Orientador: Carlos Henrique Inácio Ramos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:01:29Z (GMT). No. of bitstreams: 1 Correa_DanielHenriquedoAmaral_D.pdf: 14618006 bytes, checksum: 0865223d71dcf2f775b71ebcedecc875 (MD5) Previous issue date: 2010 / Resumo: Proteínas enoveladas incorretamente, com freqüência levam à formação de agregados fibrilares contendo extensas estruturas em folha-?, comumente denominadas de fibrilas amilóides. A hipótese acerca da capacidade de formar fibrilas amilóides com estruturas idênticas e ricas em estruturas beta, ser uma propriedade genérica de toda proteína, apoia-se no fato de até mesmo proteínas sem conexão com doenças, como a mioglobina, serem capazes de gerar estruturas fibrilares. Embora várias proteínas sejam intrinsecamente desordenadas, muitas são apropriadamente empacotadas, podendo se desenovelar totalmente ou parcialmente de maneira a expor regiões propensas à agregação, que podem converter o polipeptídeo em fibrilas amilóides. De fato, vários estudos sugerem que intermediários parcialmente enovelados estão envolvidos na fibrilogênese. A apomioglobina (apoMb) de baleia do espermacete é uma proteína bem caracterizada, que forma um intermediário durante o desenovelamento do estado nativo ou após a diluição do estado desenovelado em tampão de enovelamento. A mioglobina é uma proteína altamente solúvel, cujas propriedades do estado nativo não sugerem uma predisposição dessa em formar fibrilas amilóides, corroborado pela organização de sua seqüência de resíduos de aminoácidos em hélices-? bem definidas sem elementos de estrutura em folha-?. Contudo, até a mioglobina forma fibrilas amilóides em certas condições, sugerindo que a capacidade de formar fibrilas seja uma característica comum de toda proteína e, portanto, não estando relacionada a uma estrutura primária específica. Neste projeto, visamos mapear as potenciais interações envolvidas na agregação e formação de fibrilas amilóides em apomioglobinas. Para tanto, apresentamos aqui os estudos dos efeitos de 19 mutantes de apoMb na cinética amiloidogênica da mesma. A indução de fibrilas amilóides foi realizada através da incubação das apoMbs em tampão borato de sódio 50 mM, pH 9 e aquecidas a 65°C. O processo de agregação foi acompanhado por medidas da emissão de fluorescência de Tioflavina T (ThT) e espectroscopia de dicroísmo circular (CD). Outras propriedades morfo-fisicoquímicas das amilóides de apoMb foram também estudadas: energia de ativação da formação de fibrilas, organização estrutural, citotoxicidade, efeito de semeadura, desmontagem por uréia. Nossos resultados mostram que alguns mutantes (7 no total) afetaram a cinética de formação das amilóides, e surpreendentemente, esses efeitos correlacionam-se bem com o efeito que a mutação tem sobre a estabilidade do estado nativo, mas não com o efeito sobre a estabilidade do estado intermediário do enovelamento. As estruturas globais (investigadas por difração de raios-X) das fibrilas formadas pelas ampomioglobinas selvagem e mutantes mostram-se indistinguíveis. Experimentos de citotoxicidade, utilizando um modelo de células neuronais N2A (neuroblastoma de camundongo), e semeadura, confirmam que as diferentes formas de agregados das proteínas são capazes de diminuir a viabilidade celular e de acelerar a formação das fibrilas. Generalizando, nossos resultados suportam a hipótese de que, embora o desenovelamento parcial preceda a formação de fibrilas em apomioglobina, a formação do intermediário de enovelamento não parece ser um passo obrigatório no processo e assim, o enovelamento e a formação de agregados/fibrilas são aparentemente distintos para essa proteína. / Abstract: Protein misfolding usually leads to the formation of fibrillar aggregates with extensive ?-sheet structure, commonly termed amyloid fibrils. The hypothesis that the ability to form ordered ?-rich amyloid fibers with identical structures is a generic property of proteins is supported by the fact that even proteins with no connection to disease, such as myoglobin, are able to generate fibrillar structures. Although several proteins are intrinsically disordered, many are properly packed and should unfold totally or partially exposing aggregation-prone regions that can convert the polypeptide into amyloid fibrils. Actually, several studies suggest that partially folded intermediates are involved in fibrillogenesis. Sperm whale apomyoglobin (apoMb) is a well-characterized protein that forms an intermediate after either unfolding from the native state or dilution of the unfolded protein in a folding buffer. Mb is a highly soluble protein whose native state properties do not suggest a predisposition to form amyloid fibrils, corroborated by its amino acid residues sequence organization in well-defined ?-helices with no ?-sheet elements. However, even Mb forms amyloid fibrils under certain conditions, suggesting that the ability to form fibrils is a common feature of all proteins and is not related to a specific primary structure. In this work, we aim to map potential interactions involved in apomioglobin aggregation and fibrils formation. To such aim, we present here the studies of effects on amiloidogenic kinetics of 19 apoMb mutants. The induction of amyloid fibrils was performed by incubating apoMb proteins on 50 mM sodium borate buffer at pH 9 and heat to 65°C. The aggregation process was monitored both by thioflavin T (ThT) emitted fluorescence and circular dichroism (CD) spectroscopy measurements. Other morph-physicochemical properties of apoMb amyloids were also studied: activation energy of fibril formation, structure organization, cytotoxicity, seeding effect, disassembly by urea. Our results show that some mutants (7 in total) affect the amyloid formation kinetics, and surprisingly, these effects are well correlated with the effect that the mutation has on the stability of the native state but not with the effect on the stability of the folding intermediate. The overall structures, probed by X-ray diffraction, of fibers formed by mutants and wild-type apomyoglobin are indistinguishable. Cytotoxicity experiments, using a neuronal cell line model N2A (murine neuroblastoma), and seeding experiments, confirm that different aggregated forms of proteins are capable of decreasing the cell viability and to speed up the formation of fibrils. Generally, our results support the hypothesis that although partially unfolding precedes fibril formation in apomyoglobin, formation of the folding intermediate is not an obligatory step in the process and thus folding and aggregation/fibril formation are apparently distinct in this protein. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Este estudo é uma pesquisa do tipo exploratório-descritiva que objetivou identificar como os alunos de um Curso Técnico de Enfermagem de uma Escola de Educação Profissional na cidade de São Paulo avaliam a estratégia de ensino estudo dirigido aplicada na Disciplina Saúde do Adulto. A população constituiu-se de 17 alunos e a amostragem 14, com formação básica profissional de auxiliar de enfermagem que concluíram o Curso de Habilitação Profissional de Técnico de Enfermagem. O instrumento de coleta de dados utilizado foi um questionário, contendo 11 questões, aplicado em um único momento, após autorização do Comitê de Ética da mantenedora da Escola e da diretoria da Instituição, os preceitos éticos que envolvem os sujeitos participantes foram seguidos.A metodologia para análise dos resultados ocorreu de acordo com a natureza das questões, quantificados em porcentuais simples e as demais analisadas pelo conteúdo de acordo com a proposta de Bardin (1977). Os resultados foram apresentados em quadros, tabelas e gráficos. A análise dos resultados foi realizada à luz do referencial de Freire (2000). Os alunos conceituaram como boa (86,0%) e excelente (14,0%) a estratégia utilizada, e foram identificadas 83 (100%) unidades semânticas na análise das respostas que justificaram os conceitos, sendo categorizados em: Vantagens, 44 (53,0%); Não “Legal”, 13 (16,0%); Dificuldades, 6 (7,0%); Sentimentos, 10 (12,0%) e Sugestões, 10 (12,0%). Os sujeitos aferiram notas de 1 a 4 em nível crescente de aceitação aos propósitos didático-pedagógicos, conferidos ao estudo dirigido inspirado em Nérici (1992), os proósitos didático-pedagógicos receberam maiores notas que permitem o desenvolvimento de temas de interesse do aluno; estimulam o prazer para estudar e incentivam o aluno a ser o sujeito de seu aprendizado. Considerando que o estudo dirigido foi usado dentro de uma pedagogia interacionista, dialética, materialista e progressista, concluiu-se que os alunos justificaram o conceito atribuído ao estudo dirigido de forma competente e responsável, contribuindo para a melhoria dos resultados do processo ensino-aprendizado em sala de aula, trazendo importantes reflexões docentes para o desenvolvimento de habilidades e competências no uso do estudo dirigido. / This is a descriptive exploratory study that aimed to identify how students attending a practical nurse course at a vocational school in the city of São Paulo evaluate the teaching strategy of direct study applied to the course Health of Adults. The population was comprised by seventeen students and the sample by fourteen of them. Students had a basic professional training as nursing assistants, and had finished the practical nurse course by the time the evaluation of direct study was carried out. A questionnaire with twelve questions was used as the tool to collect data. It was applied once after the authorization of the Ethics Committee of the entity that supports the school and the school management team. Ethical concepts with regards to the subjects were followed. Different methodology was applied to study the results based on the type of questions: quantified in simple percentages or content analysis, according to what was proposed by Laurence Bardin (1977). Results are presented in charts, tables and graphs. Result analysis was performed according to Freire (2000). Students rated the strategy used as good (86%) and excellent (14%). Eighty-three (100%) semantic units were identified to examine the answers that justified the rates. The units were separated in cathegories: advantages, 44 (53%); not “nice”, 13 (16%); difficulties, 6 (7%); feelings, 10 (12%), and suggestions, 10 (12%). Subjects graded the didatic educational proposals from 1 to 4, in crescent order of acceptance regarding directed study as proposed by Nérici (1992); the educational proposals that received higher rates were those that allowed the development of issues that interest the student, that increases the pleasure in studying, and that encourage the student to be the subject of his learning process. Considering that directed study was used in a progressive materialist dialectical interactive educational style, we conclude that the students justify the concept given to directed study in a competent and responsible manner, contributing to the improvement of results of learning-teaching process in the classroom, and raising important thoughts on teacher regarding development of skills and competences to use directed study.

As fosfolipases A2 (PLA2s, ou fosfatidil-acil hidrolases EC 3.1.1.4) catalisam especificamente a hidrólise das ligações ácido-éster na posição sn-2 de glicerofosfolipídios liberando, como produto da catálise, ácidos graxos e lisofosfolipídio. São encontradas em plantas, mamíferos e em veneno de animais vertebrados e invertebrados e estão envolvidas em uma ampla variedade de processos fisiológicos. A fosfolipase A2 secretada humana do grupo IIA (hsPLA2 gIIA) é uma proteína de fase aguda da resposta imunológica, pois sua expressão é induzida por endotoxinas e citocinas via processos autócrinos e/ou parácrinos durante processos inflamatórios de relevância clínica. A hsPLA2 gIIA mostra efeito bactericida contra infecção por Staphylococcus aureus, e tem marcada preferência por fosfolipídios aniônicos tais como fosfatidilglicerol (PG) encontrados em membranas bacterianas. Uma grande variedade de inibidores de PLA2 do grupo IIA foi descrita na literatura, incluindo substâncias polianiônicas que atuam contra os efeitos inflamatórios destas enzimas. Suramina é um derivado de naftiluréia polissulfonado que recentemente mostrou ligação com os resíduos catiônicos no sítio de reconhecimento interfacial de Bothropstoxina-I (BthTX-I), uma PLA2-Lys49 isolada do veneno de Bothrops jararacussu, inibindo a atividade miotóxica da proteína. Devido ao tipo de interação diferenciada da suramina com BthTX-I em relação aos inibidores competitivos de PLA2, nós avaliamos a especificidade de ligação da suramina na hsPLA2 gIIA como um modelo para estudar este novo tipo de inibidor de PLA2s. O efeito da suramina nas atividades biológicas e de membranas artificiais da hsPLA2 gIIA foi avaliado. A suramina aboliu tanto a atividade hidrolítica da hsPLA2 gIIA quanto a atividade de danificação de membranas artificiais Ca2+ independente. Embora a suramina não tenha inibido a atividade bactericida da hsPLA2 gIIA contra a linhagem Micrococcus luteus, a ativação de macrófagos foi abolida pela mesma de maneira dependente de hidrólise. Além disso, técnicas de simulação de dinâmica molecular, calorimetria de titulação isotérmica e mutagênese sítio dirigida foram utilizadas para mapear os sítios de ligação da suramina na proteína. A interação da suramina com a hsPLA2 gIIA resultou de interações eletrostáticas entre grupos sulfonados com cadeias laterais de aminoácidos da região do sítio ativo e dos resíduos em torno das posições 15 e 116 localizados, respectivamente, na N- e Cterminal. Portanto, estes resultados permitem sugerir que a suramina pode atuar como inibidor de sPLA2s / Suramin is a polysulphonated napthylurea used as an antiprotozoal drug that presents inhibitory activity against a broad range of enzymes. We have evaluated the effect of suramin against the artificial and biological activities of the secreted human group IIA phospholipase A2 (hsPLA2 gIIA), a protein involved in inflammatory processes. To map the suramin binding sites on the hsPLA2 gIIA, proteins with mutations in the active site region and in the protein surface that makes contact with the phospholipids membrane were expressed in E. coli and refolded from inclusion bodies. The activation of macrophage cell line RAW 264.7 by hsPLA2 gIIA was monitored by nitric oxide release, and bactericidal activity of the protein against Micrococcus luteus was evaluated by colony counting and by flow cytometry. The hydrolytic activity of the hsPLA2 gIIA against lipossomes composed of a mixture of dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) was inhibited by a concentration of 100 nM suramin. The activation of macrophages by hsPLA2 gIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA2 gIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 M. The affinity of interaction of the suramin with hsPLA2 gIIA was evaluated by suramine fluorescence and the mutants K15A, K38A, R54A and K123A presented a reduced affinity. The binding of the suramin/hsPLA2 gIIA complex was investigated by molecular dynamics simulations, which indicated two conformations of the bound inhibitor, which involve cationic amino-acid side chains in the active-site region and residues around positions 15 and 116 located in the N- and C-termini respectively in the substrate recognition surface. These results were correlated with isothermal titration calorimetry data, which demonstrated 2.7 suramin-binding sites on the hsPLA2 gIIA. These results suggested that suramin represents a novel class of phospholipase A2 inhibitor

O notável desenvolvimento da indústria eletrônica observado nos últimos tempos tem permitido aplicações de conjuntos integrados de sensores em ambientes sem fio, conhecidos por wireless sensor networks (WSN), que passam por sensoriamento de processos industriais, ambientes tóxicos, projetos militares de monitoração de variáveis de segurança, até observação de fenômenos físicos naturais. Uma das principais especificações de uma rede WSN, o consumo de energia afeta diretamente a capacidade e tempo de vida útil do sistema, pois, na maioria dos casos, seus módulos possuem baterias independentes e sua substituição nem sempre é tarefa simples. Motivado pela necessidade de oferecer robustez e economia de energia nas redes WSN, o protocolo difusão direcionada se baseia na centralização de dados e a identificação de seus módulos é feita por meio de pares valor-atributo. Sua estrutura permite a adição de componentes de software que podem atuar na análise e modificação dos dados recebidos com o objetivo de alterar o protocolo original. O objetivo principal deste trabalho é investigar um método para otimização do uso de energia disponível em redes WSN com intuito de prolongar seu tempo de vida útil. O metódo consiste em analisar os valores das energias atribuídas aos módulos componentes da rede por meio de incorporação de um algoritmo de rotas baseado em energia ao protocolo difusão direcionada. Comparações de desempenho da rede em relação ao seu tempo de vida e energia dos módulos são realizadas utilizando o simulador NS-2. As simulações feitas em diversos cenários indicaram melhoria de desempenho em relação ao protocolo difusão direcionada original. Os cenários onde o protocolo original foi alterado apresentaram um número maior de rotas descobertas e possibilitaram um aumento de pelo menos 22% no tempo de vida da rede, em relação ao protocolo original. / The notable development of electronic industry in the last years allows the implementation of sensor integrated circuits in wireless environments, known as wireless sensor networks (WSN), which leads to industrial process sensing, toxic environments, military security monitor projects and natural physical phenomenon. As one of the main specifications of a WSN network, the energy consumption directly affects the capacity and the system useful lifetime, since most of the time its modules have independent batteries and their substitution is not always a simple task. Motivated by the need of offering robustness and energy economy for WSN networks, the directed diffusion protocol is data-centric based and its modules identification is made by attribute-value pairs. The directed diffusion structure enables the addition of software components which can act on the analysis and modification of received data with the objective of changing the original protocol. The main objective of this work is to investigate a method for optimization of available energy on WSN networks with the intention of increasing its useful lifetime. The method consists on analyzing the energy values attributed to the component modules of the network by incorporating an energy-based routing algorithm to directed diffusion protocol. Performance comparisons of the network related to its lifetime and modules energy are developed using the NS-2 simulator. Simulations performed in several scenarios indicated a better performance in relation to the original directed diffusion protocol. The scenarios where the original protocol was changed had larger number of discovered routes and allowed a rising of at least 22% on network lifetime, in relation to the original protocol.

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 tese_simone_simionatto.pdf: 333806 bytes, checksum: 6d2a7ca25216004656318115c514c721 (MD5) Previous issue date: 2008-10-10 / Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), a respiratory disease responsible for significant economic losses. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. Besides, their preparation is expensive because of fastidiously growth of M. hyopneumoniae in vitro. Therefore, the development of alternatives for EP prophylaxis is important for improving health conditions of pigs. Recombinant DNA technology can be used to overcome problems with conventional vaccines. Nevertheless, because of the use of TGA and not TGG to code for tryptophan, translation of mycoplasmal genes terminates prematurely when cloned in other bacteria, such as Escherichia coli. Because of this, the number of antigenic proteins characterized to date in vaccine formulations or immunodiagnostic tests is still restricted. In this work, 63 genetic fragments were selected, which correspond to 48 sequences coding (CDS) of M. hyopneumoniae. First, an overlap extension-PCR method for site-directed mutagenesis of M. hyopneumoniae genes was standardized, aiming at substituting TGA codon by TGG. With this improved method, site-directed mutagenesis was successfully achieved in 14 M. hyopneumoniae genes. Other selected genes were amplified by PCR and cloned into Champion pET200D/TOPO®. A total of 59 genetic fragments were efficiently cloned. From these, 49 had their proteins expressed in E. coli and 35 were purified by affinity chromatography using Ni- Sepharose columns (HisTrap ). Immunogenic and antigenic properties of these proteins were analyzed. For this, the proteins were tested against sera from hyperimmune and from convalescent pigs through ELISA (enzyme-linked immunosorbent assay) and Western blot. Nineteen recombinant proteins were specifically recognized by convalescent pig sera indicates they are expressed during infection. Immune humoral response against recombinant proteins was evaluated in BALB/c mice by ELISA. The results showed that antigens induce variable levels of antibodies, which allows inferring the immunogenicity of each antigen. Sera from inoculated mice with twenty recombinant proteins were able to recognize the native protein by ELISA. This study allowed identifying and characterizing new immunogenic proteins according to their potential for use in diagnostic tests and/or vaccine. These data represent an important contribution for the development of more efficient serological tests and a subunit vaccine for controlling M. hyopneumoniae infections in pigs. / Mycoplasma hyopneumoniae é o agente etiológico da pneumonia enzoótica suína (PES), uma doença respiratória responsável por significativas perdas econômicas. Vacinas comerciais são mundialmente utilizadas no controle desta doença, porém proporcionam apenas proteção parcial. Além disso, são caras devido ao crescimento fastidioso do M. hyopneumoniae in vitro. Desta forma, o desenvolvimento de alternativas para a profilaxia da PES é fundamental para a melhoria da sanidade dos suínos. A tecnologia do DNA recombinante pode ser usada para superar problemas com as vacinas convencionais. No entanto, pela utilização de códons TGA e não TGG para codificar o amino ácido triptofano, a tradução de genes de micoplasmas termina prematuramente quando clonados e expressos em outras bactérias, como Escherichia coli. Devido a isso, o número de proteínas antigênicas de M. hyopneumoniae caracterizadas e testadas como vacinas ou em testes de imunodiagnóstico ainda é restrito. Neste trabalho, 63 fragmentos gênicos foram selecionados, os quais correspondem a 48 seqüências codificadoras (CDS) de M. hyopneumoniae. Num primeiro momento, foi padronizado um método de PCR de sobreposição para mutagênese sítio-dirigida de genes de M. hyopneumoniae, objetivando a substituição do códon TGA por TGG, na seqüência do DNA. Com esse método, a mutagênese sítio-dirigida foi realizada com sucesso em 14 genes de M. hyopneumoniae. Os demais genes selecionados foram amplificados por PCR e clonados no vetor Champion pET200D/TOPO®. No total, 59 fragmentos gênicos foram clonados eficientemente. Destes, 49 tiveram suas proteínas expressas em E. coli e 35 foram purificadas por cromatografia de afinidade em coluna de Ni- Sepharose (HisTrap ). As propriedades antigênicas e imunogênicas destas proteínas foram analisadas. Para isso, as proteínas foram testadas contra soro de suínos convalescentes e soro hiperimune através de ELISA (enzyme-linked immunosorbent assay) e Western blot. Dezenove proteínas recombinantes foram reconhecidas especificamente por soro de suínos convalescentes, indicando que elas são expressas durante a infecção. Resposta imune humoral contra as proteínas recombinantes foi avaliada em camundongos BALB/c através de ELISA. Os resultados demonstraram que os antígenos induzem um nível variável de anticorpos, o que nos permite inferir quanto à capacidade imunogênica de cada antígeno. O soro dos camundongos inoculados com 20 proteínas foi capaz de reconhecer as proteínas nativas através de ELISA. Este estudo permitiu identificar e caracterizar novas proteínas imunogênicas de acordo com o seu potencial para uso em testes de diagnóstico e/ou vacina. Estes dados representam uma importante contribuição para o desenvolvimento de testes sorológicos mais efecientes e vacinas de subunidade para o controle da infecção causada por M. hyopneumoniae em suínos.

Abstract The mitochondrial oxidative phosphorylation system is composed of five multisubunit enzyme complexes. Complex I is the first and largest of these, containing 46 subunits, seven encoded by mitochondrial DNA (mtDNA) and the rest by nuclear DNA. Isolated complex I deficiency is a major cause of metabolic errors in infancy and childhood, presenting as encephalomyopathies or multisystem disorders. Due to the bigenomic origin of complex I, the genetic causes of these defects can be either mitochondrial or nuclear. The object of the present work was to identify the underlying genetic cause in cases of children with complex I deficiency and to obtain more information on the structurally and functionally important sites of complex I subunits. The complete coding region of mtDNA was analysed by conformation-sensitive gel electrophoresis and subsequent sequencing. In addition, nine nuclear genes encoding conserved subunits of complex I were sequenced. The structural and functional consequences of the new sequence variants were further elucidated using mutagenesis of homologous residue in bacterial NDH-1 or by studying complex I assembly and expression in patient cell lines. Analysis of the mtDNA coding region in 50 children revealed four definitely pathogenic mutations, 3460G>A, 10191T>C, 11778G>A and 14487T>C, in seven patients. In addition, two novel mtDNA base pair substitutions were identified, 3866T>C in a patient with muscle weakness and short stature and 4681T>C in a patient with Leigh syndrome. The latter mutation causes a Leu71Pro amino acid exchange in the ND2 subunit. Cybrid clones harbouring this mutation retained the complex I defect, and reduced amounts of fully assembled complex I were detected in patient cell lines. The 3866T>C mutation leads to a Ile187Thr amino acid substitution in the ND1 subunit, and functional studies of the homologous amino acid substitution in E. coli showed that this had an effect on the assembly or stability of the NDH-1 holoenzyme. Sequencing of the nine nuclear-encoded complex I genes revealed only one novel base pair substitution with pathogenic potential. Further studies are needed, however, to establish the role of the Arg18Cys substitution in the mitochondrial leading peptide of the TYKY subunit. The above findings emphasize the contribution of mtDNA mutations to the aetiology of pediatric patients with complex I deficiency. Furthermore, two LHON primary mutations were identified in the present cohort of patients, although the clinical signs differed considerably from the classical symptoms of LHON. This suggests that the phenotype caused by primary LHON mutations is more variable than has so far been thought.

Der Weg hin zum kompetenzorientierten Lehren und Lernen und damit die Einführung von Bildungsstandards steht im Zentrum gegenwärtiger schultheoretischer Diskussionen. In der vorliegenden Arbeit wird dieser Diskurs aufgegriffen und gezeigt, dass die schulpraktische Umsetzung dieser „Outputorientierung“ eine vielschichtige und komplexe ist. Im Fokus der theoretischen Betrachtungen der Arbeit stehen die Themen Selbststeuerung und Selbstorganisation beim Lernen, Lernziele, Bildungsstandards und Kompetenzorientierung. Die empirische Studie analysiert, basierend auf den theoretischen Erörterungen, die Möglichkeiten der Steigerung der Fremdsprachenkompetenz von Schülerinnen und Schülern. Erforscht wird dies am Beispiel des webbasierten adaptiven Systems des Englisch-Assistenten bzw. Englisch.Digital. Es wurden fünf Explorationen auf Basis von drei Fragebogenuntersuchungen sowie Informelle Kompetenzmessungen (IKM) mit 75 Schüler/‑innen zweier österreichischer Hauptschulen der achten Schulstufe durchgeführt. Die Untersuchung richtete sich auf die hypothesentestende Exploration von vier Konstrukten (Allgemeine Selbstwirksamkeitserwartung – Selbsteinschätzung, Spezielle Selbstwirksamkeitserwartung im Unterrichtsfach Englisch im Bereich Lesen – Selbsteinschätzung, Selbstregulation – Selbsteinschätzung und spezielle Fremdsprachenkompetenz im Bereich Lesen – Selbsteinschätzung und Informelle Kompetenzmessung). Die Untersuchung zeigt ein differenziertes Bild in Bezug darauf, welche Schülergruppen vom digitalen Medienangebot des Englisch-Assistenten (Englisch.Digital) am stärksten profitieren, durchgehend signifikante Ergebnisse konnten in den fünf Explorationen nicht erzielt werden, was unterschiedliche Gründe haben kann (z. B. Unterschiede in den individuellen Wirkungen oder in den Unterrichtsprozessen oder auch Beschränkungen des Studiendesigns). Zusammenfassend kann festgestellt werden, dass die Frage nach den Wirkungen von digitalen Medien einer weiteren Ausdifferenzierung bedarf und keine allgemeingültigen Aussagen diesbezüglich getroffen werden können, dass digitale Medien per se eine bestimmte Lernwirkung erzeugen. Es ist festzuhalten, dass es nicht ausreichen wird, neue Technologien zu den traditionellen Lehr- und Lernmethoden lediglich hinzuzufügen, es hat auch ein Wandel der Unterrichtsstruktur zu erfolgen (vgl. Dittler, 2003, S. 193 und Lembke & Leipner, 2015, S. 181). Eine Möglichkeit für dies und für die Umsetzung bzw. Anwendung von Informationskompetenz bietet der in dieser Arbeit analysierte Englisch-Assistent (Englisch.Digital). / The road to competency-based teaching and learning, and thus the introduction of educational standards, is at the heart of current educational debate. This doctoral dissertation takes up this discussion and shows that the practical implementation in schools is a multi-layered, multi-faceted and complex one. The focus of the theoretical discussion of the current paper is the fields of self-control and self-organization of learning, learning objectives, educational standards and competence orientation. The empirical study analyses, based on the theoretical considerations, the opportunities for developing pupils’ skills in the foreign language (English). This is explored using the web-based adaptive version of the “English Assistant” or “Englisch.Digital”. Five explorations based on three questionnaires and informal competence measurements (IKM) with 75 pupils (grade eight) were carried out at two Austrian secondary schools. The empirical study focused on (the hypothesis testing) exploration of four constructs (self-assessment of general self-efficacy, self-assessment of special self-efficacy in reading, self-assessment of self-regulation and self-assessment and informal competence measurements of specific skills in reading in the foreign language). The study shows a mixed picture with respect to which groups of students benefit the most from the digital media offer of Englisch.Digital. Consistent significant results in the five explorations could not be obtained, which may be due to any of a number of different reasons (e.g. differences in individual effects/reactions, the teaching processes or limitations of the study design). To summarise, it can be stated that the question of the effects of digital media requires further differentiation and no general conclusions can be drawn with respect to the supposition that digital media produce a certain learning effect per se. It should be noted that it will not suffice to merely add new technologies to the traditional teaching and learning methods, changes to teaching structures will also have to be made (cf. Dittler, 2003, p. 193 and Lembke & Leipner, 2015, p. 181). The English Assistant (Englisch.Digital) analysed in this dissertation offers one possible solution for this and also the problem of the transposition or implementation of information literacy.

Lean Startup lacks a method specifically designed to evaluate whether a HCI-design is fulfilling the promises delivered by the unique value proposition (UVP). At the time of writing, concurrent think aloud protocols during usability testing and interviews are used to that end. There are, however, a couple of issues bundled with the use of such methods. Using a case study conducted on two iterations of high-fidelity prototypes of a meal planning website currently in development, a method called Directed Product Reaction (DPR) is proposed. DPR is a result of the adaptation of Product Reaction Cards, and the addressing of issues uncovered during case study iterations. DPR aspires to be that specifically designed method needed for assessment of user experience in relation to UVP in Lean Startup, and while the resulting method proposed has far from achieved that goal, it serves as a foundation moving forward.

The diploma thesis discusses family communications during Mealtimes. The first part defines the basic theoretical concepts of the theory of children's speech and research associated with it, it defines preschool child development with an emphasis on language and further defines the concept of family communication and child directed speech. At the end of the theoretical part the thesis explains the importance of food/meals in family life. The theoretical part is mainly based on professional literature. The second part consists of five case studies that focus on the analysis of video communications during family meals. All families that participated on the recording signed a consent. The information was also obtained with the help of sociologically oriented survey about adult participants. All recordings were transcribed according to the CHAT system guidelines, which is part of the CHILDES database. The results are compared with the findings of the professional literature. Keywords communication, pre-school child, child directes speech, food/meals

Abstract Osteoporosis is a disease characterized by abnormalities in the amount and architectural arrangement of bone tissue, which leads to impaired skeletal strength and increased susceptibility to fractures. Type 5 tartrate-resistant acid phosphatase (TRACP, AcP5) has been suggested to participate directly in bone resorption. In this study, baculovirus expression vector system in insect cells was used to gain large amounts of recombinant type 5 acid phosphatase for structure determination, structure-based functional studies and production of monoclonal antibodies. Active and inactive forms of the enzyme were separated from each other by cation-exchange chromatography, and characterized. The enzyme was crystallized and the three-dimensional structure was determined. Based on the three-dimensional structure of the active site five different enzyme variants were constructed, produced in insect cells, and purified. The wild type enzyme and the mutated forms were characterized, and their kinetic parameters were determined. The importance of amino acids that were expected to be essential for the acid phosphatase activity was confirmed. The acid phosphatase activity and reactive oxygen species generating activity of this dual enzyme proved to exploit different amino acids in their reaction mechanisms. Further studies are needed to clarify the physiological substrates of TRACP in vivo. The findings of this study could form a base for construction of inhibitors for TRACP that could be useful therapeutic agents for osteoporosis and related bone disorders.