werden sollte, nicht trivial ist, da die Ratios beider Berechnungsmethoden nur eine sehr geringe Übereinstimmung zeigen. Die Tatsache, dass sich die Ratios unter Verwendung verschiedener LNAA-Kombinationen bei Anwendung der Km-normalisierten Berechnungsmethode kaum unterschieden, lässt die Km-normalisierte Berechnungsmethode gegenüber der regulären robuster erscheinen. Was die zu verwendende LNAA-Kombination anbelangt, wäre anzuraten, dass in zukünftigen Studien eine umfassende LNAA-Kombination auswählt wird, da das Ratio sich so stabiler gegen Ausreißer der einzelnen LNAAs darstellt.

Transient receptor potential (TRP) channels are expressed throughout the central nervous system and have a unique ability to detect a wide range of stimuli including changes in voltage, temperature, pH, lipid environment, small molecule agonists, and mechanical stress. While it is known that TRP channels contain the same six transmembrane helix (S1-S6), tetrameric architecture as voltage-gated channels, the degree to which functional and structural analogies are relevant remains poorly understood. This thesis describes a multidisciplinary approach toward understanding the structure and function of TRP channel transmembrane domains by focusing on the S1-S4 transmembrane helices of the TRPV1. This focus is inspired by the voltage-sensor domain (VSD) of the S1-S4 helices of voltage-gated channels, for which a range of studies show functional and structural independence. While some TRP channels are voltage-sensitive, their S4 helix does not contain the positive string of amino acids of canonical VSDs. However, the S1-S4 helices are functionally significant as the binding site of small molecule ligands in both TRPV1 and TRPM8 (for capsaicin and menthol, respectively). The question of TRP channel transmembrane domain modularity is addressed in this thesis by expression and purification trials as well as radioligand-binding assays. It is demonstrated that the S1-S4 and S1-S6 helices of TRPV1 can be properly inserted, overexpressed, and show signs of stability upon detergent-extraction from Saccharomyces cerevisiae membranes. However the TRPV1 S1-S4 and S1-S6 helices do not show wildtype (WT)-like binding in [³H]-RTX binding assays. These results indicate that the TRPV1 transmembrane domains are likely structural but not functional domains. The S. cerevisiae expression system remained promising for the overexpression of TRP transmembrane domains as well as the production of functional, though not stable upon detergent-extraction, WT TRPV1. This WT TRPV1 was subsequently found to functionally bind both RTX, used in ligand binding assays, as well as the double-knot toxin (DkTx), targeted to the pore domain (the S5-S6 helices). An effect of DkTx on RTX binding affinity demonstrates an allosteric interaction indicative of a possible tighter packing between the two transmembrane domains than is seen in voltage-gated channels containing the canonical VSD. Computational approaches additionally allowed for the investigation of the intramembrane capsaicin binding site in the TRPV1 S1-S4 helices, crucial to the initial motivations of this study. While the literature locates the capsaicin binding site to the TRPV1 S1-S4 helices, a `binding pocket' has yet to be defined, with regards to the orientation of bound capsaicin and its access route to the site via the bilayer. Using molecular dynamics (MD) simulations the preferred location of capsaicin within the bilayer is defined, as well as the elucidiation of capsaicin flip-flop between bilayer leaflets as a key event prior to TRPV1 binding. A transient binding was also observed between a homology model of the TRPV1 S1-S4 helices and capsaicin, possibly encouraging the idea that the S1-S4 helices still contain a partial binding site, though of too low affinity to be observed in the binding experiments performed here.

EN The growing emergence of bacteria resistant to conventional antibiotics is very alarming. This has prompted an intensive search for alternative antimicrobial agents which kill bacteria with different modes of action than do traditional antibiotics and do not develop drug resistance. Among these, antimicrobial peptides (AMPs) are considered as promising compounds against resistant pathogens. These positively charged peptides permeabilize or disrupt bacterial cell envelope which leads to leakage of cytoplasmic components and cell death. The aim of my dissertation thesis was the study of the action mechanism of novel antimicrobial peptides which I have isolated from the venom of different wild bees. I identified six novel AMPs which were named panurgines (PNG), codesane (COD) and antapines (ANTPs). These peptides were isolated from the venom of three different bee species (Panurgus calcaratus, Collete daviesanus and Anthophora plumipes). I was also involved in the structural studies of lasiocepsin (Las), the antimicrobial peptide identified in the venom earlier in our laboratory. All studied peptides possess activity against various strains of bacteria and low or moderate hemolytic activity. We prepared series of PNG, COD and ANTP analogs in order to study the effect of physicochemical properties...