<p>The pathological hallmarks of sickle cell disease originate from a single mutation of the beta hemoglobin gene resulting in a valine at position 6 instead of the canonical glutamic acid. This small change perpetuates many factors, manifesting into chronic embolic processes in the microvasculature, causing painful vaso-occlusive episodes and eventual organ failure. There have been numerous therapies developed to reduce the mortality of sickle cell ranging from agents to induce production of fetal hemoglobin to chronic blood transfusions. Although each of these options are effective at improving the quality of life for sickle cell patients, they only treat one aspect of the disease and, for some, become ineffective over time. In the hope of producing a better therapy, a better understanding of the pathogenesis of vaso-occlusive episodes is needed. While many models have been offered to account for these vaso-occlusive events, one recently proposed mechanism stems from the elevated tyrosine phosphorylation of the cytoplasmic domain of the major erythrocyte membrane protein, Band 3. Band 3 serves as a hub for many critical proteins in the red cell. It binds ankyrin, which associates the spectrin cortical cytoskeleton to the red cell membrane, deoxygenated hemoglobin, the kinases Wnk1 and OSR1, which regulate cation transport, and a glycolytic enzyme metabolon that regulates the production of ATP and glutathione. When Band 3 is tyrosine phosphorylated, each of these proteins dissociate, causing significant changes to red cell homeostasis. These changes include an accumulation of reactive oxygen species, vesiculation and release of prothrombotic microvesicles, leakage of cell free hemoglobin, and a decrease in cell volume. Normally, Band 3 exists in a predominantly unphosphorylated state, however, in sickle cell disease, Band 3 is abundantly tyrosine phosphorylated. Reduction in the tyrosine phosphorylation of Band 3 has been documented to prevent the release of microvesicles and hemoglobin from sickle cell red blood cells. Because these microvesicles and cell free hemoglobin contribute to the vaso-occlusive episodes in sickle cell patients, inhibiting the mechanism for their release offers a potential therapeutic option. But to accomplish this, the molecular cause for the elevated tyrosine phosphorylation in sickle cell disease must be identified. Since tyrosine phosphorylation is performed by a tyrosine kinase and removed by a tyrosine phosphatase, the elevation in phosphorylation must be due to changes in both of these processes. Unfortunately, the identity and nature of these kinases and phosphatases are poorly understood. In this dissertation, I identified the tyrosine kinases Syk, Lyn, and Src attributed to Band 3</p> <p>15</p> <p>phosphorylation that facilitates the release of microvesicles and hemoglobin in sickle cell red blood cells. Inhibition of Syk or one of the two Src family kinases is sufficient to prevent the destabilization of the red blood cell membrane. These kinases function in a hierarchy, where one of the three Src family kinase, Lyn phosphorylates Syk, activating it, and promoting the phosphorylation of Band 3 at tyrosines 8 and 21. Prevention of either phosphorylation event prevents the release of microvesicles and cell free hemoglobin. I also report the identification of PTP1B as the tyrosine phosphatase responsible for maintaining Band 3 in an unphosphorylated state. Interestingly, in sickle cell disease, this tyrosine phosphatase is proteolytically cleaved, resulting in a reduction in dephosphorylating potential. It has been reported previously that PTP1B is a substrate of the calcium dependent protease, calpain and that calpain inhibitors improve the cell morphology of sickle erythrocytes. Inhibition of this proteolytic process may offer an additional therapeutic option for the treatment of sickle cell disease.</p>

<p>The following thesis will focus on understanding the downstream behavioral effects of δORmediated β-arrestinsignaling. δORagonists have been implicated as effective targets for a variety of diseases, however detrimental side effects of opioid-targeting agonists limit their clinical use. δORagonists specifically can induce seizures, however the underlying mechanism contributing to this  behavior  is  unknown.  We  review  this  phenomenon  in  more  detail,  highlighting  current agonists known to induce seizures and potential circuits and pathways involved. Our work suggests β-arrestinsignaling  is  involved,  specifically β-arrestin2  mediated  signaling  may  be  largely contributing  to δORagonist-induced  seizure  behavior.  As  it  is  possible  the β-arrestinisoforms have unique roles in seizure behavior, we also analyzed methods in which to provoke β-arrestinisoform bias of δORtargeting compounds. Though the full mechanism relating δORagonists with seizures remains unknown, our work provides foundational detail of this behavior, implicating the importance of β-arrestinisoform signaling through δOR; allowing for future studies to full define this seizure pathway and develop δORsafer agonists.  </p>

This dissertation presents research in the development and use of a multi-modal visual and haptic virtual model in higher education. The model, named Chemical Force Feedback (CFF), represents molecular recognition through the example of protein-ligand docking, and enables students to simultaneously see and feel representations of the protein and ligand molecules and their force interactions. The research efforts have been divided between educational research aspects and development of haptic feedback techniques. The CFF model was evaluated in situ through multiple data-collections in a university course on molecular interactions. To isolate possible influences of haptics on learning, half of the students ran CFF with haptics, and the others used the equipment with force feedback disabled. Pre- and post-tests showed a significant learning gain for all students. A particular influence of haptics was found on students reasoning, discovered through an open-ended written probe where students' responses contained elaborate descriptions of the molecular recognition process. Students' interactions with the system were analyzed using customized information visualization tools. Analysis revealed differences between the groups, for example, in their use of visual representations on offer, and in how they moved the ligand molecule. Differences in representational and interactive behaviours showed relationships with aspects of the learning outcomes. The CFF model was improved in an iterative evaluation and development process. A focus was placed on force model design, where one significant challenge was in conveying information from data with large force differences, ranging from very weak interactions to extreme forces generated when atoms collide. Therefore, a History Dependent Transfer Function (HDTF) was designed which adapts the translation of forces derived from the data to output forces according to the properties of the recently derived forces. Evaluation revealed that the HDTF improves the ability to haptically detect features in volumetric data with large force ranges. To further enable force models with high fidelity, an investigation was conducted to determine the perceptual Just Noticeable Difference (JND) in force for detection of interfaces between features in volumetric data. Results showed that JNDs vary depending on the magnitude of the forces in the volume and depending on where in the workspace the data is presented.

La RMN du solide a récemment émergé en tant que technique très puissante en biologie structurale, permettant de caractériser au niveau atomique des systèmes qui ne peuvent être étudiés par d’autres méthodes. Des protocoles spécifiques à la RMN du solide sont à présent bien établis pour la préparation des échantillons, l’attribution des spectres et l’acquisition de contraintes structurales. Ensemble, ces protocoles ont ouvert la voie aux premières déterminations de structures tridimensionnelles de molécules biologiques à l’état solide avec une résolution atomique, et ce non seulement pour des échantillons protéiques microcristallins, mais également pour des systèmes plus complexes tels que des fibrilles ou des protéines membranaires.La détermination structurale de tels systèmes est cependant encore loin d’être une routine, et des avancées de plus large ampleur sont attendues grâce à des développements aux niveaux méthodologique et matériel. Pour cette raison, une majeure partie du travail présenté dans cette thèse a été consacrée au développement d’expériences à la fois nouvelles et sophistiquées pour améliorer la sensibilité et la résolution des méthodes déjà existantes pour attribuer les spectres et élargir les possibilités offertes par la RMN du solide en vue d’étudier la structure de systèmes protéiques plus larges. Ces développements reposent notamment sur l’utilisation de champs magnétiques très intenses et sur la rotation des échantillons à l’angle magique dans la gamme des très hautes vitesses angulaires. Nous montrons que dans ce cadre, il est possible de concevoir des expériences utilisant uniquement des champs radiofréquences à faible puissance ainsi que d’utiliser des transferts sélectifs, l’acquisition de corrélations à travers les liaisons chimiques et la détection proton.En particulier, nous montrons que des expériences de corrélation homonucléaire reposant sur des transferts scalaires deviennent une alternative compétitive aux expériences basées sur des transferts dipolaires. Deux nouvelles séquences d’impulsion permettant de détecter des corrélations 13C-13C à travers les liaisons chimiques avec une excellente résolution sont présentées; couplées à des transferts 15N-13C, elles permettent l’attribution des résonances de la chaîne principale des protéines avec une grande sensibilité.De plus, nous démontrons qu’il est possible d’obtenir des raies très fines pour les résonances de protons dans des protéines complètement protonées à l’état solide grâce à la rotation à l’angle magique à ultra-haute vitesse, sans avoir recours à la deutération. Dans ce contexte, nous avons développé de nouvelles stratégies permettant d’attribuer rapidement et de façon fiable les résonances des spins 1H, 15N, 13CO, 13CA et 13CB dans différentes classes de protéines, ainsi que pour mesurer des contraintes structurales à partir des distances entre protons. L’approche proposée repose sur la haute sensibilité des protons et accélère donc considérablement les processus d’attribution et de détermination structurale des protéines à l’état solide, comme illustré sur la protéine beta-2-microglobuline.Enfin, nous avons appliqué cette nouvelle approche pour réaliser l’attribution et l’étude structurale et fonctionnelle de trois catégories de complexes protéiques: les fibrilles amyloidogènes formées par beta-2-microglobuline, les nucléocapsides du virus de la rougeole, et les nucléocapsides d’Acinetobacter phage205. Nous avons également utilisé la technique de Polarisation Nucléaire Dynamique pour obtenir des informations sur l’ARN des nucléocapsides du virus de la rougeole.Nous considérons que les résultats présentés dans cette thèse représentent une avancée substantielle dans le domaine de la RMN du solide appliquée à la biologie structurale. Grâce aux progrès actuels dans ce domaine, l’impact de la RMN biomoléculaire à l’état solide promet d’augmenter dans les prochaines années. / Solid-state NMR has recently emerged as a key technique in modern structural biology, by providing information at atomic level for the characterization of a wide range of systems that cannot be investigated by other atomic-scale methods. There are now well established protocols for sample preparation, resonance assignment and collection of structural restraints, that have paved the way to the first three-dimensional structure determinations at atomic resolution of biomolecules in the solid state, from microcrystalline samples to fibrils and membrane-associated systems. These determinations are however still far from being routine, and larger breakthroughs are expected with further methodological and hardware developments. Accordingly, most of the work presented in this thesis consists of the development of new, sophisticated NMR experiments to improve the sensitivity and resolution of the currently existing schemes for resonance assignment and to extend the capabilities of solid-state NMR in terms of structural investigation of proteins for the analysis of large substrates. These developments notably rely on the use of very high magnetic fields and ultra-fast magic-angle spinning (MAS). We show the great potential of this particular regime, which enables the use of low-power experiments and the acquisition of selective cross-polarization transfers, through-bond correlations and 1H-detected correlations.In particular, we show that homonuclear correlation experiments based on through-bond transfers become competitive alternatives to dipolar transfer schemes. Two new pulse sequences that detect sensitive and resolved 13C-13C through-bond correlations are introduced, which coupled to 15N-13C dipolar transfer steps provide sensitive routes for protein backbone resonance assignment.Furthermore, we demonstrate that narrow 1H NMR line widths can be obtained for fully protonated proteins in the solid state under ultra-fast MAS, even without perdeuteration. In this context, we have developed new strategies for extensive, robust and expeditious assignments of the 1H, 15N, 13CO, 13CA and 13CB resonances of proteins in different aggregation states, and new schemes for the measurements of site-specific 1H-1H distance restraints. This approach relying on the very high sensitivity of 1H spins remarkably accelerates the processes of assignment and structure determination of proteins in the solid state, as shown by the assignment and de novo structure determination of native beta-2-microglobulin. Finally, we apply this new approach to perform resonance assignment and to study structural and dynamic features of three complex protein aggregates: amyloid fibrils formed by native and D76N beta-2-microglobulin, Acinetobacter phage 205 nucleocapsids and measles virus (MeV) nucleocapsids. We also used Dynamic Nuclear Polarization to obtain the first information about RNA in MeV nucleocapsids.We believe that the results presented in this thesis represent a substantial step forward for solid-state NMR in structural biology. With all the current advances in the field, the impact of biomolecular solid-state NMR is likely to increase in the next years.

La recherche sur le cancer se tourne vers le développement de la nanomédecine, c’est-à-dire l’utilisation de nanoparticules pour augmenter l'efficacité thérapeutique et réduire la toxicité du traitement. Dans ce contexte, ces travaux ont été consacrés à la conception de nano objets pour des applications en thérapie anticancéreuse. Ces systèmes ont été élaborés à partir d'un châssis moléculaire cyclodécapeptidique présentant plusieurs ligands peptidiques RGD ciblant l’intégrine αvβ3, récepteur transmembranaire jouant un rôle clé dans l’angiogenèse. Dans un premier temps, nous avons synthétisé plusieurs composés en faisant varier la structure du châssis peptidique afin de moduler la présentation des ligands RGD lors de leur interaction avec l’intégrine αvβ3. L’évaluation biologique in vitro des différents composés synthétisés suivi d’une étape de simulation de dynamique nous a permis de déterminer une présentation optimale des ligands RGD. Nous avons ensuite développé une nouvelle voie de synthèse combinant deux réactions orthogonales catalysées au cuivre permettant l’accès à ces vecteurs peptidiques avec de meilleurs rendements et avec un temps de synthèse réduit par rapport à la voie classique. Finalement, nous avons greffé les clusters de RGD sur des nanoparticules lipidiques, puis polymériques, afin d’apporter un élément de ciblage. Ces deux projets ont été réalisés à travers des collaborations, respectivement avec le laboratoire du Prof. Patrick Couvreur (nanoparticules de squalène) et avec le laboratoire du Dr Marie-Thérèse Charreyre (nanoparticules de copolymères NAM/NAS). Ces systèmes ont ensuite été étudiés in vitro pour des applications en thérapie et en imagerie / Cancer research is now taking advantage of nanomedicine that is to say the use of nanoparticles to increase treatment efficiency and reduce toxicity. In this context, this work has been devoted to the design and synthesis of nano objects for applications in cancer therapy. These systems are based on a cyclodecapeptidic molecular scaffold presenting several RGD peptidic ligands targeting the αvβ3 integrin, a transmembrane receptor playing a key role in angiogenesis. We first synthesized several compounds by modifying the structure of the peptidic scaffold in order to alter the presentation of the RGD ligands during their interaction with the αvβ3 integrin. Biological in vitro evaluation of the different synthesized compounds followed by dynamics simulation allowed us to identify an optimal presentation of the RGD ligands. We then developed a new synthesis combining two orthogonal copper-catalyzed reactions yielding those peptidic vectors with lower reaction times and better yields compared to the classic synthesis. We finally grafted the RGD clusters on lipidic, then polymeric nanoparticles to add targeting moieties. Both projects were realized through collaborations, respectively with Prof. Patrick Couvreur’s lab (squalene nanoparticles) and Dr. Marie-Thérèse Charreyre’s lab (NAM/NAS copolymers). Those systems were then evaluated in vitro for applications in therapy and imaging.

La recherche sur le cancer se tourne vers le développement de la nanomédecine, c’est-à-dire l’utilisation de nanoparticules pour augmenter l'efficacité thérapeutique et réduire la toxicité du traitement. Dans ce contexte, ces travaux ont été consacrés à la conception de nano objets pour des applications en thérapie anticancéreuse. Ces systèmes ont été élaborés à partir d'un châssis moléculaire cyclodécapeptidique présentant plusieurs ligands peptidiques RGD ciblant l’intégrine αvβ3, récepteur transmembranaire jouant un rôle clé dans l’angiogenèse. Dans un premier temps, nous avons synthétisé plusieurs composés en faisant varier la structure du châssis peptidique afin de moduler la présentation des ligands RGD lors de leur interaction avec l’intégrine αvβ3. L’évaluation biologique in vitro des différents composés synthétisés suivi d’une étape de simulation de dynamique nous a permis de déterminer une présentation optimale des ligands RGD. Nous avons ensuite développé une nouvelle voie de synthèse combinant deux réactions orthogonales catalysées au cuivre permettant l’accès à ces vecteurs peptidiques avec de meilleurs rendements et avec un temps de synthèse réduit par rapport à la voie classique. Finalement, nous avons greffé les clusters de RGD sur des nanoparticules lipidiques, puis polymériques, afin d’apporter un élément de ciblage. Ces deux projets ont été réalisés à travers des collaborations, respectivement avec le laboratoire du Prof. Patrick Couvreur (nanoparticules de squalène) et avec le laboratoire du Dr Marie-Thérèse Charreyre (nanoparticules de copolymères NAM/NAS). Ces systèmes ont ensuite été étudiés in vitro pour des applications en thérapie et en imagerie / Cancer research is now taking advantage of nanomedicine that is to say the use of nanoparticles to increase treatment efficiency and reduce toxicity. In this context, this work has been devoted to the design and synthesis of nano objects for applications in cancer therapy. These systems are based on a cyclodecapeptidic molecular scaffold presenting several RGD peptidic ligands targeting the αvβ3 integrin, a transmembrane receptor playing a key role in angiogenesis. We first synthesized several compounds by modifying the structure of the peptidic scaffold in order to alter the presentation of the RGD ligands during their interaction with the αvβ3 integrin. Biological in vitro evaluation of the different synthesized compounds followed by dynamics simulation allowed us to identify an optimal presentation of the RGD ligands. We then developed a new synthesis combining two orthogonal copper-catalyzed reactions yielding those peptidic vectors with lower reaction times and better yields compared to the classic synthesis. We finally grafted the RGD clusters on lipidic, then polymeric nanoparticles to add targeting moieties. Both projects were realized through collaborations, respectively with Prof. Patrick Couvreur’s lab (squalene nanoparticles) and Dr. Marie-Thérèse Charreyre’s lab (NAM/NAS copolymers). Those systems were then evaluated in vitro for applications in therapy and imaging.

No description available.

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings. In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.