Influenza A is involved in hundreds of thousands of deaths globally every year resulting from viral infection-related complications. Previous efforts to subdue the virus by preventing proper function of wild-type (WT) neuraminidase (N), and M2 proteins using oseltamivir and amantadine (AMT) or rimantadine (RMT), respectively, exhibited success initially. Over time, these drugs began exhibiting mixed success as the virus developed drug resistance. M2 is a proton channel responsible for the acidification of the viral interior which facilitates release of the viral RNA into the host. M2 has a His37-tetrad that is the selective filter for protons. This protein has been demonstrated to be a feasible target for organic compounds. However, due to a mutation from serine to asparagine at residue 31 of M2, which is found in the majority of influenza strains circulating in humans, AMT and RMT block is insufficient. From simulations, it is unclear whether the insensitivity results from weak binding or incomplete block. The question of how the S31N mutation caused MT and RMT insensitivity in M2 is addressed here by analyzing the binding kinetics of AMT and RMT using the two-electrode voltage clamp electrophysiology method. The dissociation rate constant (k2) is dramatically increased compared to WT for both AMT and RMT, by 1500-fold and 17000-fold respectively. Testing of AMT at 10 mM demonstrates complete block, albeit weak, of the S31N M2 channel. At 10 mM, RMT does not reach complete block even though the binding site is saturated. When RMT is in the bound state, it is not blocking all the current, and is binding without block. These results motivated the development of novel M2 blockers using copper complexes focusing on the His37 complex in M2. I hypothesized that copper complexes would bind with the imidazole of a histidine in the His37 complex and prevent proton conductance. The His37 complex is highly conserved in the M2 channel and, therefore, would be important target for influenza therapeutics. By derivatizing the amines of known M2 blockers, AMT and cyclooctyalmine, to form the iminodiacetate or iminodiacetamide, we have synthesized Cu(II) containing complexes and characterized them by NMR, IR, MS, UV–vis, and inductively coupled plasma mass spectroscopy (ICP-MS). The copper complexes, but not the copper-free ligands, demonstrated H37-specific blocking of M2 channel currents and low micromolar anti-viral efficacies in both Amt-sensitive and Amt-resistant IAV strains with, for the best case, nearly 10-fold less cytotoxicity than CuCl2. Isothermal titration calorimetry was used to obtain enthalpies that showed the copper complexes bind to one imidazole and curve fitting to the electrophysiology data provided rate constants for binding in the M2 channel. Computational chemistry was used to obtain binding geometries and energies of the copper complexes to the His37-tetrad. The results show that the copper complexes do bind with the His37 complex and prevent proton conductance and influenza infection.

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[pt] Tradicionalmente, os testes de formação em poços de petróleo buscam caracterizar o campo de permeabilidades a partir da interpretação dos transientes de pressão (PTA) nos períodos de fluxo e estática baseados em modelos isotérmicos de escoamento em meios porosos. Com o avanço da instrumentação dos testes, registros mais precisos de temperatura passaram a estar disponíveis e fomentaram a pesquisa baseada em modelos não isotérmicos que possibilitaram a análise a partir dos transientes de temperatura (TTA). Além da caracterização de parâmetros do reservatório como permeabilidade e porosidade com a interpretação dos transientes de temperatura, os dados de pressão obtidos a partir de um modelo não isotérmico representa de forma mais fidedigna o fenômeno físico sobretudo quando os testes são submetidos a maiores diferenciais de pressão. Este trabalho consiste no desenvolvimento de um simulador para teste de formação que considera a modelagem não isotérmica de reservatório unidimensional radial acoplado a um poço produtor e na utilização deste simulador, associado a métodos de otimização multivariável, para resolução do problema inverso da caracterização de parâmetros do reservatório. Alguns métodos de otimização foram testados e o algoritmo do Simplex de Nelder-Mead apresentou melhor eficácia. Foram estabelecidos três tipos de problemas e utilizados em três casos hipotéticos considerando inclusive a imposição artificial de ruídos nos sinais de pressão e temperatura utilizados para resolução do problema inverso. / [en] Traditionally, oil well formation tests aim to characterize the reservoir permeability field from pressure transient analysis (PTA) of drawdown and build up based on isothermal flow models in porous media. With the advancement of well test instrumentation, more accurate temperature records became available and have encouraged researches based on non-isothermal models that made possible the temperature transient analysis (TTA). In addition to the characterization of reservoir parameters such as permeability and porosity by TTA, the pressure data obtained from a non-isothermal model represent better the physical phenomenon, especially when the tests are subjected to greater drawdowns. This work consists in the development of a simulator for formation test that considers non-isothermal modeling of a unidimensional radial reservoir coupled to a production well and in the use of this simulator, associated with multivariable optimization methods, to solve the inverse problem of reservoir parameters characterization. Some optimization methods were tested and the Nelder-Mead Simplex algorithm presented better efficiency. Three types of problems were established and used in three hypothetical cases, including artificially imposed noise in pressure and temperature signals used to solve the inverse problem.

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[pt] Nesse trabalho foi realizado um estudo cinético do revenido de reações do aço de baixa liga (AISI 1050) usando a dilatometria não isotérmica. Os parâmetros cinéticos do primeiro e terceiro estado do revenido (aqui foram nomeados como processos I e II ) foram calculados assumindo que as reações obedecem ao modelo cinético de Johnson--Mehl--Avrami--Kolmogorov (JMAK) . Os formalismos mediante os quais os parâmetros cinéticos (E, n, Ko) são calculados é apresentado. Foram usados cinco formalismos para realizar o estudo . Três destes formalismos estão embasados em diferentes aproximações da integral da temperatura, um na regra de adição e um destes não usa nenhuma aproximação para o calculo. Os intervalos de confiança dos parâmetros também foram calculados. O resultado mostra que os valores calculados coincidem independentemente do método usado. Além disso, não dependem da temperatura ou da fração transformada. Conclui-se que neste caso ocorreu um processo com saturação de sítios. / [en] In this paper we present a kinetic study of the reactions of tempering in low-alloy steel (AISI 1050), using the non-isothermal dilatometry. The kinetic parameters of the first and third state of the tempering (here were named as processes I and II) were calculated assuming that the reactions follow the kinetic model of Johnson - Mehl - Avrami - Kolmogorov (JMAK). The calculation of the parameters was not done by setting any model. The formality by which the kinetic parameters (E, n, Ko) are calculated was presented. Five formalisms have been used mainly for the study. Three of them are based on different approximations of the integral of temperature. Another method were based on addition rule .Finally the last method does not use any other approach to the calculation. The result shows that the calculated values are very similar and these values are independent of the method used. Also, do not depend on temperature or transformed fraction .In this study it was concluded that this case happened a process with saturation of sites. During the study the confidence intervals of the parameters were calculated.

No / The effect of matrix polymer and filler content on the rheological behavior of hydroxyapatite-filled injection molding grade high-density polyethylene (HDPE) has been studied. Studies of the flow curves revealed that the matrix and the composite exhibit three distinct regions in the flow curve, namely, a pseudoplastic region at low to moderate shear rates, a plateau and a second pseudoplastic region at high shear rates. The shear stress corresponding to the plateau (Tc) is dependent on both the filler concentration and the melt temperature. Addition of HA in the HDPE matrix increases the value of Tc and decreases compressibility of the melt. An increase in temperature also raises the value of Tc. From the nature of flow curves it is concluded that the matrix polymer largely decides the rheology of the composite.

<p dir="ltr">The gastrointestinal tract harbors a diverse range of microorganisms, collectively constituting the gut microbiome. <a href="" target="_blank">The maintenance of a symbiotic relationship between the host and these microorganisms is essential to gastrointestinal health. Disruption of the ecological balance within the gut microbiome can result in discomfort or pathological conditions.</a> <a href="" target="_blank">This dissertation explores these alterations within the gastrointestinal tract as potential indicators for specific gastrointestinal diseases.</a> <a href="" target="_blank">In pursuit of this, I collaborated with others to develop a smart ingestible capsule that offers a non-invasive method for enhancing the effectiveness of differential diagnosis and treatment strategies for Inflammatory Bowel Disease (IBD). </a>My contributions encompassed conducting <i>in vitro</i> protein sampling and extraction experiments, as well as enteric coating dissolution tests. Following thorough characterization of the capsule, I advanced to <i>ex vivo</i> sampling experiments. As a proof of concept, the capsule's sampling capabilities have been rigorously validated both <i>in vitro</i> and <i>ex vivo</i> using calprotectin, a key biomarker for monitoring and managing IBD. Future research may explore integrating this technology with other sensors for diverse chemical and gas sensing capabilities, aiming to refine the differential diagnostics of Irritable Bowel Syndrome (IBS) and IBD.</p><p dir="ltr">Simultaneously, the potential transmission of pathogenic microorganisms from the gastrointestinal tract to the environment through fecal matter can lead to substantial public health implications if adequate surveillance is not in place. These pathogens can contaminate water and food sources from various origins, exacerbating the problem. Furthermore, conventional laboratory-based assays, while effective, have extensive turnaround times and require skilled scientists to operate them. In response to this challenge, I have undertaken the development of point-of-care assays, aiming to streamline the detection of fecal contamination. This innovation is designed to mitigate the limitations associated with traditional methods by offering a more rapid and user-friendly approach. The primary objective is to enhance the accessibility of these assays, enabling on-site personnel with varying levels of expertise to utilize them effectively. Through the widespread adoption of these point-of-care assays, the overarching goal is to ensure the consistent provision of safe and reliable water and food supplies to the public.</p>

La génération des cellules hématopoïétiques, aussi connue sous le nom d'hématopoïèse, est contrôlée par l’activité conjuguée de facteurs de transcription lignée-spécifiques permettant l’expression, en temps et lieu, de gènes spécifiques nécessaires pour le développement cellulaire. Dans le cadre de notre étude, nous avons étudié les facteurs de transcription KLF2 et KLF4 qui jouent des rôles cruciaux dans la formation des lymphocytes B et T. KLF2 et KLF4 activent la transcription de gènes spécifiques via leur interaction avec le co-activateur (CBP). Leurs interactions avec CBP requièrent le domaine de transactivation (TAD) qui est localisé dans la région N-terminal des facteurs KLF2 et KLF4. Des études préalables ont montré que des domaines TAD sont aussi présents chez la protéine suppresseur de tumeur p53 et que ces domaines sont requis pour les interactions entre la protéine p53 et le co-activateur CBP. Récemment, plusieurs structures des TADs de p53 en complexe avec les domaines TAZ2 et KIX de CBP ont permis de démontrer que ces TADs sont de nature acide et contiennent un motif ΦΧΧΦΦ crucial pour la formation des interactions. De plus, il s’avère que ces TADs sont similaires aux TADs de KLF2 et KLF4. L’étude présentée dans ce mémoire relate la caractérisation structurelle et fonctionnelle des interactions formées par les facteurs de transcription KLF2 et KLF4 avec leur partenaire d'interaction, CBP, pour activer la transcription de gènes spécifiques. Nos analyses ont été faites en utilisant différentes techniques telles que le titrage calorimétrique isotherme (ITC), la résonance magnétique nucléaire (RMN) ainsi que des expériences de transactivation chez la levure. Notre étude permet une meilleure compréhension des rôles opposés mais complémentaires qu'ont les protéines KLF2 et KLF4 au cours du développement et de la différentiation des lymphocytes B et T en plus de fournir les détails mécanistiques à la base de leurs interactions. Ces informations seront potentiellement utiles pour le développement d'outils à des fins thérapeutiques dans le cadre des leucémies, notamment. / Hematopoietic development is regulated through a combinatorial interplay between lineage-specific activators and the general transcription factors that enables cell-specific patterns of gene expression. In this study, the transcription factors KLF2 and KLF4 play crucial roles in lymphocytes B and T development by activating transcription of specific genes through interactions with the co-activator (CBP). These interactions involve the transactivation domains (TAD) localized in the N-terminal region of KLF2 and KLF4 factors. Previous studies have shown that TADs are also found in the tumor suppressor protein p53 and these TADs are responsible for the interactions between the p53 protein and the coactivator CBP. Recently, several structures of p53TADs in complex with the TAZ2 and KIX domains of CBP have shown that these TADs are acidic and possess a ΦΧΧΦΦ motif crucial for the formation of the interaction. Interestingly, these TADs are similar to the ones found on KLF2 and KLF4. This thesis provides a structural and functional characterization of the interactions formed by the transcription factors KLF2 and KLF4, which have opposing roles, and competes for the same interacting partner CBP to activate transcription. The analysis is done using isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy and a yeast activation assay. This study brings a greater understanding on the opposing roles yet complementary of KLF2 and KLF4 proteins involved in B and T lymphocytes specific lineages selection and also provides information for potential therapeutic research regarding disease such as leukemia.

Le facteur de transcription IIH (TFIIH) joue un rôle crucial dans la transcription et dans la réparation de l’ADN. La sous-unité Tfb1/p62 (levure et humain) de TFIIH interagit avec de nombreux facteurs de transcription (p53, NFκB, TFIIEα) et de réparation (Rad2/XPG and Rad4/XPC) (1). La majorité des interactions avec Tfb1/p62 requiert le domaine d’homologie à la Pleckstrin (PH) localisé dans la région N-terminal de la protéine (2, 3). Ce domaine PH forme des complexes avec des domaines de transactivation acide provenant de protéines cibles impliquées dans la transcription et la réparation de l’ADN. De récentes études ont montré que Tfb1/p62 est une cible pour les protéines virales telles que la protéine VP16 du virus de l’herpès simplex (HSV) de type 1, la protéine E1 du virus du papillome humain (VPH) et la protéine EBNA-2 du virus Epstein-Barr (EBV) (4, 5). Ces protéines virales interagissent avec la sous-unité Tfb1/p62 par un domaine de transactivation acide suggérant une interaction similaire à ce qui est observé chez les facteurs de transcription humains comme p53. Ce mémoire présente une caractérisation structurelle et fonctionnelle du complexe formé par la protéine virale EBNA2 et la protéine humaine Tfb1/p62. L’analyse est faite en utilisant le titrage calorimétrique isotherme (ITC), la résonance magnétique nucléaire (RMN) et une expérience de transactivation chez la levure. Cette étude amène une plus grande compréhension des protéines impliquées dans les maladies comme le lymphome de Burkitt et le lymphome de Hodgkin qui sont souvent associées à l’infection à l’EBV (revue dans (6)) et caractérise une cible potentielle pour un antiviral. / The general transcription factor IIH (TFIIH) plays crucial roles in both transcription and DNA repair. Tfb1/p62 (yeast and human), one of the ten/eleven subunits of TFIIH, has been shown to interact with several important transcription (p53, NFκB, TFIIEα) and repair factors (Rad2/XPG and Rad4/XPC) (1). Most of the interactions with Tfb1/p62 require the Pleckstrin homology (PH) domain located at the amino-terminal end of the protein (2, 3). This PH domain in particular forms complexes with highly acidic domains from target proteins involved in both transcriptional activation and DNA repair. Recent studies has shown that the Tfb1/p62 subunit of TFIIH is also targeted by a number of viral proteins including the Herpes Simplex virus (HSV) protein VP16, the Human papillomavirus (HPV) protein HPV E1 and the Epstein-Barr virus (EBV) protein EBNA-2 (4, 5). These viral proteins interact with the Tfb1/p62 subunit via acidic domain which suggests that they are forming similar interactions as the one observed with human transcription and repair factors. This thesis provides a structural and functional characterization of the complex formed by the viral proteins EBNA2 and the human protein Tfb1/p62 subunit of TFIIH. The analysis is done using isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy and a yeast activation assay. This study brings a greater understanding of proteins implicated in diseases such as the Burkitt’s lymphoma directly linked to an EBV infection (review in (6)) and shows a viable target for antiviral drug.