Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-03-09T22:56:49Z No. of bitstreams: 1 KyviaBezerraMota_TESE.pdf: 5202658 bytes, checksum: 44dd9aeba607843c325d09b4d48f945d (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-03-13T23:39:17Z (GMT) No. of bitstreams: 1 KyviaBezerraMota_TESE.pdf: 5202658 bytes, checksum: 44dd9aeba607843c325d09b4d48f945d (MD5) / Made available in DSpace on 2017-03-13T23:39:17Z (GMT). No. of bitstreams: 1 KyviaBezerraMota_TESE.pdf: 5202658 bytes, checksum: 44dd9aeba607843c325d09b4d48f945d (MD5) Previous issue date: 2016-10-07 / Essa tese trata de duas pesquisas com diferentes naturezas conceituais no campo da modelagem molecular, por?m baseadas em semelhantes princ?pios te?ricos da f?sica qu?ntica e da qu?mica computacional. No primeiro estudo, as estabilidades dos sistemas ER?-OHT (Receptor ? de Estr?geno-Hidroxitamoxifeno), ER?-RAL (Receptor ? de Estr?geno-Raloxifeno) e Integrina ?v?3-Cilengitide foram determinadas em termos energ?ticos a partir da t?cnica de fracionamento molecular com capuzes conjugados (MFCC) e no escopo da teoria do funcional da densidade (DFT) seguindo o esquema GGA-PBE-Grimme para calcular a intera??o energias dos sistemas estudados. No primeiro estudo, foi poss?vel predizer a relev?ncia individual dos amino?cidos Glu, Asp, Arg, Lys no sistema com o Tamoxifeno e Glu, Asp e Lys com o Raloxifeno. Para isso ser poss?vel, as for?as de atra??o e repuls?o dos res?duos que comp?em os sistemas foram calculadas. Cada um desses res?duos apresentou uma energia de intera??o que variou dependendo da natureza qu?mica da sua cadeia lateral, do ambiente de microsolvata??o que o envolve e dos contatos intermoleculares que estabelece. O estudo pioneiro da estabilidade conformacional em termos energ?ticos de uma regi?o livre de amino?cidos incitar? pesquisas centradas no desenvolvimento e s?ntese de novos antagonistas utilizados no tratamento de diversas patologias comuns na popula??o geral. No segundo estudo, foram determinadas as propriedades energ?ticas do sistema Integrina ?V?3-Cilengitide, cuja estrutura cristalogr?fica apresenta c?tions Mn+2. Observou-se a import?ncias dos amino?cidos Ser, Asp, Leu, Arg, Lys, Ala e Pro na intera?ao da integrina com o fragmento RGD (R: arginina; G: glicina; D: ?cido asp?rtico) do cilengitide. A import?ncia da integrina com o fragmento RGD no mecanismo de ades?o de diversas c?lulas envolvidas nas doen?as cancer?genas e tamb?m na implanta??o embrion?ria permitir? o desenvolvimento de novas terapias impactantes na sobrevida de pacientes portadores de diversas neoplasias e na reprodu??o humana. / This thesis deals with two researches with different conceptual nature in the field of molecular modeling based on similar theoretical principles of quantum physics and computational chemistry. In the first study the stabilities of ER?-OHT (Receptor ? Estrogen co-crystalyzed with Hydroxytamoxifen), ER?-RAL (?-Estrogen Receptor co-crystalyzed with Raloxifene) and integrin ?v?3 co-crystalyzed with cilengitide systems were determined in terms of energy from the Molecular Fractionation Technique Caps Conjugates (MFCC), and the scope of the Density Functional Theory (DFT) following GGA-PBE-Grimme scheme for calculating the interaction energy of the systems studied. In the first study, it was possible to predict the relevance of individual amino acids Glu, Asp, Arg, Lys in the system with Tamoxifen and Glu, Asp and Lys in Raloxifene system. For this to be possible, the attraction and repulsion forces of the residues that comprise the systems were calculated. Each of these residues had an energy of interaction that varied depending on the chemical nature of the side chain, the microsolvation environment that surrounds and intermolecular contacts establishing. The pioneer study of conformational stability in terms of energy of free amino acid region will encourage research focusing on the development and synthesis of new antagonists used in the treatment of several common diseases in the general population. In the second study, we determined the energetic properties of Integrin ?V?3-cilengitide system, whose crystallographic structure has Mn+2 cations. It was noted the importance of amino acids Ser, Asp, Leu, Arg, Lys, Ala and Pro in integrin interaction with the RGD fragment (R: arginine; G: glycine; D: aspartic acid) of cilengitide. The importance of integrin with the RGD fragment in the adhesion mechanism of various cells involved in cancers and in embryo implantation allows the development of new therapies impacting on survival of patients with various forms of neoplasms and in human reproduction.

Avanços na tecnologia introduziram o laser como um grande aliado na remoção seletiva do tecido cariado. Entretanto, o efeito dos lasers e seus diferentes parâmetros nos tecidos duros dentais e fibrilas colágenas não foi completamente estudado. O objetivo deste estudo foi avaliar os efeitos da irradiação com os lasers de Er:YAG e Er,Cr:YSGG na morfologia e na resistência de união da dentina irradiada à resina composta. Sessenta e quatro hemi-discos de dentina obtidos de terceiros molares humanos foram aleatoriamente divididos em 9 grupos (n=7): G1 Controle (não irradiado); G2 Laser de Er:YAG 250 mJ, 4 Hz; G3 Laser de Er:YAG 200 mJ, 4 Hz; G4 Laser de Er:YAG 180 mJ, 10 Hz; G5 Laser de Er:YAG 160 mJ, 10 Hz; G6 - Laser de Er,Cr:YSGG 2W, 20 Hz; G7 - Laser de Er,Cr:YSGG 2,5W, 20 Hz; G8 - Laser de Er,Cr:YSGG 3W, 20 Hz; G9 - Laser de Er,Cr:YSGG 4W, 20 Hz. Destes, vinte e sete amostras (n=3) foram processadas e destinadas à avaliação em Microscopia Eletrônica de Varredura (MEV) para análise morfológica de superfície e em corte transversal da dentina irradiada. Nove fragmentos (n=1) foram submetidos à Microscopia Eletrônica de Transmissão (MET) e vinte e sete (n=3) foram restaurados para análise da interface adesiva. Para o teste de resistência de união, 45 superfícies dentinárias planificadas foram restauradas e, após 24 horas de armazenamento em água, foram submetidas ao teste de microtração. O teste de ANOVA (=5%) foi realizado e verificou-se que o G1 apresentou os maiores valores de resistência de união quando comparado aos demais grupos experimentais. Não houve diferença estatisticamente significativa entre os grupos irradiados, e a análise morfológica revelou para estes grupos uma superfície irregular, com aparência escamosa, sem smear layer e com os túbulos dentinários abertos. Nos cortes transversais foi possível observar que esses efeitos se estendem à subsuperfície dentinária e resultam na formação de um padrão modificado dos tags durante o processo de hibridização. Os resultados deste estudo in vitro sugerem que a irradiação com os lasers de Er:YAG e Er,Cr:YSGG promovem um padrão morfológico específico da superfície dentinária, os quais podem interferir negativamente nos valores de resistência de união à resina composta. / Improvements on technology have introduced the laser as a great ally in the selected removal of dental caries. However, the different laser parameters and their effects on dental hard tissues and collagen fibrils have not yet been completely studied. The aim of the present study was to evaluate the effect of Er:YAG and Er,Cr:YSGG laser irradiation on dentin morphology and on microtensile bond strength of resin composite to the irradiated dentin. Sixty-four dentin hemi-disks obtained from sound human third molars were randomly divided into 9 groups (n=7): G1 Control, G2 Er:YAG Laser 250 mJ, 4 Hz; G3 Er:YAG Laser 200 mJ, 4 Hz; G4 Er:YAG Laser 180 mJ, 10 Hz; G5 Er:YAG Laser 160 mJ, 10 Hz; G6 - Er,Cr:YSGG Laser 2W, 20 Hz; G7 - Er,Cr:YSGG Laser 2,5W, 20 Hz; G8 - Er,Cr:YSGG Laser 3W, 20 Hz; G9 - Er,Cr:YSGG Laser 4W, 20 Hz. Twenty seven specimens (n=3) were processed for Scanning Electron Microscopy (SEM) for surface and cross-sectional morphological analysis. Nine specimens (n=1) were processed for Transmission Electron Microscopy (TEM) and twenty seven (n=3) were restored to evaluate the adhesive interface. For the microtensile bond strength (^TBS) test, 45 flat occlusal dentin surfaces were restored and after storage in water for 24h, were submitted to ^TBS test. ANOVA (=5%) showed that G1 (control group) presented the highest ^TBS values when compared with the irradiated groups, which did not differ from each other. SEM analysis showed an irregular scaly surface, free of smear layer and with open dentinal tubules. The cross-sectional dentin micrographs showed that the effects of laser extended to the dentin subsurface resulting in a pattern of tags modified in the hibridization process. The findings of this in vitro study showed that both Er:YAG and Er,Cr:YSGG laser irradiation resulted in a specific morphological pattern of dentin that negatively affects the bond strength to resin composite to such dentin.

O objetivo deste estudo é avaliar a influência da irradiação com laser de Er,Cr:YSGG no espaço protético de condutos radiculares preparados para a fixação de pinos em fibra de vidro reforçados (FRC Postec®), avaliando-se o comportamento de três diferentes técnicas adesivas (self-etching= Panavia F®; total-etching= Excite DSC® + Variolink II® e self-adhesive= RelyX Unicem Aplicap®). Observaram-se as orientações da norma ISO-TS 11405 para a preparação das raízes dentais unirradiculares humanas. As amostras foram separadas na altura da junção amelo-dentinaria, tratadas endodonticamente com instrumentação rotatória e seladas com técnica de condensação lateral. Foram utilizados dois tipos diferentes de pontas de safira para irradiação intracanal o axial (Z4, 400 m de diâmetro) e o radial ou side firing (RFT4, 415 m de diâmetro) com densidade de energia de 40 J/cm2. Esta densidade de energia foi obtida como parte deste experimento, analisando-se diversos parâmetros de energia com os resultados morfológicos e térmicos obtidos. Nesta densidade, obtive-se grande quantidade de túbulos dentinários abertos e variação de temperatura segura para uso clínico. Cada raiz foi irradiada em cinco movimentos circulares, de apical para cervical, com intervalo de 20 segundos, entre elas. Os FRC Postec foram cimentados imediatamente após a irradiação e divididos randomicamente em nove grupos (n=10). Cada raiz foi seccionada em 6 discos de 1 mm de espessura para ensaio do tipo push-out, para a obtenção da força máxima de ruptura, e posterior cálculo da resistência de união adesiva em função da área de superfície do núcleo deslocado, resultando na pressão em MPa. A resistência de união adesiva nos grupos irradiados foi estatisticamente superior do que os grupos controle. Foram avaliadas as diferenças na resistência de união adesiva em diferentes porções do núcleo (cervical, médio e apical), onde o sistema adesivo tipo self-adhesive (RelyX Unicem Aplicap) teve o melhor resultado para os grupos irradiados. Avaliou-se por técnica de FTIR as diferenças químicas ocorridas na dentina intrarradicular irradiada com laser de Er,Cr:YSGG, em diferentes porções (cervical, médio e apical) em função da orientação dos túbulos. Foram observadas nos espectros de FTIR alterações na intensidade das bandas de algumas moléculas, indicando variação na quantidade da parte orgânica (amida I e amida II) após a irradiação, nas diferentes regiões dentais em função do feixe laser sobre a orientação dos túbulos dentinários, sem causar alterações na sua composição. Essas diferenças podem interferir no resultado da resistência de união adesiva. A técnica de utilização de laser de Er,Cr: YSGG em dentina radicular pré-cimentação de núcleo protético aumenta a resistência de união adesiva, na dependência do cimento utilizado. / The aim of this study was to evaluate the influence of the Er,Cr:YSGG laser irradiation in the prosthetic post space before FRC Postec placement using three different adhesive systems (self-etching- Panavia F; total-etching- Excite DSC + Variolink II e self adhesive - RelyX Unicem Aplicap). According to ISO-TS 11405, sectioned at the CEJ, human dental single roots were endodontically treated with rotatory instruments and filled with gutta-percha points and sealer. Were used at 40 J/cm2 an axial Z4 laser tip (400 m diameter) and a radial (side firing) RFT4 laser tip (415 m diameter). This density of energy was experimentally determinated observing morphological changes in SEM images due to an increase range in the energy applied to dentinal root canal surface. At the length between 32 and 49 J/cm2 most of the intracanal dentinal tubules were open. The temperature increase was within the biological safe suggesting a feasible clinical use. Each root canal was irradiated five times with helicoidal movements from apical to cervical, with 20 seconds interval. FRC Postec were immediately cemented and randomly divided in nine groups (n=10). Each root canal was cut in six discs for push out test, were the maximum failure load was obtained and the bond strength was calculated as a function of post bonded area. The bond strength for the irradiated groups was superior among all control groups. The evaluation of bond strength among root region (cervical, middle and apical) was performed and differences were observed. The self-adhesive (RelyX Unicem Aplicap) system was noted superior among all irradiated groups and all regions. Chemical changes were analyzed by FTIR due to dental root portion and dentinal tubules orientation to laser beam direction. The FTIR spectra obtained indicated changes due to tubules orientation and root canal portion irradiated such as the proportion among the compositional ratio without causing chemical changes. These compositional ratio alterations may affect the bond strength results. The irradiation with Er,Cr:YSGG laser on dentinal walls of a prosthetic post space preparation before the post cementation, as suggested in this study, may increase the bond strength between the post and root canal dentin depending upon the luting cement that is used.

A evidência científica da cariologia atual vem apontando para a promoção de saúde bucal, técnicas preventivas, diagnóstico precoce da doença cárie e suas lesões nos elementos dentais, remineralização de lesões de cárie incipientes e a intervenção restauradora célere como procedimentos minimamente invasivos. Durante o ato de remoção de lesão de cárie, é comum remover-se inadvertidamente também tecido dental hígido durante a fase final, por ser um tanto quanto difícil precisar clinicamente os limites entre tecido dental comprometido e tecido dental viável. Usando-se a tecnologia com lasers de Érbio, uma dica clínica subjetiva é a mudança da percepção do som emitido pela ablação do tecido dental ao passar-se de substrato cariado para hígido (de grave para agudo), como uma forma adicional de saber que a ablação naquele ponto deve ser interrompida. Este estudo visa classificar estas diferenças sonoras em dentina e torná-las um parâmetro objetivo para odontologia de mínima intervenção ao usar-se lasers de Érbio. Para tanto, foram realizadas três fases do estudo: primeiramente, utilizaram-se vinte dentes posteriores humanos, sendo dez cariados e dez hígidos. A dentina foi irradiada com laser de Er:YAG sob os mesmos parâmetros, distância e refrigeração e um microfone monodirecional foi posicionado a 10cm da área operatória para captação e gravação dos sons produzidos pela ablação ao se operar tanto em dentina hígida quanto cariada. Dez pulsos por arquivo foram então analisados em um software (200 análises). Foram encontradas diferenças entre os padrões sonoros produzidos dos grupos cariados e hígidos e encontrado um ponto de corte para estas freqüências sonoras, que seria testado a seguir. Em outra segunda etapa, foi desenvolvido, testado e aplicado um software limitador de freqüência sonora, para que, em tempo real, avisasse o operador se o tecido dentinário que estava sendo ablacionado pelo laser de Érbio (Er:YAG e Er,Cr:YSGG) era cariado ou viável, e este evitasse o sobrepreparo, ou seja, desgastar o tecido sadio inadvertidamente, de acordo com os preceitos de mínima intervenção. Finalmente, em uma terceira etapa, o método foi validado através dos critérios visual-tátil e de fluorescência a laser; ilustrações representativas dos espécimes testados foram obtidas através de microscopia de luz e de microscopia eletrônica de varredura. Conclui-se que o som da ablação dentinária é um parâmetro objetivo que pode ser utilizado como um recurso adicional de orientação no processo de remoção de dentina cariada com lasers de Érbio e que a ferramenta desenvolvida para tanto foi efetiva, devendo ser aprimorada. O método foi validado exitosamente pelos critérios propostos. / Studies in cariology have been struggling for the development of health promotion, caries prevention techniques, precocious diagnoses of lesions, re-mineralization of incipient carious lesions and early restorative intervention with minimally invasive procedures. When removing caries lesion, healthy dental structure is often removed inadvertently during its final phase, for being quite difficult to clinically precise the limits between viable and decayed dental tissues. With laser technologies, a subjective clinical hint, often used to indicate when tissue ablation should be stopped is that different sounds are perceptive whether in carious (bass) or in healthy (treble) dental structure; when sound produced by ablation turned treble it would mean that healthy tissue was reached. This study aims to classify those audio differences and to turn them into objective parameters for a conservative operative dentistry with minimally invasive tissue removal when using Erbium lasers. For so, three phases of this study were needed: at first, twenty freshly extracted posterior human teeth were used (10 decayed and 10 sound teeth). Dentine was irradiated with Er:YAG laser under the same parameters, distance and refrigeration and a mono directional microphone was set 10cm far from the operative area in order to capture and record the ablation produced sounds when working either on carious or healthy dentine. Ten pulses per file were then analyzed in a computer software (200 analyses).It was permitted to draw differences between decayed and healthy produced sounds and also to establish a cut-off value for these sound frequencies, that would be tested later on. On a second phase, a piece of software which was able to border the sound frequency was then developed and tested. This tool was meant to warn the operator, in real-time basis, if sound dentine was reached, while it was ablated by Erbium lasers (either Er:YAG or Er,Cr:YSGG) and so one could avoid over-treatment, which means not to remove sound tissue inadvertently, according to minimal intervention dentistry concepts. Finally, on a third part of the study, the proposed guiding method for dentine caries removal was validated through visuo-tactile and laser fluorescence criteria. Representative illustrations of the tested specimens were obtained by light microscopy and scanning electron microscopy. As a conclusion, audio analysis came out to be a technical reliable objective parameter to determine whether laser ablated dentine substrates are decayed or sound; therefore it can be proposed as an additional conservative parameter to guide the clinician during dentine caries removal process, and that the tool developed for so was effective and should be sharpened. This method was successfully validated by the proposed criteria.

O objetivo deste estudo foi avaliar os efeitos dos lasers de Nd:YAG, CO2 e Er,Cr:YSGG no tratamento de superfície da cerâmica Y-TZP antes e depois da sinterização final, por meio de Microscopia Eletrônica de Varredura (MEV), mensuração da rugosidade de superfície (Ra) e teste de resistência adesiva por microcisalhamento (RAD). Para tanto, barras de Y-TZP foram confeccionadas e distribuídas entre as 3 fases do experimento, sendo as duas primeiras fases seletivas para a identificação dos parâmetros lasers a serem utilizados na fase 3 de análise da RAD. Na fase 1 MEV - foram testados parâmetros que variavam entre 50 e 100 mJ para o laser de Nd:YAG, 2 a 4W para o laser de CO2 e 1 a 3,5W para o laser de Er,Cr:YSGG, testados antes ou após a sinterização da Y-TZP. Observou-se que a maioria dos tratamentos foram capazes de promover alterações na cerâmica com diferentes intensidades dependendo do parâmetro utilizado. Para a fase 2 Ra foram selecionados apenas os tratamentos com os lasers de Nd:YAG e Er,Cr:YSGG aplicados antes da sinterização da cerâmica, sendo que cada laser foi avaliado individualmente e comparados ao tratamento de superfície com o sistema Rocatec. Os tratamentos com os maiores valores de Ra foram: para o laser de Nd:YAG - 1,6W-20Hz (11.623±3,419) e para o laser de Er,Cr:YSGG - 3W (6,051±1,991). O sistema Rocatec apresentou os menores valores de rugosidade (1,297±0,026). Para a fase 3 do estudo RAD foram selecionados os parâmetros que apresentaram os maiores valores de Ra para cada laser, além do parâmetro com características favoráveis à adesão observado na fase 1 MEV. Sendo assim, o teste de microcisalhamento foi realizado entre a cerâmica Y-TZP tratada com os lasers antes da sinterização final, nos parâmetros de 1,6W e 1,2W para o laser de Nd:YAG e 3W para o laser de Er,Cr:YSGG; e com o sistema Rocatec, utilizando-se dois cimentos resinosos de dupla ativação (Panavia e RelyX U100). A análise estatística ANOVA Split-Plot dos dados de RAD obtidos revelou que não houve diferença estatística entre os cimentos estudados. Porém, no fator tratamentos de superfície observou-se que os maiores valores de RAD foram obtidos para o sistema Rocatec (20,92±3,4MPa). Os dois parâmetros utilizados para o laser de Nd:YAG foram semelhantes entre si com valores de RAD intermediários (1,2W=9,79±4 MPa e 1,6W=11,35±3,64 MPa) e o tratamento com o laser de Er,Cr:YSGG apresentou os menores valores de RAD (1,43±1,21MPa). Pôde-se concluir que os tratamentos de superfície com todos os lasers testados nos parâmetros de irradiação após a sinterização, provocaram danos à cerâmica Y-TZP. Os tratamentos com os lasers de Nd:YAG e Er,Cr:YSGG antes da sinterização, apesar de apresentarem características micromorfológicas sugestivas de aumento da área de superfície, não foram suficientes para aumentar a resistência adesiva entre a Y-TZP e os cimentos resinosos testados. / The aim of this study was to evaluate the effects of Nd:YAG, CO2 and Er,Cr:YSGG lasers on the surface treatment of Y-TZP ceramic before and after the final sintering by means of Scanning Electron Microscope (SEM), surface roughness measurement (Ra) and microshear bond strength adhesive resistance test (RAD). In order to do so, Y-TZP bars were produced and distributed among the three experimental phases, being the two first ones selective in what regards identifying parameters for the lasers used in phase 3 of the RAD analysis. In phase 1 SEM parameters varying from 50 to 100 mJ were tested for the Nd:YAG laser, from 2 to 4W for the CO2 laser and from 1 to 3,5W for the Er,Cr:YSGG laser, either before or after the Y-TZP sintering. It was observed that most treatments were able to promote changes in the ceramic with varied intensity according to the parameter used. In phase 2 Ra only treatments with Nd:YAG and Er,Cr:YSGG lasers applied before ceramic sintering were selected, and each laser was individually evaluated and compared to the surface treatment with Rocatec system. The treatments which had the highest Ra values were: 1,6W-20Hz (11.623±3,419) for the Nd:YAG laser and 3W (6,051±1,991) for Er,Cr:YSGG. Rocatec system showed the lowest roughness levels (1,297±0,026). As for phase 3 of the study RAD the highest Ra parameters for each laser were selected as well as the parameter with favorable characteristics to adherence observed in phase 1 SEM. Therefore, the microshear bond strength test was done not only in the Y-TZP ceramic treated with lasers before the final sintering (using 1,6W and 1,2W parameters for the Nd:YAG laser and 3W for Er,Cr:YSGG), but also in the Rocatec system, making use of two double activation resin cements (Panavia e RelyX U100). The ANOVA Split-Plot statistical analysis of the RAD data showed that there were no statistical variations concerning the studied cements. However, in what regards the surface treatments, it was observed that the highest RAD values were obtained for the Rocatec system (20,92±3,4MPa). Both parameters used for the Nd:YAG laser were similar, presenting intermediate RAD values (1,2W=9,79±4 and 1,6W=11,35±3,64 MPa). The treatment which used Er,Cr:YSGG laser presented the lowest RAD values (1,43±1,21MPa). It was possible to conclude that surface treatments with all tested lasers in the irradiation parameters after sintering caused damage to the Y-TZP ceramic. Treatments making use of Nd:YAG and Er,Cr:YSGG lasers before the sintering were not enough in order to increase the adhesive resistance between Y-TZP and the resin cements tested, even though they presented micromorphological characteristics that indicated growth in the surface area.

Abstract The delta opioid receptor (δOR) is a member of the G protein-coupled receptor family. This transmembrane receptor has an important role in the regulation of pain. The OPRD1 gene that encodes the human δOR (hδOR) contains at least 11 single-nucleotide polymorphisms (SNPs). The only nonsynonymous SNP resides in the amino-terminal (N-terminal) domain of the receptor and it replaces Phe at position 27 with Cys, thus introducing an unpaired Cys residue on the extracellular surface of the receptor. The Cys27 variant has been shown to have an allelic frequency of about 10% in Caucasian populations. The polymorphic site is flanked by two putative N-glycosylation sites at Asn18 and Asn33. In this study, the folding, maturation and trafficking of hδOR was assessed using the hδORPhe27 and hδORCys27 variants and the N-glycosylation deficient forms of the latter as models in a heterologous expression system. The effects of N-glycosylation and the unpaired Cys-residue were studied with various biochemical, pharmacological and cell biological methods. In addition, protein-protein interactions of the intracellular hδOR precursors were assessed. The hδORCys27 and hδORPhe27 variants differed significantly in their subcellular localization and maturation efficiency. The newly synthesized hδORCys27 was found to accumulate in the endoplasmic reticulum (ER) prior to its ER-associated degradation in proteasomes. Although a slow maturation rate was characteristic for both variants, only the hδORCys27 had poor maturation efficiency. The cell surface expression of hδORCys27 was further decreased because the constitutive internalization of this receptor was enhanced compared to hδORPhe27. N-linked glycosylation was not required for hδOR function or ligand binding, but was important for the expression of the correctly folded receptor species at the cell surface. The mutant non-N-glycosylated receptor was shown to traffic to the cell surface with enhanced kinetics, but some of the plasma membrane receptors were in a nonnative conformation. Also, the overall levels of the non-N-glycosylated hδORCys27 were decreased as the receptor was efficiently internalized for lysosomal degradation in a constitutive fashion. The hδORCys27 and hδORPhe27 precursors were found to interact with several ER localized proteins, such as calnexin (CNX), protein disulfide isomerase (PDI) and ERp72. The receptors also associated with the sarco(endo)plasmic reticulum calcium ATPase 2b (SERCA2b), which was shown to occur during translocation of the receptor to the ER membrane or immediately thereafter. The interaction was not receptor N-glycan dependent and the normal functional activity of SERCA2b was shown to be required for proper cell surface expression of hδOR. / Tiivistelmä δ-opioidireseptori kuuluu G-proteiinikytkentäisiin reseptoreihin, ja sillä on tärkeä rooli kivun säätelyssä. Ihmisen δ-opioidireseptoria koodaavassa OPRD1 geenissä on havaittu ainakin 11 yhden nukleotidin polymorfiaa. Vain yksi tunnetuista polymorfioista aiheuttaa muutoksen proteiinin aminohapposekvenssiin. Se sijaitsee reseptorin aminoterminaalisessa osassa ja se muuttaa fenyylialaniinin (Phe) kohdassa 27 kysteiiniksi (Cys), joka on pariton. Cys27-variantin yleisyys eurooppalaisessa väestössä on noin 10 %. Polymorfisen kohdan molemmilla puolilla on N-glykosylaatiokohdat asparagiineissa Asn18 ja Asn33. Tämän työn tavoitteena oli tutkia δ-opioidireseptorin laskostumista, maturaatiota ja kuljetusta heterologisessa solumallissa käyttämällä Phe27- ja Cys27-variantteja sekä Cys27-variantin N-glykosyloimatonta mutanttia. Cys27-polymorfian ja N-glykosylaation vaikutuksia tutkittiin useilla biokemiallisilla, farmakologisilla sekä solubiologisilla menetelmillä. Lisäksi työssä tutkittiin solunsisäisen δ-opioidireseptorin esiasteen vuorovaikutusta muiden proteiinien kanssa. Phe27- ja Cys27-varianttien sijainti solun sisällä ja maturaatiotehokkuus eroavat toisistaan merkittävästi. Vastasyntetisoitu Cys27-variantti kerääntyy endoplasmakalvostoon, josta se ohjautuu proteasomihajoitukseen. Molemmat variantit kulkeutuvat solun pintaan hitaasti. Cys27-variantin prosessointi on huomattavasti tehottomampaa ja sen määrää solun pinnalla vähentää myös lisääntynyt ohjaaminen solunsisäiseen lysosomihajotukseen. N-glykosylaatiolla ei havaittu olevan vaikutusta reseptorin toimintaan tai ligandin sitomiseen, mutta sillä on tärkeä merkitys oikein laskostuneiden reseptorien kuljetukselle solun pinnalle, koska osa pintaan päässeistä N-glykosyloimattomista reseptoreista on muodossa, johon reseptorispesifinen ligandi ei sitoudu. Vaikka mutanttireseptori kulkeutuukin solun pintaan nopeammin, sen määrä solun pinnalla on alhaisempi, koska mutanttireseptori ohjataan huomattavan nopeasti solun pinnalta lysosomihajotukseen. Phe27- ja Cys27-varianttien havaittiin olevan myös vuorovaikutuksessa eräiden endosomaalisen kalvoston proteiinien kanssa, kuten kalneksiinin, proteiinidisulfidi-isomeraasin ja ERp72-proteiinin. Kumpikin reseptori havaittiin yhteisessä rakenteessa sarko(endo)plasmakalvoston kalsium-ATPaasi 2b -pumpun (SERCA2b) kanssa N-glykosylaatiosta riippumattomalla tavalla. Nämä proteiiniryhmät muodostuvat, kun reseptori liitetään synteesin aikana endoplasmakalvostoon tai heti sen jälkeen. Vuorovaikutus toiminnallisen SERCA2b:n kanssa havaittiin tärkeäksi toimintakykyisen δ-opioidireseptorin esiintymiselle solun pinnassa.

This master thesis dives a brief introduction to the languages for creating web information systems and languages for creating web pages. It describes existing modular systems for publication (also known as content management systems, CSM), their advantages  and disavantages. Requirements for the Content Management System are specified and the  general scheme (ERD) for such a system is proposed. Presented system is realised in PHP and database system MySQL. Implemented user-friendly aplication running on the server communicates developed system that provides content management functions.

Der Prozess der lexikalischen Selektion, d.h. die Auswahl der richtigen Wörter zur Übermittlung einer intendierten Botschaft, ist noch nicht hinreichend verstanden. Insbesondere wurde kaum erforscht, inwiefern Bedeutungsaspekte, welche in sensomotorischen Erfahrungen gründen, diesen Prozess der Sprachproduktion beeinflussen. Die Rolle dieser Bedeutungsaspekte wurde mit zwei Studien untersucht, in denen Probanden Sätze vervollständigten. In Studie 1 wurde der visuelle Eindruck der Satzfragmente manipuliert, so dass die Sätze auf- oder absteigend am Bildschirm erschienen. In Studie 2 mussten die Probanden Kopfbewegungen nach oben oder unten ausführen, während sie die Satzfragmente hörten. Wir untersuchten, ob räumliche Aspekte der produzierten Wörter durch die räumlichen Manipulationen sowie die räumlichen Eigenschaften der präsentierten Satzfragmente beeinflusst werden. Die vertikale visuelle Manipulation in Studie 1 wirkte sich nicht auf die räumlichen Attribute der produzierten Wörter aus. Die Kopfbewegungen in Studie 2 führten zu einem solchen Effekt – bei Kopfbewegungen nach oben waren die Referenten der produzierten Wörter weiter oben im Raum angesiedelt als nach Bewegungen nach unten (und anders herum). Darüber hinaus war dieser Effekt stärker, je ausgeprägter die interozeptive Sensibilität der Probanden war. Zudem beeinflussten die räumlichen Aspekte der Satzfragmente die räumlichen Eigenschaften der produzierten Wörter in beiden Studien. Somit zeigt diese Arbeit, dass in der Erfahrung basierende Bedeutungsanteile, welche entweder in Sprache eingebettet sind oder durch körperliche Aktivität reaktiviert werden, die Auswahl der Wörter beim Sprechen beeinflussen und dass interindividuelle Unterschiede diesen Effekt modulieren. Die Befunde werden in Bezug zu Theorien der Semantik gesetzt. Darüber hinaus wird das Methodenrepertoire erweitert, indem mit Studie 3 ein Ansatz für die Durchführung von Online-Sprachproduktionsexperimenten mit Bildbenennung vorgestellt wird. / The process of lexical selection, i.e. producing the right words to get an intended message across, is not well understood. Specifically, meaning aspects grounded in sensorimotor experiences and their role during lexical selection have not been investigated widely. Here, we investigated the role of experientially grounded meaning aspects with two studies in which participants had to produce a noun to complete sentences which described sceneries. In Study 1, the visual appearance of sentence fragments was manipulated and they seemed to move upwards or downwards on screen. In Study 2, participants moved their head up- or downwards while listening to sentence fragments. We investigated whether the spatial properties of the freely chosen nouns are influenced by the spatial manipulations as well as by the spatial properties of the sentences. The vertical visual manipulation used in Study 1 did not influence the spatial properties of the produced words. However, the body movements in Study 2 influenced participants’ lexical choices, i.e. after up-movements the referents of the produced words were higher up compared to after downward movements (and vice verse). Furthermore, there was an increased effect of movement on the spatial properties of the produced nouns with higher levels of participants’ interoceptive sensibility. Additionally, the spatial properties of the stimulus sentences influenced the spatial properties of the produced words in both studies. Thus, experientially grounded meaning aspects which are either embedded in text or reactivated via bodily manipulations may influence which words we chose when speaking, and interindividual differences may moderate these effects. The findings are related to current theories of semantics. Furthermore, this dissertation enhances the methodological repertoire of language production researchers by showing how language production studies with overt articulation in picture naming tasks can be run online (Study 3).

Positive-strand RNAviruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear endoplasmic reticulum (ER) membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that ER-vesicle protein of 14 kD (Erv14), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, the perinuclear ER localization of BMV 1a is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coat components (Sec13, Sec24 or Sec31). The requirement of Erv14 for the localization of BMV 1a is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by both Erv14 and Sec24 for the proper localization of BMV 1a. The COPII pathway is well known for being involved in protein secretion; our data suggest that a subset of COPII coat proteins have an unrecognized role in targeting proteins to the perinuclear ER membrane.

Integral membrane proteins of the DP1 and reticulon families are responsible for maintaining the high membrane curvature required for both smooth ER tubules and the edges of ER sheets. Mutations in these proteins lead to motor neurone diseases such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain Reticulon Homology Domains (RHD) that have unusually long (≈30 aa) hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilise membrane curvature. I have uncovered the secondary structure and dynamics of the DP1 protein Yop1p and identified a C-terminal conserved amphipathic helix that on its own interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming amphipathic helices. Together, these results indicate that amphipathic helices play a previously unrecognised role in RHD membrane curvature stabilisation. This work paves the way towards full structure determination of Yop1p by solution state NMR and marks the first high structural resolution study on an RHD protein.