A good ecological status of streams and rivers is crucial for maintaining ecological functionality of running waters. Worldwide eutrophication threatens to change structure and function of freshwater ecosystems (Dodds et al., 2008). To reduce the symptoms of eutrophication in streams and rivers an additional approach, besides the reduction of external nutrient inputs from catchment areas, is needed. Therefore the goal has been set to transfer the approach of biomanipulation, which is widely accepted as tool in water quality management in lakes and reservoirs, to streams. The objective of this study was accordingly to analyse and evaluate some crucial preconditions for top-down control of stream food webs. For that purpose the present thesis examined effects of fish predation (stone loach and gudgeon) on grazer-periphyton interaction in small streams by assessing predator avoidance by benthic grazers, effects of benthic grazers on periphyton community composition during fish presence/absence and the possibility of top-down control on algal biomass by benthivorous fish.

This thesis is a comparative study, which compare two selected countries, Finland and the Czech Republic in the organization of basic education. The benchmark of equity has been chosen as a comparative criterion. This criterion was further divided into single indicators. The key indicator in this respect was the selectivity of educational systems. For this reason, the thesis presents especially the form and the extent of differentiation of students by each level of basic education in both chosen counties and try to find the number of children who are educated in them. The results of comparative analysis showed that five educational programs with different degree of quality exist in the Czech Republic, while there are only two types of schools in Finland. Finland has used the differentiation of pupils in schools, even in clases in much smaller scale in comparison with the Czech Republic. In Finland, there are no classes with extended teaching of some subjects. The next difference between selected countries is about the age when the students start to be selectived. The students are not differentionated before 16-years in Finland. On the other hand, students of the Czech Republic are divaded in various educational programs after completion of primary school, that means in 11-years. Only 4 % of...

Finding economical and environmentally friendly processes to recover heavy metals (HMs) from spent batteries is a research priority to move toward sustainability. Adsorption seems an acceptable procedure to replace the current separation/purification stage of hydrometallurgical techniques. Chitosan is an efficient adsorbent for HM uptake from aqueous solutions. Nevertheless, in practice, chitosan modification is unavoidable to improve its physicochemical properties. Sodium tripolyphosphate is an environmentally benign crosslinker that can be used for chitosan modification. In addition, ion-imprinting technique could potentially enhance the adsorption efficiency and selectivity of crosslinked chitosan. Considering the above, the primary purposes of this research were: investigating the adsorption efficiency of chitosan for heavy metals uptake from synthetic solutions; modifying chitosan by crosslinking alone and combined with ion-imprinting techniques to improve the physicochemical properties as well as adsorption capacity and selectivity of chitosan; evaluating and comparing the adsorption efficiency of modified chitosan beads for the adsorption of Cd(II), Ni(II) and Co(II) in single and multicomponent batch adsorption systems. Chitosan and sodium tripolyphosphate crosslinked chitosan beads were prepared to remove Cd(II) from aqueous solution in the first phase. FTIR and XRD of the synthesized beads showed partial consumption of chitosan amine groups and a decrease in crystallinity of chitosan structure over crosslinking reaction. The isotherm and thermodynamic studies showed that Langmuir isotherm was the best fit to the experimental data of Cd(II) adsorption on crosslinked chitosan and all the adsorption reactions were endothermic and spontaneous. A reduced quadratic model, constructed by the Response Surface Methodology (RSM), indicated that the Cd(II) adsorption uptake of 99.87 (mg/g) was achieved at 55 °C and 2.92 % (w/v) crosslinking degree. Then, chitosan and crosslinked chitosan beads by sodium tripolyphosphate were used for Ni(II) adsorption from aqueous media in the second phase. The BET characterization showed that increasing the crosslinking degree reduced the chitosan beads' surface area and their total pore volume. The Langmuir model described the experimental results best and showed that the maximum adsorption capacity of chitosan (80.00 mg/g) decreased after crosslinking (52.36 mg/g). In addition, a reduced quadratic model with a correlation coefficient of 0.96 was established to correlate the adsorption uptake of Ni(II) with pH and crosslinking degree. In the third phase, the adsorption of Ni(II) and Cd(II) ions from single and binary metal ions solutions onto chitosan and crosslinked chitosan beads was studied. The extended Freundlich model fitted the adsorption equilibrium data in the binary system, implying the existence of preference in the order of Ni(II) > Cd(II). Desorption studies with a mixture of NaCl and H2SO4 were also conducted during this phase, demonstrating a desorption efficiency of greater than 85 %. In the fourth phase, the removal of cadmium from aqueous solution was examined using a novel Cd(II)-imprinted crosslinked chitosan. SEM, FTIR, TGA, and BET characterizations revealed that the ion-imprinted chitosan beads had better physicochemical properties than chitosan beads and superior potential adsorption properties than non-imprinted crosslinked chitosan beads. The isotherm and thermodynamic studies revealed that the Langmuir isotherm fitted the Cd(II) experimental data the best, and the adsorption reactions were spontaneous and endothermic. The kinetics data were also best fitted by the pseudo-second-order equation. Finally, the ion-imprinted crosslinked chitosan beads were employed for the selective adsorption of Cd(II) in a competitive adsorption system of Cd(II)-Ni(II)-Co(II) in phase five. The characterization of the prepared adsorbents was performed using XRD and BET, showing a higher surface area of ion-imprinted crosslinked chitosan than non-imprinted crosslinked chitosan beads. The Extended Langmuir model fitted the experimental results obtained from the multi-component system, indicating that ion-imprinted crosslinked chitosan had a higher total metal uptake with better selectivity toward Cd(II) uptake compared to non-imprinted crosslinked chitosan. Studying the adsorption mechanism in a ternary system showed that the adsorption was governed by chemical binding and ion exchange mechanisms in the ternary system. In conclusion, crosslinking by sodium tripolyphosphate improved chitosan physiochemical properties; however, it resulted in a decrease in HM adsorption uptake. The RSM was used to assess the effect of pH, temperature, and crosslinking degree and optimize the adsorption uptake of chitosan. Also, ion-imprinting was effective in enhancing the adsorption capacity and selectivity of crosslinked chitosan for the ion used as a template (Cd(II)) in preparing ion-imprinted crosslinked chitosan.

L’objectif de ce travail est de remplacer les catalyseurs organométalliques à base d’étain et de mercure utilisés lors de la synthèse de polyuréthane via la réaction isocyanate – alcool. Une étude bibliographique a montré que la basicité et la nucléophilie d’un catalyseur organique gouvernent son activité et le mécanisme réactionnel qu’il induit. Pour les catalyseurs organométalliques, ces propriétés catalytiques s’expliquent par des considérations électroniques ainsi que par le principe HSAB. D’autre part, une étude approfondie du catalyseur organomercuriel a été menée. Elle a révélé l’intérêt d’ajouter à l’espèce catalytique organométallique un acide carboxylique, de préférence à longue chaîne carbonée. Celui-ci ralentit l’hydrolyse du catalyseur tout en augmentant sa sélectivité envers la réaction isocyanate – alcool. De nouveaux catalyseurs ou systèmes catalytiques originaux ont ensuite été évalués grâce à un test simplifié. Trois catalyseurs organométalliques ont ainsi été sélectionnés pour de nouveaux essais dans des conditions plus proches de celles rencontrées dans l’industrie. Ils se sont tous révélés actifs, mais seul le -dicétonate de zinc II permet d’obtenir un prépolymère incolore et transparent, deux critères essentiels pour les applications visées. Il pourrait donc remplacer le catalyseur organostannique. Enfin, l’étude de la sélectivité de systèmes catalytiques impliquant un catalyseur commercial en présence d’acide néodécanoïque a confirmé le rôle protecteur de ce dernier. La sélectivité du complexe de zinc retenu précédemment a par ailleurs été évaluée et apparaît 2,5 fois supérieure à celle du complexe organomercuriel à remplacer. / The aim of this work is to replace organotin and organomercury catalysts used for the synthesis of polyurethane via the isocyanate – alcohol reaction. A bibliographic review revealed that basicity and nucleophilicity of an organic catalyst affect its activity and the reaction mechanism it induces. For organometallic catalysts, these catalytic properties can be explained by electronic considerations and by the HSAB principle. On the other hand, a comprehensive study of the organomercury catalyst highlighted the benefit to combine it with a carboxylic acid, preferably with a long carbon backbone. This slows down hydrolysis of the catalyst while increasing its selectivity toward the isocyanate – alcohol reaction. New original catalysts or catalytic systems were then evaluated using a simplified experiment. Three organometallic catalysts were selected for further testing in conditions closer to industrial ones. They all showed appropriate catalytic activity, but the zinc II -diketonate is the only one to provide a colorless and transparent prepolymer, two essential criteria for the intended applications. This complex could therefore replace the organotin catalyst. Finally, the study of the selectivity of catalytic systems involving a commercial catalyst in the presence of neodecanoic acid confirmed its protective role toward hydrolysis. The previously retained zinc complex was also evaluated and revealed a selectivity 2.5 times greater than that of the organomercurial complex to replace.

[pt] A presente tese apresenta uma análise do Sistema de Justiça Juvenil brasileiro, com foco nos aminhos percorridos pelos adolescentes na fase de apuração do ato considerado infracional. A pesquisa foi construída com uma abordagem qualitativa e fundamentou-se no materialismo histórico-dialético, com base nos estudos da criminologia crítica. Foram utilizadas como fontes de investigação: observação participante; análise de relatórios técnicos sobre as unidades socioeducativas de acautelamento, elaborados pela equipe técnica de Serviço Social do Ministério Público do RJ e entrevista com profissional do Sistema Socioeducativo do DEGASE. O objetivo geral da pesquisa é analisar o Sistema de Justiça Juvenil, a fim de compreender como o funcionamento, condições de atendimentos, rotinas, fluxos, decisões e demais ações são operacionalizados pelos órgãos do Sistema de Justiça Juvenil, à luz dos instrumentos normativos vigentes. Os resultados demonstraram como o Estado, mesmo com o avanço dos instrumentos normativos de proteção à infância e juventude, criminaliza os adolescentes mais pobres e impõe uma seletividade punitiva racializada para absorvê-los no Sistema de Justiça Juvenil. Uma vez inseridos nas engrenagens do sistema, foi possível constatar a dimensão da violência institucional que atravessa todos os caminhos da apreensão, internação provisória, até a realização das audiências - período em que ainda estão sob a garantia constitucional de presunção de inocência. Nesses casos, opera-se uma punição antecipada de uma infração ainda não julgada, que se naturaliza no cotidiano dos órgãos que compõem esse sistema e se materializa em um cenário de violação de direitos individuais e coletivos, que reforça o processo de desumanização e a banalização de suas vidas. / [en] This Thesis presents an analysis of the Brazilian Juvenile Justice System, focusing on the paths taken by teenagers in the investigation phase of the act considered infraction. The research was built with a qualitative approach and was based on historical-dialectical materialism, based on critical criminology studies. The following research sources were used: participant observation; analysis of technical reports on the socio-educational precautionary units, prepared by the technical team of Social Service of the Public Ministry of RJ and interview with a professional from the Socio-educational System of DEGASE. The general objective of the research is to analyze the Juvenile Justice System, to understand how the functioning, conditions of services, routines, flows, decisions, and other actions are operated by the Juvenile Justice System bodies, in the light of the normative instruments in force. The results showed how the State, even with the advance of normative instruments to protect children and youth, criminalizes the poorest adolescents and imposes a racialized punitive selectivity to absorb them in the Juvenile Justice System. Once inserted into the gears of the system, it was possible to verify the dimension of institutional violence that crosses all paths from apprehension, provisional internment, until the holding of hearings - a period in which they are still under the constitutional guarantee of presumption of innocence. In these cases, there is an early punishment of an infraction not yet judged, which is naturalized in the daily life of the bodies that make up this system and materializes in a scenario of violation of individual and collective rights, which reinforces the process of dehumanization and the trivialization of their lives.

[pt] A implementação de medidas que reduzam as emissões de gases de efeito estufa ganha importância no cenário atual. Um importante método para captura de CO2 consiste nos processos de separação por membranas. Visando melhorar a eficiência seletiva na separação de gases, este trabalho estudou a síntese de membranas poliméricas de matrizes mistas a fim de aumentar os valores de permeabilidade, utilizando líquidos iônicos em sua estrutura, que apresentam elevada solubilidade de CO2 e seletividade. A síntese do líquido iônico foi realizada a partir do cátion imidazólico e do ânion NTf2(-) , em reações de 3 etapas. Os filmes poliméricos de matrizes mistas foram sintetizados por diferentes tipos de polímeros comerciais, PEBAX 1657, PEBAX2533 e PU 1185A10; com concentrações de 0 por cento, 20 por cento e 60 por cento (m/m) do líquido iônico. A técnica de ressonância magnética nuclear (RMN) de (1)H e (13)C foi utilizada para validar a composição do líquido iônico. As caracterizações de membranas compósitas se deram pelas técnicas de microscopia eletrônica de varredura (MEV), análise termogravimétrica (TGA) e espectroscopia de infravermelho com transformada de Fourier (FTIR). Na presença do líquido iônico, a seletividade relativa de CO2/N2 apresentou um aumento considerável para as membranas de PU e PEBAX2533, enquanto a permeabilidade de CO2 aumentou nas membranas de PU e PEBAX1657. / [en] The measures to reduce greenhouse gas emissions, gains more importance in the current scenario. Processes involving membrane separation are an important method for CO2 capture which are widely used. In order to improve the selective efficient in the gas separation this paper studies the development and synthesis of composite polymeric membranes that will be able to increase the permeability using ionic liquids in your structure, which have high CO2 solubility and selectivity. The ionic liquid synthesis was obtained using imidazolium cation and the NTf2(-) anion, on 3 steps reactions. The composite polymeric membranes were synthesized by different types of commercial polymers, PEBAX1657, PEBAX2533 and PU 1185A10; with 0 percent, 20 percent and 60 percent (wt.) concentrations of ionic liquid. The nuclear magnetic resonance (NMR) technique for 1H and 13C was used to validate the ionic liquid structure. The composite membrane characterizations were obtained by those techniques: scanning electron microscope (SEM), thermogravimetric analysis (TGA) and Fourier transform infrared (FTIR). In the presence of ionic liquid, the selectivity of CO2/N2 increased for the PU and PEBAX2533 membranes, and the permeability of CO2 increased for the PU and PEBAX1657 membranes.

<p> We developed some universal platforms to overexpress the minimal functional entities of ion channels. The modular property of ion channels have been demonstrated from many aspects, such as crystal structures, chimeric channel experiments and discovery of similar modules in distantly related protein families. Thus it should be feasible to express each module independent of other channel modules. The pore-forming module of ion channels has multiple important properties as selectivity, conductivity and drug-binding. If it can be overexpressed, it will provide valuable information about channel selectivity to different ions and structural bases for drug binding as well as important application in drug screening and rational drug design. </p> <p> To test this, we first used the model channel KcsA to identify the minimal requirements for a pore-forming domain to functionally exist independently. Chapter 2 of this thesis explains in detail how the wild type C-terminal cytoplasmic domain of KcsA functions. We found that this domain has dual function as pH-sensor and tetramerization domain, and it is essential for the expression of the pore-forming domain of KcsA. Once we knew the physiological role of the cytoplasmic domain, the scenario was set to answer the question of how to make it better for the application of structural and functional studies. </p> <p> In chapter 3 and chapter 4, we replaced the wild type C-terminal domain with non-native tetramerization domains. We identified the direct correlation between protein expression level and overall thermostability of pore-forming domains. The C-terminal tetramerization domains stabilize channels in a cooperative way and play a critical way in in vivo channel assembly. The selection of the linker between pore-forming domain and tetramerization domain, the splicing motif, and the handedness of C-terminal tetrameric coiled coils all affect channel expression level and stability. </p> <p> We applied our finding in KcsA to a wide range of ion channels in chapter 5, including voltage-gated potassium channels, Ca2+-gated potassium channels, inwardrectifying potassium channels, cyclic nucleotide-gated potassium channels and voltagegated sodium channels. We managed to express similar minimal structural modules from these more structurally complicated channels with the assistance of different cytoplasmic tetramerization domains. Several minimal channels expressed well and showed similar biophysical and functional property as the wild type channels. </p> <p> These studies demonstrate that the pore-forming modules of ion channels can be expressed independently while retaining the proper structure and drug-binding properties as their wild type predecessors when using our universal expression platform. The potential application in structural studies and drug-screening is promising. </p> / Thesis / Doctor of Philosophy (PhD)

La douleur chronique est une maladie fréquente et grave qui, pour de nombreuses personnes, ne peut pas être entièrement traitée avec les choix thérapeutiques actuels. Des agonistes des récepteurs opioïdes delta (DORs) ont été proposés comme interventions thérapeutiques pour ces maladies. Des recherches précliniques ont montré que l'activation des DOR produit des effets anti-hyperalgiques et antidépresseurs avec moins d'effets secondaires associés aux analgésiques opioïdes disponibles sur le plan clinique. Cependant, de nombreux agonistes DOR induisent une tolérance analgésique, entravant ainsi leur développement en tant que médicaments. Les travaux de cette thèse visent à mieux comprendre les causes cellulaires et moléculaires de la tolérance ainsi que ce qui rend certains agonistes plus résistants à la tolérance que d'autres. Dans le premier projet, nous nous sommes concentrés sur la superactivation de l'adénylyl cyclase induite par un ligand, un modèle de réponse adaptative médiée par les isoformes de l'adénylyl cyclase (AC). La superactivation de l'adénylyl cyclase (SA) a été associée à l’hyperalgésie, la tolérance analgésique et à des symptômes de sevrage. Ainsi, nous étions curieux de voir si les profils de signalisation cellulaire créés pour la découverte de médicaments pouvaient nous fournir des informations sur la capacité d'un ligand à induire la SA. Pour répondre à cette question, nous avons généré des profils de signalisation complets pour six agonistes différents du DORs (Met-enképhaline, deltorphine II, DPDPE, SNC-80, ARM390 et TIPP) tout en surveillant 12 différents résultats de signalisation avec des biocapteurs à base de BRET. L'analyse des profils de signalisation a montré une sélectivité fonctionnelle remarquable parmi les ligands étudiés. Ensuite, nous avons pu classer les agonistes DOR en fonction de la similarité de leurs profils en utilisant l'approche que nous avons adaptée de notre laboratoire. Nous avons par la suite démontré que, à l'exception de TIPP, dont la réponse SA était Ca2+-indépendante, les catégories de médicaments résultant du regroupement sont corrélées avec la capacité du ligand à provoquer une SA. Une investigation plus approfondie des mécanismes a révélé que Gαi/o était essentiel tant pour la SA déclenchée par TIPP que par Met-Enkepkaline, mais les mécanismes en aval étaient assez distincts pour ces ligands. Ensemble, nos résultats indiquent que les mécanismes sous-jacents à la tolérance cellulaire induite par les agonistes DOR sont spécifiques au ligand. Dans le deuxième projet, nous nous sommes principalement intéressés aux mécanismes de tolérance aux agonistes DOR qui peuvent être en partie expliqués par la désensibilisation et la régulation négative des récepteurs. Il a été établi que, les ligands qui induisent le recyclage du récepteur après l'internalisation ont été trouvés pour fournir une analgésie de longue durée. Par conséquent, les expériences menées dans cette étude ont été menées pour révéler davantage les déterminants moléculaires sous-jacents au recyclage du récepteur et sur la manière dont l'interaction agoniste-récepteur pourrait produire des modèles distincts de régulation des récepteurs. Nous avons évalué l'activation de l'agoniste et la désensibilisation du signal DOR-Gαi1. Nos données ont rapporté que le DPDPE était pratiquement sans effet sur la désensibilisation de l'activation de Gαi1, tandis que la désensibilisation par la deltorphine II était plus importante que celle induite par le DPDPE mais moins que celle induite par l'ARM390 et le SNC-80. Ensuite, nous avons établi que les DORs stimulés par le DPDPE se recyclaient de manière plus efficace que ceux activés par la deltorphine II. De plus, nous fournissons des preuves phénoménologiques que des interventions similaires ont des effets distincts sur le recyclage évoqué par chaque ligand. En particulier, la truncation du DOR ou la surexpression de βarr2 avaient des effets différentiels sur le recyclage par le DPDPE et la deltorphine II. Il est admis que les mécanismes sous-jacents à ces différences restent à être pleinement décrits, mais la phénoménologie de nos observations soutient l'idée que le DPDPE et la deltorphine II mettent en œuvre des processus de recyclage distincts. / Chronic pain is a common and severe disease that, for many people, cannot be fully treated with current therapeutic choices. Agonists of the delta opioid receptor (DOR) have been proposed as therapeutic interventions for this illness. Preclinical research has shown that DORs produce antihyperalgesic and antidepressant-like effects with fewer side effects than the ones associated with clinically available opioid analgesics. However, numerous DOR agonists induce analgesic tolerance, hampering their development as medications. Thus, further investigations are needed to understand the mechanisms underlying the tolerance associated with chronic opioid use. This thesis aimed to further understand the cellular and molecular mechanisms that causes tolerance as well as what makes some agonists more resistant to tolerance than others. In the first project, we focused on ligand-induced cyclase superactivation (SA), a pattern of adaptive response mediated by adenylyl cyclase (AC) isoforms. Cyclase SA has been associated with hyperalgesia, analgesic tolerance, and withdrawal symptoms. Therefore, we were curious to assess weather cell-based signalling profiles created for drug discovery could provide us with information on the ability of a ligand to induce cyclase SA. To address this question, we generated comprehensive signalling profiles for six different DOR agonists (Met-enkephalin, deltorphin II, DPDPE, SNC-80 and ARM390) while monitoring 12 different signalling outcomes with BRET-based biosensors. Analysis of the signalling profiles showed remarkable functional selectivity among the investigated ligands. Next, we were able to classify DOR agonists based on the similarity of their profiles using the approach we adapted from our lab. We subsequently demonstrated that except for TIPP, whose SA response was Ca2+-independent, the drug categories resulting from clustering are correlated with ligand capacity to cause SA. Further investigation of the mechanisms revealed that Gαi/o was essential for both TIPP and Met-Enkepkalin-driven cyclase SA. However, downstream mechanisms were quite distinct for these two ligands. Altogether, our findings indicate that mechanisms underlying cellular tolerance induced by DOR agonists are ligand-specific. In the second project, we were primarily concerned with the mechanisms of tolerance to DOR agonists that may be, in part, explained the receptor desensitization and downregulation. Obviously, ligands that induce receptor recycling after internalization have been found to provide long-lasting analgesia. Therefore, the objectives of the experiments in this project were to assess the molecular determinants affecting receptor recycling and how agonist-receptor interaction can result in different patterns of receptor regulation. We assessed agonist inducing activation and desensitization of DOR-Gαi1 signal. Our data showed that DPDPE was efficient in activating the receptor without noticeable desensitization effect. On the other hand, deltorphin II exerted a significant desensitization effect. However, this effect was low when compared to ARM390 and SNC-80. Then, we established that DORs stimulated by DPDPE recycle more efficiently than those activated by deltorphin II. We also provided phenomenological evidence on receptor recycling elicited by each ligand. In particular, DOR truncation or the overexpression of βarr2 had differential effects on receptor recycling by DPDPE and deltorphin II. While our data shed light on the mechanism underlying these differences, further investigation is needed for the mechanism to be fully elucidated. Admittedly, our observations support the notion that DPDPE and deltorphin II engage distinct recycling processes.

L'autophagie maintient l'homéostasie cellulaire en dégradant les composants cellulaires. Chez l'humain, les protéines LC3 jouent un rôle central dans l'autophagie en interagissant avec d'autres facteurs contenant des régions d'interaction LC3 (LIR). Cette thèse porte sur le rôle de différents facteurs contenant des LIR, tels que le facteur nucléaire DOR et la protéine NSs du virus de la fièvre de la vallée du Rift (VFVR). Les protéines LC3 sont principalement présentes dans le noyau des cellules au repos normales, et leur passage au cytosol en réponse au stress nécessite une interaction avec DOR. Récemment, il a été démontré que cette interaction entre DOR et LC3B dépend de la désacétylation de deux résidus lysine conservés (K49/K51 de LC3A et K46/K48 de GABARAP). Cependant, les détails mécanistiques du rôle des résidus lysine individuels dans le transfert d'autres protéines LC3 demeurent inconnus. De plus, la caractérisation de l'interaction NSs-LC3 ainsi que son impact sur l'autophagie lors de l'infection par le RVFV demeurent évasives. Par conséquent, l'objectif de ces études est d'investiguer les différences structurelles et fonctionnelles des protéines humaines LC3 à différents stades de l'autophagie via leur interaction avec DOR et NSs. Nos études biophysiques et structurales ont permis d’identifier des éléments clés déterminant la spécificité de la région d'interaction LC3 de DOR (DORLIR) pour GABARAP. Nos études structurales ont défini une conformation en feuillet  chez DORLIR lorsqu'elle est en complexe avec GABARAP, ce qui joue un rôle important dans l'établissement de cette spécificité. Les études structurales ont également montré que l'acétylation de la deuxième Lys de GABARAP ou LC3A perturbe des interactions clés du W35 de DORLIR, ce qui conduit à une diminution de l'affinité qui est cohérente avec nos résultats ITC. Ces résultats ont été confirmés grâce à des expériences cellulaires en utilisant des substitutions K-en-Q pour imiter l'acétylation des Lys. En cellules, les substitutions K-en-Q à la deuxième Lys ont entravé le transfert cytoplasmique de GABARAP et de LC3A, ainsi que leur colocalisation avec DOR, tandis que les substitutions K-en-Q à la première Lys se comportent comme des protéines de type sauvage. Dans l'ensemble, la désacétylation de la deuxième Lys conservée est cruciale pour le transfert cytoplasmique de GABARAP et LC3A lors de l'autophagie, ce qui diffère de ce qui a été observé auparavant avec LC3B, où la désacétylation des deux Lys était nécessaire. Cette étude fournit également des informations sur les interactions entre la protéine NSs du VFVR et les protéines LC3, ainsi que l'impact de NSs sur l'autophagie lors de l'infection par le VFVR. Nous avons identifié quatre motifs potentiels d'interaction LC3 (NSs1-4) dans la protéine NSs, et des études d’ITC ont démontré que NSs4 interagit avec une affinité sous micromolaire-micromolaire avec les protéines LC3 humaines. De plus, nous avons confirmé que les protéines LC3 interagissent avec NSs dans les cellules, et que chez les cellules infectées par le RVFV, LC3A colocalise avec NSs. Dans l'ensemble, les résultats indiquent que la protéine NSs joue un rôle clé dans la modification de l'autophagie lors des infections par le VFVR. / Autophagy maintains cellular homeostasis through catabolism of cellular components including organelles, proteins, and pathogens. In humans, the six LC3 (Microtubule-associated protein 1 light chain 3) protein (LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in autophagy through interactions with other factors that contain LC3-interacting regions (LIRs). This study focuses on the role of different factors that contain LIRs such as the nuclear factor DOR and the NSs protein from the RVFV. LC3 proteins are predominantly present in the nucleus of normal resting cells and their shuttling to the cytosol in response to stress requires interaction with DOR. Recently, this interaction between DOR and LC3B was shown to depend on the deacetylation of two conserved Lys residues (K49/K51in LC3 subfamily proteins and K46/K48 in GABARAP subfamily proteins). However, the mechanistic details of the role of the individual Lys residues in the shuttling other LC3 proteins is unknown. In addition, the characterization of NSs-LC3 interaction as well as its impact on RVFV (Rift Valley fever virus) infection on autophagy remains elusive. Therefore, the goal of these studies is to investigate the structural and the functional differences of the six human LC3 proteins in different stages of autophagy through their interaction with DOR and NSs. Our biophysical and structural studies identified key elements determining the specificity of the LIR from DOR (DORLIR) for the GABARAP subfamily. Our structural studies defined a -sheet conformation in DORLIR when complexed with GABARAP, which is important role for establishing this specificity. ITC studies with acetylated versions of LC3A and GABARAP demonstrated that acetylation of the second Lys significantly decreases binding to the DORLIR whereas acetylation at the first Lys has little to no effect. Our structural studies also demonstrate that acetylation at the second Lys of either GABARAP or LC3A disrupts key interactions between W35 of the DORLIR, which leads to the decreased affinity. The in vitro results were verified in cellular experiments using K-to-Q substitutions to mimic Lys acetylation. In cells, K-to-Q substitutions at the second Lys impaired the cytoplasmic shuttling of both GABARAP and LC3A from the nucleus as well as their colocalization with DOR, whereas K-to-Q substitutions at the first Lys behaved like wild-type proteins. Taken together, the deacetylation of the second conserved Lys is critical for the cytoplasmic shuttling of GABARAP and LC3A during autophagy, which is in contrast to what was observed with LC3B where deacetylation of both Lys was required. This study also provides insights into interactions between the NSs protein of RVFV and LC3 proteins and the impact of NSs on autophagy during RVFV infection. We identified four potential LIR motifs (NSs1-4) in the NSs protein and ITC studies demonstrated that NSs4 interacts with submicromolar-micromolar affinity with the human LC3 proteins. In addition, we confirmed that LC3 proteins interact with NSs in cells and that in RVFV infected cell LC3A colocalizes with NSs. Taken together, the results indicate that the NSs protein plays a key role in altering autophagy during RVFV infections.

Ionenkanäle spielen eine wichtige Rolle in vielen physiologischen Prozessen. Während manche Kanäle hochspezifisch für eine Ionensorte sind, sind andere Kanäle weniger selektiv. Tetramere Ionenkanäle weisen eine gemeinsame Grundarchitektur auf, von der nur ionotrope Glutamatrezeptoren (iGluRs) abweichen, deren Struktur im Vergleich zu den anderen invertiert ist. Eine Untergruppe der iGluRs stellen α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Rezeptoren (AMPARs) dar, welche im Zentralnervensystem von Wirbeltieren die Mehrheit der exzitatorischen Neurotransmission übernehmen. Eine einzelne posttranskriptionelle Veränderung (Glutamin zu Arginin) an der Spitze des Selektivitätsfilters (SF, Q/R Stelle) von AMPAR-Typ 2 (GluA2) macht den Kanal quasi undurchlässig für Ca2+. Das Ziel dieser Arbeit war das Erstellen einer GluA2-Kanalchimäre mit Hilfe des bakteriellen Kanals NaK. Mutation rund um die Q/R Stelle wurden nicht toleriert von der Chimäre, während C-terminale Mutationen des SF stabil waren und für strukturelle Studien verwendet wurden. Die Entfernung einer Aminosäure im Vergleich zum NaK Wildtyp erzeugte eine Begradigung des SF und eine Erweiterung der wassergefüllten Ausbuchtung. Überraschenderweise kristallisierten die NaK Chimären überwiegend in zweifach symmetrischer Anordnung. Vor allem im SF kam es zu ausgeprägter lokaler Asymmetrie, unabhängig von der Ionenzusammensetzung. Um die Flexibilität des SF näher zu untersuchen, wurden Aminosäuren von NaK in GluA2 integriert und mithilfe von Patch-clamp Elektrophysiologie untersucht. Gleichsam wie in NaK, wurden Mutationen an der Filterspitze nicht toleriert, wohingegen die C-terminale Hälfte problemlos ausgetauscht werden konnte. Die funktionelle Integrität der NaK Chimäre wurde mithilfe von Einzelkanalmessungen in Bilayern überprüft. Zusätzlich wurden, auf Basis der gelösten Strukturen, Molekulardynamiksimulationen durchgeführt, welche einen dynamischen Einblick in den Permeationsmechanismus erlauben. / Ion channels play an important role in many physiological processes, for example the generation of action potentials. While some channels display high selectivity for one ion species, others are more promiscuous. All tetrameric cation channels share the same principal architecture, but the transmembrane domain of ionotropic glutamate receptors (iGluRs) is inverted relative to the other members. A subfamily of iGluRs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), mediates the vast majority of excitatory neurotransmission in the vertebrate central nervous system. In AMPA-type 2 iGluRs (GluA2), a single post-transcriptional modification (glutamine to arginine) at the tip of the selectivity filter (SF, Q/R site) renders the channel Ca2+ impermeable. The aim of this thesis was therefore to create an AMPAR pore mimic by using the bacterial channel NaK. Unfortunately, mutations around the Q/R site abolished expression of the NaK-GluA2 chimera, while C-terminal mutations of the SF were stable and could be used for structural studies. The shortening of the SF by one amino acid caused a straightening and opened an extended water-filled vestibule. Strikingly, most of the tested chimeras exhibited twofold symmetry with strong local asymmetry in the mutated part of the SF independent of the ion type present. For functional tests, residues from NaK wt were also swapped into GluA2. N-terminal mutations abolished the current response, whereas C-terminal mutations behaved wt-like. Single-channel bilayer experiments confirmed the functional integrity of the NaK-GluA2 chimera. Additionally, extensive molecular dynamics simulations, based on the solved structures, were carried out alongside. In summary, it could be demonstrated that the SF of the non-selective NaK-GluA2 chimera is highly flexible and accommodated all tested ions. Ion binding is accompanied by local asymmetric rearrangements, possibly creating an energetically simple way to allow permeation.