Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2014. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2014-07-25T15:51:49Z No. of bitstreams: 1 2014_MaritzaOcampoBetancur.pdf: 2785011 bytes, checksum: 1e7f6e8c3bda3abce6c1882650617a39 (MD5) / Approved for entry into archive by Guimaraes Jacqueline(jacqueline.guimaraes@bce.unb.br) on 2014-07-29T14:39:53Z (GMT) No. of bitstreams: 1 2014_MaritzaOcampoBetancur.pdf: 2785011 bytes, checksum: 1e7f6e8c3bda3abce6c1882650617a39 (MD5) / Made available in DSpace on 2014-07-29T14:39:53Z (GMT). No. of bitstreams: 1 2014_MaritzaOcampoBetancur.pdf: 2785011 bytes, checksum: 1e7f6e8c3bda3abce6c1882650617a39 (MD5) / A levedura Pichia pastoris tem sido amplamente utilizada na produção de proteínas recombinantes devido a características como fácil manipulação, rápido crescimento e capacidade de fazer modificações pós-traducionais mais similares às dos mamíferos. Apesar do vasto uso desta levedura como sistema de expressão, há poucas marcas de seleção disponíveis para sua manipulação genética. As marcas existentes podem ser auxotróficas (genes de vias biossintéticas como HIS4, ARG4, URA3, ADE1, dentre outros) ou dominantes (geralmente genes que conferem resistência a drogas). O limitado número de marcas seletivas atualmente disponíveis restringe a construção de cepas de P. pastoris contendo mais de uma modificação genética. Nesse contexto, o objetivo deste estudo foi interromper, pela primeira vez, o gene LEU2 no genoma de P. pastoris com o fim de gerar uma linhagem auxotrófica para o aminoácido leucina que pudesse ser utilizada como hospedeira para vetores contendo este gene como marca de seleção. A ruptura gênica foi obtida pela transformação da levedura com um cassete de expressão contendo os genes kanR (resistência a kanamicina/G418) ou Sh ble (resistência a zeocina) flanqueados por regiões 5´ e 3´ do gene LEU2 para promover a recombinação homóloga neste locus. Para construir esse cassete de deleção o gene clonado LEU2 teve um fragmento de aproximadamente 400 pb excisado após digestão com enzimas de restrição sendo substituído pelo cassete de expressão flanqueado por sequências loxP. Após transformação e seleção de mutantes auxotróficos, a marca dominante foi removida. Para tanto, a levedura foi transformada com um vetor contendo o gene que codifica para a proteína CreA, uma recombinase sítio-específica que catalisa a reação de recombinação entre duas sequências loxP. Finalmente, para testar esta nova linhagem, foi construído um vetor contendo eGFP como gene repórter e o gene LEU2 como marca de seleção. A transformação da linhagem auxotrófica leu2 com esse vetor confirmou a recuperação da prototrofia e a capacidade da levedura para produzir a proteína heteróloga intracelularmente. ______________________________________________________________________________ ABSTRACT / The yeast Pichia pastoris has been widely used for the production of recombinant proteins due to several characteristics such as easy genetic manipulation, fast growth and the ability to carry out post-translational modifications similarly to mammals. Despite the wide use of Pichia pastoris as expression system there are few selectable markers available for its genetic manipulation. The existing markers can be auxotrophic (genes of biosynthetic pathways, for example HIS4, ARG4, URA3, ADE1, among others) or dominant (mainly genes that confer drug resistance). The limited number of available selectable markers restricts the construction of strains with more than one recombinant modification. The aim of this study was to interrupt, for the first time, the LEU2 gene in the P. pastoris genome to generate an auxotrophic strain for the amino acid leucine which could be used as host strain for vectors carrying the LEU2 gene as selectable marker. The disruption was achieved transforming the yeast with an expression cassette that contained the kanR (kanamycin/G418 resistance) or Sh ble genes (zeocin resistance) flanked by regions from the LEU2 gene to promote homologous recombination at that locus in the P. pastoris genome. To construct that deletion cassette the cloned LEU2 gene had an excised fragment of approximately 400 bp after digestion with restriction enzymes. That fragment was substituted by the expression cassette flanked by loxP sequences. After transformation and selection of auxotrophic mutants the dominant marker was removed. To accomplish this, the yeast was transformed with a vector carrying the gene that codes for the CreA protein, a site-specific recombinase that catalyzes the recombination reaction between two loxP sequences. Finally, to test the new strain, it was constructed a vector containing eGFP as a reporter gene and the LEU2 gene as a selectable marker. Transformation of P. pastoris leu2 with that vector confirmed the recovery of the prototrophy and the ability of the yeast to produce the intracellular heterologous protein. ______________________________________________________________________________ RESUMEN / La levadura Pichia pastoris ha sido ampliamente utilizada para la producción de proteínas recombinantes debido a características como fácil manipulación, rápido crecimiento y la capacidad de realizar modificaciones postraduccionales similares a las de los eucariotas. A pesar del gran uso de esta levadura como sistema de expresión hay pocas marcas de selección disponibles para su manipulación genética. Las marcas existentes pueden ser auxotróficas (genes de vías biosintéticas como HIS4, ARG4, URA3, ADE1, entre otros) o dominantes (principalmente genes que confieren resistencia a drogas). El limitado número de marcas de selección disponibles actualmente restringe la construcción de cepas de P. pastoris con más de una modificación recombinante. En este contexto, el objetivo de este estudio fue interrumpir, por primera vez, el gen LEU2 en el genoma de P. pastoris con el fin de generar una cepa auxotrófica para el aminoácido leucina que pudiera ser utilizada como hospedera para vectores que tengan el gen LEU2 como marca de selección. La ruptura se logró al transformar la levadura con un casete de expresión que contenía el gen kanR (resistencia a kanamicina/G418) o el gen Sh ble (resistencia a zeocina) flanqueado por regiones del gen LEU2 para promover recombinación homóloga en ese locus del genoma de P. pastoris. Para esto se clonó el gen LEU2 y se digirió con enzimas de restricción para retirar un fragmento de aproximadamente 400 pb el cual se reemplazó por el casete de expresión flanqueado por secuencias loxP. Después de la transformación y selección de mutantes auxotróficos se removió la marca dominante. Para esto se transformó la levadura con un vector que contenía el gen que codifica para la proteína CreA, una recombinasa sitio-específica que cataliza la reacción de recombinación entre dos secuencias loxP. Finalmente para evaluar la nueva cepa se construyó un vector que contenía el gen eGFP como reportero y el gen LEU2 como marca de selección. La transformación de P. pastoris leu2 con este vector confirmó la recuperación de la prototrofía y la capacidad de la levadura para producir la proteína heteróloga intracelularmente.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-05-30T15:01:16Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Maria do Perpétuo S.L.Vasconcelos.pdf: 1475698 bytes, checksum: 5ac01487cbd277bea7d8f0b3803546fb (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-05-30T15:03:01Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Maria do Perpétuo S.L.Vasconcelos.pdf: 1475698 bytes, checksum: 5ac01487cbd277bea7d8f0b3803546fb (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-05-30T15:03:51Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Maria do Perpétuo S.L.Vasconcelos.pdf: 1475698 bytes, checksum: 5ac01487cbd277bea7d8f0b3803546fb (MD5) / Made available in DSpace on 2017-05-30T15:03:51Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Maria do Perpétuo S.L.Vasconcelos.pdf: 1475698 bytes, checksum: 5ac01487cbd277bea7d8f0b3803546fb (MD5) Previous issue date: 2016-11-04 / The components of system Confea/Crea/Mútua always discuss in your events the importance of professional appreciation which In turn for the most people is limited to the focus of professional remuneration. However, when it is really value by the labor market, the money it's natural. The objective of this work is to Point out the indicators for real professional appreciation, like a manner to contribute to this issue taking effect of effective ways not only in documents and burocratics formalities, but directly performed in the daily life of each professional in the technological area. It is also part of the objectives of the study to analyze the actions taken by the Crea/AM in aspect qualification versus valuation. This work has carried out by means of a case study using bibliographic and field research through the definition of a sample restricted to the incorporating companies that demonstrate more clearly how happen the process of professional valuation of Confea/Crea/Mútua system, seen by the optics of the users of the system itself; until then professionals that integrate the technical staff of the selected companies. This process took place through the elaboration, application and tabulation of data from a semi-open questionaire. In last instance the analysis of the data recorded in the research was done which will allow the continuity of the studies in future works. With the results, was observed that isn`t happening the correct disclosure for the technological community of Confea/Crea system initiatives about programs of partnerships and with this create a certain rejection by part of the users. Finally, the conclusion is that the question of professional valuation of the technological area basically begins with the professional himself, improving in continuous qualification with the awareness of your responsibilities and your professional profile; of the education institutions in establishing interaction with the class entities; of the Confea/Crea system in supporting the professional for the continued education, spreading how much the Crea/AM is directed in your mission, and that can measuring an indicator of quality of the degree of satisfaction of the professionals adhering to the Confea/Crea/Mútua system. / Os integrantes do Sistema Confea/Crea/Mútua sempre discutem em seus eventos a importância da Valorização Profissional, que por sua vez para a maioria das pessoas se limita ao enfoque da remuneração profissional: “ganhar mais”. No entanto, quando se é realmente valorizado pelo mercado de trabalho, ganhar mais é uma conseqüência natural. Objetiva-se com este trabalho, apontar os indicadores para uma verdadeira “Valorização Profissional”, de forma a contribuir para que esta questão passe a vigorar de forma efetiva não apenas em documentos e trâmites puramente burocráticos, mas diretamente realizado no cotidiano de cada profissional da área tecnológica. Também faz parte dos objetivos do trabalho analisar as ações adotadas pelo Crea/AM no aspecto qualificação versus valorização. Este trabalho foi realizado por meio de estudo de caso, utilizando pesquisa bibliográfica e de campo, através da definição de uma amostra restrita às empresas incorporadoras que venham a demonstrar de forma mais elucidativa como se dá o processo de valorização profissional dentro do Sistema Confea/Crea/Mutua, visto pela ótica dos próprios usuários do sistema; até então profissionais que integram o quadro técnico das empresas selecionadas. Esse processo se deu através da elaboração, aplicação e tabulação dos dados oriundos de um questionário semi-aberto. Em última instância foi feita a análise dos dados registrados na pesquisa o que possibilitará a continuidade dos estudos em trabalhos futuros. Diante dos resultados, verificou-se que não está havendo a devida divulgação para a comunidade tecnológica das iniciativas do Sistema Confea/Crea dos programas de parcerias e com isso gera uma certa rejeição por parte dos usuários. Por fim, conclui-se que a questão da valorização profissional da área tecnológica fundamentalmente começa com o próprio profissional, se aperfeiçoando em qualificação continuada com a consciência de suas responsabilidades e seu perfil profissional; das instituições de ensino em firmar interação com as entidades de classe; do Sistema Confea/Crea em apoiar o profissional para a educação continuada, divulgar o quanto o Crea/AM se encontra direcionado em sua missão e que o Crea/AM possa mensurar um indicador de qualidade do grau de satisfação dos profissionais afetos ao Sistema Confea/Crea.

Cyril Navrátil FF UK Universita Karlova Románská studia - NMgr. Hispanistika 2013/2014 Abstract The purpose of this essay is to study neoclassical composition. The denomination of this method of word-formation already caused disagreements among the linguists. Many authors name it prefixation (affixation), while others believe that it is a special type of composition, whose components (or at least one of them) take their origins from Latin or Greek. Even though the majority of the Spanish lexicon comes from these languages (mainly from Latin), this type of composition follows some particular rules. In this essay, we do not intend to say who is right and who is wrong, but to demonstrate the current situation of the neoclassical composition in modern Spanish, using the corpus. Nevertheless, it is not possible to completely ignore the different points of view of these authors, who are acclaimed in the academic world. Therefore we decided to divide this study in a theoretic part and a practical one. In the theoretic part, we will recapitulate the terminology used and we will try and choose the most convenient definition for this method. In the practical part, we will see twenty examples of Greco-Latin elements: auto, cida/cidio, cracia/crata (arca/arquía), cripto, crono, eco, euro, filo/filia, foto, narco, neo,...

Carbon catabolite repression (CCR) is a mechanism by which micro-organisms preferentially utilize more easily metabolizable carbon sources in comparison to less easily metabolizable carbon sources. It prevents the organisms from unnecessary expenditure of energy and enables them to exploit the nutrients in appropriate manner. It represents a complex system of gene regulation. The main aim of this study was to study the intracellular localization of proteins involved in CCR including CreA, CreB, CreC and CreD in A. nidulans in repressing and derepressing conditions. The major regulatory protein involved in CCR in A. nidulans is CreA. It is a DNA-binding repressor, but very little is known about the molecular events that allow CreA function to result in appropriate regulation in response to carbon source. To determine the amount and localization of CreA in different carbon sources, strains were made over-expressing GFP and HA tagged CreA. Western analysis showed that high levels of full length CreA can be present in cells that show normal responses to carbon catabolite repression, whether they are grown in repressing or derepressing media. Hence the amount of CreA is similar in both the conditions and thus degradation of CreA is not a key step in carbon catabolite repression. Fluorescence microscopy studies have shown that CreA is in the nucleus under repressing and derepressing carbon conditions and this is not affected by the absence of CreB or CreD, the other important proteins in A. nidulans. Thus mere localization of CreA in nucleus is not sufficient to cause carbon catabolite repression and there is some modification process involved for CreA to act as a repressor protein in CCR. CreB is a deubiquitinating protein and CreC is a protein containing five WD 40 repeats, a putative nuclear localization signal (NLS) and a proline rich region and both the proteins are present in the cell in a complex. CreB was localized using strains that over-expresses GFP tagged CreB and fluorescence microscopy. CreB is present mainly in the cytoplasm in both repressing and derepressing conditions. Moreover, intracellular localization of CreB is unaffected by the presence or absence of CreD. However, the amount of CreB was higher in a creD+ background as compared to a creD34 mutant background, implying that the presence of CreD affects the amount of CreB in the cell. CreC was localized by using strain that over-expresses YFP tagged CreC and it is also present mainly in the cytoplasm. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p from S. cerevisiae. CreD is proposed to be involved in ubiquitination process in CCR in A. nidulans. Localization studies have shown that CreD is present throughout the cell in a punctate pattern with more in the cytoplasm than in the nucleus. CreB and CreD co-localize in some regions of the cell whereas in other regions either CreB or CreD is present. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346526 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Carbon catabolite repression (CCR) is a mechanism by which micro-organisms preferentially utilize more easily metabolizable carbon sources in comparison to less easily metabolizable carbon sources. It prevents the organisms from unnecessary expenditure of energy and enables them to exploit the nutrients in appropriate manner. It represents a complex system of gene regulation. The main aim of this study was to study the intracellular localization of proteins involved in CCR including CreA, CreB, CreC and CreD in A. nidulans in repressing and derepressing conditions. The major regulatory protein involved in CCR in A. nidulans is CreA. It is a DNA-binding repressor, but very little is known about the molecular events that allow CreA function to result in appropriate regulation in response to carbon source. To determine the amount and localization of CreA in different carbon sources, strains were made over-expressing GFP and HA tagged CreA. Western analysis showed that high levels of full length CreA can be present in cells that show normal responses to carbon catabolite repression, whether they are grown in repressing or derepressing media. Hence the amount of CreA is similar in both the conditions and thus degradation of CreA is not a key step in carbon catabolite repression. Fluorescence microscopy studies have shown that CreA is in the nucleus under repressing and derepressing carbon conditions and this is not affected by the absence of CreB or CreD, the other important proteins in A. nidulans. Thus mere localization of CreA in nucleus is not sufficient to cause carbon catabolite repression and there is some modification process involved for CreA to act as a repressor protein in CCR. CreB is a deubiquitinating protein and CreC is a protein containing five WD 40 repeats, a putative nuclear localization signal (NLS) and a proline rich region and both the proteins are present in the cell in a complex. CreB was localized using strains that over-expresses GFP tagged CreB and fluorescence microscopy. CreB is present mainly in the cytoplasm in both repressing and derepressing conditions. Moreover, intracellular localization of CreB is unaffected by the presence or absence of CreD. However, the amount of CreB was higher in a creD+ background as compared to a creD34 mutant background, implying that the presence of CreD affects the amount of CreB in the cell. CreC was localized by using strain that over-expresses YFP tagged CreC and it is also present mainly in the cytoplasm. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p from S. cerevisiae. CreD is proposed to be involved in ubiquitination process in CCR in A. nidulans. Localization studies have shown that CreD is present throughout the cell in a punctate pattern with more in the cytoplasm than in the nucleus. CreB and CreD co-localize in some regions of the cell whereas in other regions either CreB or CreD is present. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346526 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Dissertação (mestrado)—Universidade de Brasília, Faculdade UnB Gama, Programa de Pós-Graduação em Engenharia Biomédica, 2017. / Submitted by Raquel Almeida (raquel.df13@gmail.com) on 2017-11-13T16:47:10Z No. of bitstreams: 1 2017_JoãoGabrielMartinDelSolar.pdf: 5876444 bytes, checksum: ebc352deb85c718277b4fd93bfa4638b (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2017-11-24T20:32:53Z (GMT) No. of bitstreams: 1 2017_JoãoGabrielMartinDelSolar.pdf: 5876444 bytes, checksum: ebc352deb85c718277b4fd93bfa4638b (MD5) / Made available in DSpace on 2017-11-24T20:32:53Z (GMT). No. of bitstreams: 1 2017_JoãoGabrielMartinDelSolar.pdf: 5876444 bytes, checksum: ebc352deb85c718277b4fd93bfa4638b (MD5) Previous issue date: 2017-11-24 / Introdução. A humanidade, com os contínuos e exponencialmente crescentes avanços científicos e tecnológicos, se fortalece no combate às enfermidades e no incremento de sua longevidade e qualidade de vida. Os benefícios evidentes dessas tecnologias, porém, são acompanhados de relevantes riscos à integridade humana. A Anvisa (Agência Nacional de Vigilância Sanitária), visando maximizar esse potencial benéfico e controlar e reduzir os riscos, define requisitos mínimos em relação ao gerenciamento de tecnologias em estabelecimentos de saúde. Requisitos que exigem o exercício de atividades profissionais especializadas. Há, na sociedade brasileira, muitos questionamentos e incompreensões quanto às exigências legais para a gestão dos equipamentos de saúde e a engenharia clínica. Apesar de o Confea (Conselho Federal de Engenharia e Agronomia) afirmar a existência de normas regulamentadoras, sua difusão e aplicação no mercado é pequena. As consequências impactam toda a nação, estabelecimentos de saúde, profissionais de engenharia, instituições reguladoras, e, principalmente, usuários do sistema de saúde, vitimados pela sua reduzida efetividade. Esta dissertação objetiva esclarecer os fundamentos da engenharia clínica brasileira. Alicerçar sua identidade profissional, esclarecer seu enquadramento legal e evidenciar o perfil requerido para atuar como engenheiro clínico no Brasil. Métodos. O trabalho foi desenvolvido em três frentes: i. pesquisa de referências teóricas e normativas referentes à engenharia clínica brasileira; ii. visitas a hospitais e clínicas, e atuação como estagiário de engenharia clínica, a fim de conhecer a sua prática, como implantada e exercida no Distrito Federal do Brasil; iii. participação ativa em redes sociais de profissionais de engenharia clínica, monitorando as questões pungentes e o entendimento coletivo da regulação, avaliando a consistência e propriedade do próprio conhecimento, e difundindo os achados e resultados da pesquisa, como um formador de opinião entre os pares. Resultados. Identificada uma sólida identidade da engenharia clínica, o autor expõe o seu objetivo e missão profissional, suas funções e atividades típicas, e detalha seus alicerces jurídicos, nos matizes de defesa da vida e da integridade humana, de regulação sanitária dos serviços de saúde, e do controle governamental do exercício profissional da engenharia. Conclusão. A dissertação confirma a regulação da engenharia clínica, em seus matizes de defesa da vida e da saúde pública, de controle sanitário e de atuação profissional. Esclarece seus objetivos, responsabilidades, requisitos, num chamado ao seu ético exercício.{Introdução. } A humanidade, com os contínuos e exponencialmente crescentes avanços científicos e tecnológicos, se fortalece no combate às enfermidades e no incremento de sua longevidade e qualidade de vida. Os benefícios evidentes dessas tecnologias, porém, são acompanhados de relevantes riscos à integridade humana. / Introduction. Humanity, with the continuous and exponentially increasing scientific and technological advances, is strengthened in the fight against diseases and in the rise of its longevity and quality of life. The obvious benefits of these technologies, however, are accompanied by significant risks to human integrity. The Anvisa (National Health Surveillance Agency), in order to maximize this beneficial potential and control and reduce risks, defines minimum requirements regarding the management of technologies in health facilities. Requirements that demand the exercise of specialized professional activities. There are, in Brazilian society, many questions and misunderstandings regarding the legal requisites to exercise health equipment management and clinical engineering. Although Confea (Federal Council of Engineering and Agronomy) confirm the existence of regulatory rules, they lack diffusion and application in the market. The consequences impact entire nation, health facilities, engineering professionals, regulatory agencies, and, especially, users of the health service, victimized by their reduced effectiveness. This dissertation aims to clarify the fundamentals of Brazilian clinical engineering. To underpin its professional identity, to clarify their legal framework and to evidence the required profile to act as clinical engineer in Brazil Methods. The author developed this work in three fronts: i. research of theoretical and normative references related to the Brazilian clinical engineering; ii. visits to hospitals and clinics and actuation as a clinical engineering trainee in order to know its practice, as it is implemented in the Brazilian Federal District; iii. active participation in social networks of clinical engineering professionals, monitoring the poignant issues and the collective understanding of the sector regulations, evaluating the consistency and appropriateness of the proper knowledge and spreading the findings and results of the research, as an opinion leader among peers. Results. Identified a solid identity of the clinical engineering, the author exposes its professional objective and mission, its typical functions and activities and details its legal foundations, in the nuances of the defense of life and of the human integrity, of the regulation of the sanitary surveillance of health services and of the state control of engineering profissional actuation. Conclusion. The dissertation confirms clinical engineering regulation, in its nuances of defense of life and public health, sanitary control and professional actuation. Clarifies its goals, responsibilities, requisites, in a call to its ethical exercise. / Introducción. La humanidad, con los continuos y exponencialmente crecientes avances científicos y tecnológicos, se fortalece en el combate a las enfermedades y en el incremento de su longevidad y calidad de vida. Los beneficios evidentes de estas tecnologías, sin embargo, són acompañados de relevante riesgos para la integridad humana. La Anvisa (Agencia Nacional de Vigilancia Sanitaria), con el objetivo de maximizar ese potencial benéfico y controlar y reducir los riesgos, define requisitos mínimos en relación a la gestión de tecnologías en establecimientos de salud. Requisitos que exigen el ejercicio de actividades profesionales especializadas. Hay, en la sociedad brasileña, muchos cuestionamientos e incomprensiones cuanto a los requisitos legales para ejercer la gestión de los equipos de salud y la ingeniería clínica. A pesar del Confea (Consejo Federal de Ingeniería y Agronomía) afirmar la existencia de normas reguladoras, su difusión y aplicación en el mercado es pequeña. Las consecuencias afectan a toda la nación, establecimientos de salud, profesionales de ingeniería, instituciones reguladoras, y, principalmente, usuarios del sistema de salud, victimizados por su reducida efectividad. Esta disertación objetiva aclarar los fundamentos de la ingeniería clínica brasileña. Basar su identidad profesional, aclarar su marco legal y evidenciar el perfil requerido para actuar como ingeniero clínico en Brasil Métodos. El trabajo se desarrolló en tres frentes: i. investigación de referencias teóricas y normativas referentes a la ingeniería clínica brasileña; ii. visitas a hospitales y clínicas, y actuación como aprendiz de ingeniería clínica, a fin de conocer su práctica, como implantada y ejercida en el Distrito Federal de Brasil; iii. participación activa en redes sociales de profesionales de ingeniería clínica, monitoreando las cuestiones punzantes y el entendimiento colectivo de la regulación, evaluando la consistencia y propiedad del propio conocimiento, y difundiendo los hallazgos y resultados de la investigación, como un formador de opinión entre los pares. Resultados. Identificada una sólida identidad de la ingeniería clínica brasileña, el autor expone su objetivo y misión profesional, sus funciones y actividades típicas, y detalla sus bases jurídicas, en los matices de la defensa de la vida y de la integridad humana, de la regulación sanitaria de los servicios de salud y del control estatal del ejercicio profesional de la ingeniería. Conclusión. La disertación confirma la regulación de la ingeniería clínica, en sus matices de defensa de la vida y de la salud pública, de control sanitario y de actuación profesional. Esclarece sus objetivos, responsabilidades, requisitos, en un llamado a su ético ejercicio.

Esta investigación tiene como objetivo evaluar si los adolescentes que participaron del Programa Crea + en el ciclo académico 2018 -1 presentaron cambios en sus habilidades socio personales relacionadas a la autoestima. Se utilizó la Escala de Autoestima de Rosenberg (1989), validada para la población adolescente peruana. Se contó con una muestra de 17 adolescentes, con edades entre los 11 y 18 años de un colegio del distrito de Ventanilla. Los resultados obtenidos afirman que no existe una diferencia de los niveles de autoestima de los participantes antes y después de su participación en el programa. Se concluyó que los puntajes del pre test y post test del grupo experimental son similares. / The objective of this research is to evaluate whether the adolescents who participated in the Crea + Program in the 2018 -1 academic year presented changes in their socio-personal skills related to self-esteem. The Rosenberg Self-Esteem Scale (1989), validated for the Peruvian adolescent population, was used. There was a sample of 17 adolescents, aged between 11 and 18 years from a school in the Ventanilla district. The results obtained affirm that there is no difference in the levels of self-esteem of the participants before and after their participation in the program. It was concluded that the pre-test and post-test scores of the experimental group are similar. / Tesis

O mundo se depara atualmente com a perspectiva de um significativo aumento na demanda por etanol combustível. O bagaço de cana está entre os maiores subprodutos agroindustriais no Brasil, sendo uma das alternativas na utilização para a produção do etanol de segunda geração. A degradação do bagaço de cana requer a ação de muitas enzimas diferentes que são reguladas transcripcionalmente. Considerando-se que o custo de celulases e hemicelulases contribuem substancialmente no preço do bioetanol, novos estudos visando o entendimento da eficiência e produtividade de celulases são de grande importância. Para entender como melhorar coquetéis de enzimas que podem hidrolizar o bagaço de cana-de-açúcar pré-tratado, uitlizou-se um experimento de genômica para investigar-se quais genes e vias são transcripcionalmente moduladas durante o crescimento de A. niger em bagaço de cana-de-açúcar explodido. Neste trabalho foram identificados genes que codificam celulases e hemicelulases com aumento da expresão durante o crescimento em bagaço de cana-de-açúcar explodido. Foi também realizada a determinação do acúmulo de mRNA de diversos genes que codificam transportadores para verificar se estes eram induzidos por xilose e por depedência de glicose. Foram identificados 18 genes que corresponde a 58% de celulases preditas em A. niger e 21 genes que correponde a 58% de hemicelulases preditas em A. niger os quias foram altamente expressos durante o crescimento em bagaço de cana-de-açúcar explodido. Foi investigado também o papel central realizado pelas proteínas quinases e fosfatases não essenciais (NPKs e NPPs, respectivamente) quando em presença de celulose como fonte de carbono, no sensoriamento do estado energético e na subsequente via de sinalização no fungo filamentoso modelo Aspergillus nidulans. O estudo com A. nidulans identificou 11 quinases e 7 fosfatases não essências, NPKs e NPPs, respectivamente, envolvidas na produção de celulases e em alguns casos, na produção também de hemicelulases. O envolvimento destas NPKs identificadas na resposta induzida por avicel e na desrepressão foram acessados pela análise do transcriptoma da cepa selvagem e por microscopia de fluorescência através da cepa de fusão CreA::GFP expressa no selvagem e no background dos mutantes de NPKs. A ausência das quinases snfA e schA reduziu dramaticamente a resposta transcricional induzida por celulose incluindo a expressão de enzimas hidrolíticas e transportadores, enquanto que a ausência de snfA resultou em uma quase completa modulação gênica induzida por celulose. O mecanismo pelo qual essas duas quinases controlam a transcrição gênica foi identificado, onde os dois mutantes de quinases foram capazes de desbloquear o CreA mediante a repressão catabólica do carbono (CCR), sob condições de desrepressão, como em baixa presença de carbono ou crescimento em celulose. Desta forma, este trabalho abriu novas possibilidades para o entendimento da sacarificação do bagaço de cana-de-açúcar por hidrolases de A. niger e para a construção de coquetéis de enzimas mais eficientes para a obtenção do etanol de segunda geração. Também possibilitou a identificação de muitas quinases e fosfatases envolvidas no sensoriamento do carbono e do estado energético, as quais demonstraram papéis sobrespostos e distintos de snfA e schA na regulação da desrepressão de CreA e na produção de enzimas hidrolíticas em A. nidulans. / The world today is faced with the prospect of a significant increase in demand for fuel ethanol. Sugarcane bagasse is among the largest agro-industrial by-products in Brazil, one of the alternatives in use for the production of second generation ethanol. Degradation of sugarcane bagasse requires the action of many different enzymes which are transcriptionally regulated. Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse. We also sought to determine whether the mRNA accumulation of several steam-exploded sugarcane bagasseinduced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 genes that corresponds to 58% of A. niger predicted cellulases and 21 genes that correspond to 58% of A. niger predicted hemicellulases, that were highly expressed during growth on sugarcane bagasse. The central role performed by nonessential protein kinases (NPK) and phosphatases (NPP) when grown on cellulose as a sole carbon source, in the sensing energetic status and the subsequent signalling pathways was assessed in the model filamentous fungus Aspergillus nidulans. This study identified multiple kinases and phosphatases (NPKs and NPPs, respectively) involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans. The involvement of the identified NPKs in avicel-induced responses and CreA derepression was assessed by genome-wide transcriptomics and fluorescent microscopy of a CreA::GFP fusion proteinexpressed in the wild-type and NPK-deficient mutant backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses including the expression of hydrolytic enzymes and transporters, while the absence snfA resulted in a near complete loss of wild-typecellulose-induced gene modulation. The mechanism by which these two NPKs controlled gene transcription was identified, as neither of NPK-deficient mutants were able to unlock CreA-mediated carbon catabolite repression, under derepressing conditions, such as carbon starvation or growth on cellulose. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol. This work also enable the identification of multiple kinases and phosphatases involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans.

O mundo se depara atualmente com a perspectiva de um significativo aumento na demanda por etanol combustível. O bagaço de cana está entre os maiores subprodutos agroindustriais no Brasil, sendo uma das alternativas na utilização para a produção do etanol de segunda geração. A degradação do bagaço de cana requer a ação de muitas enzimas diferentes que são reguladas transcripcionalmente. Considerando-se que o custo de celulases e hemicelulases contribuem substancialmente no preço do bioetanol, novos estudos visando o entendimento da eficiência e produtividade de celulases são de grande importância. Para entender como melhorar coquetéis de enzimas que podem hidrolizar o bagaço de cana-de-açúcar pré-tratado, uitlizou-se um experimento de genômica para investigar-se quais genes e vias são transcripcionalmente moduladas durante o crescimento de A. niger em bagaço de cana-de-açúcar explodido. Neste trabalho foram identificados genes que codificam celulases e hemicelulases com aumento da expresão durante o crescimento em bagaço de cana-de-açúcar explodido. Foi também realizada a determinação do acúmulo de mRNA de diversos genes que codificam transportadores para verificar se estes eram induzidos por xilose e por depedência de glicose. Foram identificados 18 genes que corresponde a 58% de celulases preditas em A. niger e 21 genes que correponde a 58% de hemicelulases preditas em A. niger os quias foram altamente expressos durante o crescimento em bagaço de cana-de-açúcar explodido. Foi investigado também o papel central realizado pelas proteínas quinases e fosfatases não essenciais (NPKs e NPPs, respectivamente) quando em presença de celulose como fonte de carbono, no sensoriamento do estado energético e na subsequente via de sinalização no fungo filamentoso modelo Aspergillus nidulans. O estudo com A. nidulans identificou 11 quinases e 7 fosfatases não essências, NPKs e NPPs, respectivamente, envolvidas na produção de celulases e em alguns casos, na produção também de hemicelulases. O envolvimento destas NPKs identificadas na resposta induzida por avicel e na desrepressão foram acessados pela análise do transcriptoma da cepa selvagem e por microscopia de fluorescência através da cepa de fusão CreA::GFP expressa no selvagem e no background dos mutantes de NPKs. A ausência das quinases snfA e schA reduziu dramaticamente a resposta transcricional induzida por celulose incluindo a expressão de enzimas hidrolíticas e transportadores, enquanto que a ausência de snfA resultou em uma quase completa modulação gênica induzida por celulose. O mecanismo pelo qual essas duas quinases controlam a transcrição gênica foi identificado, onde os dois mutantes de quinases foram capazes de desbloquear o CreA mediante a repressão catabólica do carbono (CCR), sob condições de desrepressão, como em baixa presença de carbono ou crescimento em celulose. Desta forma, este trabalho abriu novas possibilidades para o entendimento da sacarificação do bagaço de cana-de-açúcar por hidrolases de A. niger e para a construção de coquetéis de enzimas mais eficientes para a obtenção do etanol de segunda geração. Também possibilitou a identificação de muitas quinases e fosfatases envolvidas no sensoriamento do carbono e do estado energético, as quais demonstraram papéis sobrespostos e distintos de snfA e schA na regulação da desrepressão de CreA e na produção de enzimas hidrolíticas em A. nidulans. / The world today is faced with the prospect of a significant increase in demand for fuel ethanol. Sugarcane bagasse is among the largest agro-industrial by-products in Brazil, one of the alternatives in use for the production of second generation ethanol. Degradation of sugarcane bagasse requires the action of many different enzymes which are transcriptionally regulated. Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse. We also sought to determine whether the mRNA accumulation of several steam-exploded sugarcane bagasseinduced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 genes that corresponds to 58% of A. niger predicted cellulases and 21 genes that correspond to 58% of A. niger predicted hemicellulases, that were highly expressed during growth on sugarcane bagasse. The central role performed by nonessential protein kinases (NPK) and phosphatases (NPP) when grown on cellulose as a sole carbon source, in the sensing energetic status and the subsequent signalling pathways was assessed in the model filamentous fungus Aspergillus nidulans. This study identified multiple kinases and phosphatases (NPKs and NPPs, respectively) involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans. The involvement of the identified NPKs in avicel-induced responses and CreA derepression was assessed by genome-wide transcriptomics and fluorescent microscopy of a CreA::GFP fusion proteinexpressed in the wild-type and NPK-deficient mutant backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses including the expression of hydrolytic enzymes and transporters, while the absence snfA resulted in a near complete loss of wild-typecellulose-induced gene modulation. The mechanism by which these two NPKs controlled gene transcription was identified, as neither of NPK-deficient mutants were able to unlock CreA-mediated carbon catabolite repression, under derepressing conditions, such as carbon starvation or growth on cellulose. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol. This work also enable the identification of multiple kinases and phosphatases involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans.

Carbon catabolite repression in A. nidulans is a regulatory system which allows the organism to utilize the most preferable carbon source by repressing the expression of genes encoding enzymes utilizing alternative carbon sources. A ubiquitination pathway was shown to be one of the key mechanisms which regulate carbon source utilization, when creB was found to encode a deubiquitinating enzyme. Strains containing mutations in creB show loss of repression for some metabolic pathways in carbon catabolite repressing conditions, and also grow very poorly on several sole carbon sources such as quinate and proline, suggesting CreB plays multiple roles in the cell. This work describes the analysis of the interaction of CreB with CreA, and with PrnB and QutD. Various epitope-tagged versions of CreA were expressed in A. nidulans, and an internally located HA-epitope tag was found to allow detection of CreA using Western analysis. A diploid strain was constructed between strains containing HA-tagged CreA and FLAG-tagged CreB. When CreB was immunoprecipitated, HA-tagged CreA was also precipitated in the diploid, indicating that CreA and CreB are present in a complex in vivo. To determine whether CreA is a ubiquitinated protein, a version of CreA that was tagged with both an HA epitope and a His-tag was expressed in A. nidulans, and protein extracts were precipitated with an UbiQapture™-Q matrix. Western analysis was used to show that CreA was present in the precipitate. These findings suggest that CreA is a ubiquitinated protein, and a target of the CreB deubiquitination enzyme. To determine whether the proline permease (PrnB) is a direct substrate of CreB, plasmids to express epitope-tagged versions of PrnB were constructed and introduced into the prnB mutant strain. No tagged protein could be detected by Western analysis, even when these constructs were over-expressed from the gpdA promoter. However, a construct to express an HA epitope tagged version of quinate permease (QutD) fully complemented the qutD mutant strain, and HA-tagged QutD could be easily detected in Western analysis when probed with the anti-HA monoclonal antibody. A diploid strain was made between a complementing transformant and a strain expressing a FLAG-tagged CreB construct. When QutDHA was immunoprecipitated, CreBFLAG was detected in the immunoprecipitate of the diploid. A proportion of QutDHA was also co-precipitated in the diploid when CreBFLAG was immunoprecipitated. Thus, CreB is present in a complex with QutD in vivo. Further results showed that the concentration of QutD in the cell is lower in a creB null mutant background than in the wild-type background, indicating that deubiquitination is required to prevent protein turnover. Northern analysis of mRNA showed that the failure of creB mutant strains to grow on quinate medium was not due to a failure of transcriptional induction of qutD, as the amount of mRNA was not lower in a creB1937 mutant background compared to the wild-type. Furthermore, experiments were undertaken that showed that QutD is a ubiquitinated protein. These findings suggest that quinate permease is regulated through deubiquitination involving the CreB deubiquitination protein in A. nidulans. In addition to the candidate protein approach asking whether CreA is a substrate of CreB, a proteomics approach was also used to identify proteins that interact with CreA. However, no clear interacting proteins were identified using this approach. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008