Synthetic biology is a nascent discipline that aims to design and construct new biological systems beyond those found in nature, ultimately using them to probe, control, or even replace existing biological systems. The success of synthetic biology depends on the assembly of a set of well-defined and modular tools. These tools should ideally be mutually compatible, reusable in different contexts, and have minimum crosstalk with endogenous proteins of the subject. The tobacco etch virus protease (TEV protease, TEVp) is a suitable candidate for such a tool due to its unique substrate specificity and high efficiency. Importantly, TEVp is capable of imitating proteolysis, a ubiquitous mechanism in nature for post-translational modifications and signal propagation. Here, TEVp is employed as a self-contained proteolytic device capable of executing biological tasks that are otherwise governed by endogenous proteins and processes. Consequently, the goal of using TEVp for synthetic manipulation of biosystems is achieved. First, a single-vector multiple gene expression strategy utilizing TEVp self-cleavage was created. This approach was used for the robust expression of up to three genes in both bacterial and mammalian cells with consistent stoichiometry. The products can then be individually purified or targeted to distinct subcellular compartments respectively. Second, a temperature-inducible TEVp was created by incremental truncation of TEVp. The 18th truncation of TEVp (tsTEVp) resulted in negligible activity at 37 °C, but retained sufficient activity at 30 °C for rapid processing of its substrates in several mammalian cell cultures. Finally, tsTEVp was applied in the context of other synthetic modules to generate a variety of biological responses. Its versatility was demonstrated as cellular processes including protein localization, cellular blebbing, protein degradation, and cell death were rewired to respond to the physical stimulus of temperature.

Synthetic biology is a nascent discipline that aims to design and construct new biological systems beyond those found in nature, ultimately using them to probe, control, or even replace existing biological systems. The success of synthetic biology depends on the assembly of a set of well-defined and modular tools. These tools should ideally be mutually compatible, reusable in different contexts, and have minimum crosstalk with endogenous proteins of the subject. The tobacco etch virus protease (TEV protease, TEVp) is a suitable candidate for such a tool due to its unique substrate specificity and high efficiency. Importantly, TEVp is capable of imitating proteolysis, a ubiquitous mechanism in nature for post-translational modifications and signal propagation. Here, TEVp is employed as a self-contained proteolytic device capable of executing biological tasks that are otherwise governed by endogenous proteins and processes. Consequently, the goal of using TEVp for synthetic manipulation of biosystems is achieved. First, a single-vector multiple gene expression strategy utilizing TEVp self-cleavage was created. This approach was used for the robust expression of up to three genes in both bacterial and mammalian cells with consistent stoichiometry. The products can then be individually purified or targeted to distinct subcellular compartments respectively. Second, a temperature-inducible TEVp was created by incremental truncation of TEVp. The 18th truncation of TEVp (tsTEVp) resulted in negligible activity at 37 °C, but retained sufficient activity at 30 °C for rapid processing of its substrates in several mammalian cell cultures. Finally, tsTEVp was applied in the context of other synthetic modules to generate a variety of biological responses. Its versatility was demonstrated as cellular processes including protein localization, cellular blebbing, protein degradation, and cell death were rewired to respond to the physical stimulus of temperature.

Caspase-7 is a member of a family of cysteine proteases that includes apoptotic initiators (caspases-8, -9 and -10) and executors (caspases-3, -6 and -7). During apoptosis, executioner caspases are cleaved by initiator caspases either by the extrinsic (death receptors) or by the intrinsic (mitochondrial) pathway of caspase activation. Caspase-7 is an obligate dimer in the cell and cleavage of the interdomain connector (IDC), which split the catalytic domain in two subunits, at either site 1 or site 2 allows the conversion of the enzyme from the zymogen (inactive) state to the active state through a conformation switch that leads to the creation of a substrate binding pocket and the catalytic site. During caspase-7 activation, a 23-residue N-terminal peptide is also cleaved. Consequently, caspase-7 displays different N-terminal residues from those of its zymogen. This can change the stability of caspase-7 according to the N-end rule, which relates the half-life of a protein with the residue presented at its N-terminus. This degradation pathway controls the ubiquitination of the protein based on the N-terminus. To replicate the different forms of caspase-7 produced during its activation process in a controlled manner, a TEV protease cleavage site [ENLYFQ[arrow down](S/A)] was strategically inserted to mimic the different possibilities of IDC cleavage: 1) cleavage at site 1 only, 2) cleavage at site 2 only, or 3) a double cleavage. This was done in order to obtain the N-terminal residues normally presented during the cleavage of caspase-7. These constructions have been also optimized to preserve the proteolytic activity of the enzyme with as little change as possible to the length of the IDC. These constructs were cleaved by TEV protease in vitro and in cellulo and allowed the activation of apoptosis. Furthermore, the cellular half-life of caspase-7 seems to be changed by its cleavage. In conclusion, we have developed an interesting tool for the study of caspase-7.

<p>A method describing the expression and purification of PSI-N together with fusion protein, using affinity chromatography and TEV protease. Although the method proved successful, optimization is still needed due to partial degradation of PSI-N.</p>

A method describing the expression and purification of PSI-N together with fusion protein, using affinity chromatography and TEV protease. Although the method proved successful, optimization is still needed due to partial degradation of PSI-N.

Orientador: Winston Bonetti Yoshida / Resumo: Introdução: O tromboembolismo venoso (TEV) e uma doença silenciosa e letal que acomete parcela importante dos pacientes hospitalizados. Com alta morbimortalidade e custo financeiro para o sistema de saúde o TEV pode ser prevenido com uso da profilaxia estabelecida pela literatura. No mundo real a profilaxia para TEV possui media de adequação inferior a 50%. Objetivo: Definir o perfil epidemiológico do doente com TEV no HC/FMB, a taxa de adequação da profilaxia para TEV no referido serviço e determinar meios para melhora-la. Material e método: Estudo transversal observacional realizado pela coleta de dados no prontuário medico dos pacientes que preencheram critérios de inclusão. Confrontado classificação de risco para TEV, segundo a SBACV, e a profilaxia para TEV prescrita. RESULTADOS: A taxa global de adequação das prescrições de profilaxia encontrada foi de 42.1% contra 57.9% de inadequação. Pacientes clínicos obtiveram taxa de adequação de 52.9% enquanto pacientes cirúrgicos obtiveram taxa de 37.5% de profilaxia para TEV realizada de maneira adequada. Discussão:As taxas encontradas são ligeiramente inferiores as relatadas na literatura. As inadequações de prescrição podem ser explicadas pelo fato de o medico assistente não lembrar da ocorrência da doença, por estratificar o risco do paciente de maneira inadequada ou por acreditar em maior potencial de sangramento em pacientes cirúrgicos que recebam a profilaxia química. Conclusão: Educação continuada, o estimulo a aplicação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Venous thromboembolism (VTE) is a silence and lethal disease most prevalent in hospitalized patients, making the most common preventable cause of inhospital death. With high morbidity, mortality and increase the cost of management VTE can be prevent use the appropriate prophylaxis for at-risk patients; this is a strong recommendation in guidelines and VTE scores. Despite the existence of this recommendation the real and appropriate use of prophylaxis is estimated fewer than 50% in our country. Methods: Observational cross-sectional epidemiologic evaluation comparing patients atrisk for VTE and the prophylaxis used. The Brazilian Society for Vascular Surgery Score for VTE was used to classify the prophylaxis. Findings: 42.1% at-risk patients (Include illness and surgery patients) received the appropriate prophylaxis against 57.9% who received inappropriate prophylaxis. Illness patients received appropriate prophylaxis in 52.9% while surgery patients received appropriate prophylaxis in 37.5%. Conclusion: The appropriate prophylaxis for VTE remains underused. Illness patients received appropriate prophylaxis more then surgery patients. More information, continued education for doctors and the correct use of VTE score can change the reality of VTE prevents. / Mestre

Les besoins en protéines recombinantes dans les diverses activités des bio-industries a considérablement augmenté ces dernières années. Cependant, les procédés de leur production sont encore limités par le manque de marqueurs permettant de suivre l'expression et la purification des protéines d'intérêt, la formation des corps d'inclusion et les faibles degrés de pureté.Afin de pallier à ces difficultés, nous avons développé et mis en œuvre un nouveau procédé de production et de purification de protéines recombinantes chez Escherichia coli. Ce procédé est basé sur l'utilisation d'une cassette d'expression appelée Multitags et sur le clivage par une TEV protéase immobilisée sur une matrice de streptavidine. Le Multitags comporte, à partir de son extrémité N-terminale, un double tag d'affinité (10xHis et SBP), le domaine de fixation de l'hème du cytochrome b5 et le site de clivage de la TEV protéase. En utilisant deux modèles différents de protéine d'intérêt (MyRIP et la Pfu DNA polymérase), nous avons montré l'efficacité du cytochrome b5 dans le suivi visuel et quantitatif par mesure d'absorbance des différentes étapes de production et de purification. Nous avons obtenu plus de 90% de chacune des deux protéines de fusion dans la phase soluble. L'application d'une chromatographie double via les deux tags d'affinité 10xHis et SBP a permis d'atteindre un degré de pureté du Multitags-MyRIP et du Multitag-Pfu de 99%. Nous avons construit des colonnes protéolytiques en produisant la TEV protéase sauvage et sa version mutée (S219V) en fusion avec le Streptag II et en immobilisant ces enzymes par affinité sur colonne de streptavidine-agarose. La caractérisation des colonnes protéolytiques et leur application aux protéines recombinantes d'intérêt modèles ont montré l'avantage de cette méthode d'immobilisation en termes d'activité protéase retenue, de stabilité des enzymes, de leur réutilisation et de simplification du schéma de purification et de récupération des protéines d'intérêt à haut degré de pureté. En conclusion, ces travaux de thèse ont permis de développer et de valider des outils innovants pour l'expression et la purification de protéines recombinantes. / In recent years, the need for recombinant proteins has substantially increased in various bio-industry activities. However, actual recombinant processes are still limited by the lack of markers allowing real-time expression and purification monitoring of target proteins, by inclusion bodies formation and by low quality of purity of the products. To overcome these difficulties, we have developed a new process for production and purification of recombinant proteins in Escherichia coli. The method combines the use of a multifunctional expression cassette, termed Multitags and an immobilized modified TEV protease on a streptavidin matrix. The Multitags contains, its N-terminus, two affinity purification tags (10xHis and SBP) and as a marker tag, the heme-binding domain of cytochrome b5 followed by the TEV cleavage site. Using two model proteins (MyRIP and Pfu DNA polymerase), we have demonstrated the visual and the quantitative monitoring capability of the cytochrome b5 during the expression and purification steps. When expressed in E. coli KRX more than 90% of both fusion proteins were produced in a soluble form. In addition, high purity (99%) of Multitags-MyRIP and Multitags-Pfu was achieved after two consecutive affinity purification steps using the dual affinity tag. We also produced the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence and realized their affinity immobilization on a streptavidin-agarose matrix. The characterization of the proteolytic columns and their application to the recombinant model proteins demonstrated the advantage of this immobilization method in terms of retaining activities, enzyme stabilities, possibility of reuse and simplification of the cleavage downstream steps.In conclusion, this study allowed the development and the validation of innovative tools for expression and purification of recombinant proteins.

Diese Dissertation beschreibt die Messung des Wirkungquerschnitts für die Produktion von ttbb in Protonkollisionen mit einer Schwerpunktsenergie von √s = 8 TeV. Der verwendete Datensatz entspricht einer integrierten Luminosität von 20.3/fb. Der Wirkungsquerschnitt wurde aus der Anzahl der Signalereignisse bestimmt, die durch harte Schnitte insbesondere auf genau 4 identifizierten b-jets, selektiert wurden, was zu einer hohen Reinheit des Signals führt. Bei der Messung wurden die präzisesten Kalibrierungen von der Jet-Energieskala und der b-jet Effizienz benutzt. Die Messung wurde in einem Referenzphasenraum durchgeführt, der daraufhin optimiert wurde, die Abhängigkeit von der Modellierung zu minimisieren. Der gemessene Wirkungsquerschnitt beträgt 18.9 ± 3.5 (stat)+5.6 (sys) ± 0.6 (Lumi) fb oder, nachdem der Beitrag von ttH(bb) und ttZ(bb) abgezogen wurde: 17.8 ± 3.5 (stat)+5.9 (sys) ± 0.6 (Lumi) fb. Das Ergebnis wurde mit einer Vielzahl von theoretischen Vorhersagen verglichen, einschließlich NLO-Berechnungen mit Partonschauern und einer Reihe von Modellen die sich in der Beschreibung der Gluon Spaltung unterscheiden. Es wurde gezeigt, dass das exstremste Model den Wirkungsquerschnitt überschätzt und dass die Messung die Vorhersagen bevorzugt, die mit einer niedrigen Renormierungs- und Faktorisierungsskala, berechnet wurden. / This thesis presents the measurement of the ttbb production cross-section, using a dataset of 20.3/fb of pp collisions collected with the ATLAS detector at 8 TeV. The measurement is based on a cut-and-count method, using a sample of events with exactly four b-tagged jets, which is shown to have a high purity in signal events. The measurement exploits the most precise jet energy scale and b-tagging calibrations and is performed in a fiducial phase space that is designed to minimize the model dependence of the measurement. The fiducial cross-section is measured to be 18.9 ± 3.5 (stat)+5.6 (sys) ± 0.6 (Lumi) fb or subtracting the contribution from ttH(bb) and ttZ(bb) final states, 17.8 ± 3.5 (stat)+5.9 (sys) ± 0.6 (Lumi) fb. The result is compared with a multitude of theoretical predictions, including different NLO calculations matched to a parton shower, which constitute the most precise predictions available to date, as well as with a series of models that differ in the description of the gluon splitting to b-quarks. It is shown that the most extreme gluon splitting model overestimates the observed rate of ttbb production and that the measurement favors calculations performed with renormalization/factorization scales which are softer than the scales usually employed in similar calculations.

Galaktische und extra-galaktische Objekte sind in der Lage geladene Teilchen (die kosmische Strahlung) zu sehr hohen Energien zu beschleunigen. Allerdings sind noch viele Fragen bezüglich dieser Objekte und der Beschleunigungsmechanismen offen. Sowohl Gammastrahlung als auch Neutrinos werden von den Quellen kosmischer Strahlung erwartet. Ihr Nachweis ermöglicht die Studie dieser kosmischen Teilchenbeschleuniger. Gammastrahlung wurde von galaktischen und extra-galaktischen Objekten beobachtet. Für viele dieser Objekte ist es jedoch nicht eindeutig ob diese Gammastrahlung ein Resultat der Beschleunigung kosmischer Strahlen ist. Für Neutrinos besteht diese Zweideutigkeit nicht, sie sind eindeutige Spuren der Beschleunigung kosmischer Strahlen. Der Südhimmel beheimatet viele galaktische Objekte von denen Gammastrahlung im GeV und TeV Bereich beobachtet wird. Die Detektion von Neutrinos wäre ein Beweis für die Beschleunigung kosmischer Strahlung. Der Nachweis dieser Neutrinos mit IceCube wird durch den großen Untergrund von atmosphärischen Myonen erschwert. Die hier verwendete Analyse ist auf die Selektion von Myonspuren aus Wechselwirkungen von Myonneutrinos im Detektorvolumen spezialisiert. Energieverlust und Richtung der resultierenden Myonspur wird rekonstruiert. Diese Informationen werden verwendet um nach potentiellen Quellen astrophysikalischer Neutrino im Rahmen einer ungebinnten Likelihoodanalyse zu suchen. Daten die zwischen 2011 und 2015 mit IceCube genommen wurden werden für diese Analyse verwendet. Der Fokus liegt auf Neutrinos mit Energien zwischen ein paar TeV und 100 TeV. In diesem Energiebereich wird die Sensitivität für die Detektion einer Neutrinopunktquelle um einen Faktor zwei (oder besser) verbessert. Die Resultate für eine Liste von 96 Quellkandidaten und für eine offene Suche am gesamten Südhimmel werden präsentiert. Es wurde keine signifikante Abweichung von der Untergrundhypothese gefunden. Daraus resultieren Limitationen für Neutrinoemissionen. / There are accelerators in the universe that can accelerate charged particles (cosmic rays) to very high energies. Many questions regarding these accelerators are still open. Gamma rays and neutrinos are particles expected from sites of cosmic ray acceleration and can be used to study the environment and acceleration mechanisms of these sites. While sources for both galactic and extra-galactic gamma rays have been observed, it is often unclear whether these gamma rays are by-products of cosmic ray acceleration. This ambiguity does not exist for neutrinos. An astrophysical neutrino flux has been measured by the IceCube detector. Single sources have not been resolved yet. The part of the sky visible from the southern hemisphere hosts many galactic sources observed in GeV and TeV gamma-rays. Detection of neutrinos from these sources would identify them as acceleration sites and lead to a better understanding of the environment of the acceleration sites and the acceleration mechanisms. However, this is difficult due to the vast background of atmospheric muons also detected in the IceCube detector. For this thesis, a data selection was developed that reduces this background by using parts of the detector as veto. This selection focuses on the selection of muon-tracks from muon-neutrino interactions inside the detector volume. The direction and the energy-profile of these tracks can be reconstructed. This information is used to search for potential sources using an unbinned likelihood method. This analysis uses data taken between 2011 and 2015. In contrast to earlier IceCube analyses this analysis is optimized for energies between a few TeV and 100 TeV and improves the sensitivity of the detector for a point-like source by factor of two (or better) in this energy range. Results for a list of 96 sources observed in TeV gamma-rays and a sky-scan are presented. No significant overfluctuation has been observed and limits on the neutrino emission of the sources are given.

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