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.

The American Cancer Society predicts that 1,665,540 people will be diagnosed with cancer, and 585,720 people will die from cancer in 2014. One of the most common types of cancer in the United States is skin cancer. Melanoma alone is predicted to account for 10,000 of the cancer related deaths in 2014. As a highly mobile and aggressive form of cancer, melanoma is difficult to fight once it has metastasized through the body. Early detection in such varieties of cancer is critical in improving survival rates in afflicted patients. Present methods of detection rely on visual examination of suspicious regions of tissue via various forms of biopsies. Accurate assessment of cancerous cells via this method are subjective, and often unreliable in the early stages of cancer formation when only few cancer cells are forming. With fewer cancer cells, it is less likely that a cancer cell will appear in a biopsied tissue. This leads to a lower detection rate, even when cancer is present. This lack of detection when cancer is in fact present is referred to as a false negative. False negatives can have a highly detrimental effect on treating the cancer as soon as possible. More accurate methods of detecting cancer in early stages, in a nonsubjective form would alleviate these problems. A proposed alternative to visual examination of biopsied legions is to utilize fluorescent nanocrystalline biomarker constructs to directly attach to the abnormal markers found on cancerous tissues. Quantum dots (QDs) are hydrophobic nanoscale crystals composed of semiconducting materials which fluoresce when exposed to specific wavelengths of radiation, most commonly in the form of an ultraviolet light source. The QD constructs generated were composed of cadmium-selenium (CdSe) cores encapsulated with zinc-sulfide (ZnS) shells. These QDs were then encapsulated with phospholipids in an effort to create a hydrophilic particle which could interact with polar fluids as found within the human body. The goal of this thesis is to develop a method for the solubilization, encapsulation, and initial functionalization of CdSe/ZnS QDs. The first stage of this thesis focused on the generation of CdSe/ZnS QDs and the fluorescence differences between unshelled and shelled QDs. The second stage focused on utilizing the shelled QDs to generate hydrophilic constructs by utilizing phospholipids to bind with the QDs. Analysis via spectroscopy was performed in an effort to characterize the difference in QDs both prior to and after the encapsulation process. The method generated provides insight on fluorescence trends and the encapsulation of QDs in polar substances. Future research focusing on the repeatability of the process, introducing the QD constructs to a biological material, and eventual interaction with cancer cells are the next steps in generating a new technique to target and reveal skin cancer cells in the earliest possible stages without using a biopsy.

<p dir="ltr">The BRG1/BRM-associated factor (BAF) complexes, also called SWI/SNF, are multi-subunit chromatin remodelers that regulate chromatin compaction in an ATP-dependent manner. In the past decade, BAF complexes have been under the spotlight in cancer research, especially after proteomic analyses revealed the genes encoding the subunits are amongst the most frequently mutated genes in cancer. The present dissertation focuses on prostate cancer (PCa), a disease in which the role of the BAF subunits is increasingly being explored but is yet to be defined as a potential therapeutic target. According to the GLOBOCAN report, PCa is the second most frequent cancer in males worldwide. Since most of the variants of PCa rely on the androgen receptor (AR) axis, surgical or chemical castration and androgen deprivation therapy (ADT) are the main treatment strategies for PCa patients. Even though these therapeutic approaches prolong survival, reduce tumor burden, and relieve symptoms, PCa patients eventually relapse and develop castration resistant PCa (CRPC). At present, the mechanisms underlying ADT resistance are not fully understood, current efforts focus on finding new targets for PCa treatment.</p><p dir="ltr">In the projects included in this dissertation we explored the function of the PBAF complex, a BAF subtype, in a variety of models of PCa and its potential as a therapeutic target by inhibiting or depleting its different subunits. To do so we (i) developed the first inhibitors for BRD7 (a subunit unique to PBAF) and (ii) established cell-based assays in multiple PCa cell lines to study BRD7 and other PBAF unique subunits.</p><p dir="ltr">Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromodomain-containing protein 7 (BRD7) has been implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a series of ligands designed to occupy this binding region and identified two inhibitors with increased selectivity towards BRD7, 1-78 and 2-77, which bind with submicromolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands occupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.</p><p dir="ltr">There are three BAF complexes that have been biochemically characterized up to date: canonical BAF (cBAF), polybromo-associated BAF (PBAF) and GLTSCR1/like-containing BAF (GBAF or ncBAF). All BAF complexes are characterized by containing an ATPase and accessory subunits that may be shared between them or unique to each subtype. PBAF, the BAF subtype of interest of this dissertation, contains four unique subunits: BRD7, PBRM1, ARID2 and BAF45A. We showed that knocking down BRD7 and ARID2 leads to reduction of cell viability in PCa cells with ligand-dependent and independent AR signaling, while knocking down PBRM1 leads to reduction in viability of cells with only ligand-dependent AR signaling. We also performed a chromatin immunoprecipitation assay with BAF45A and observed that it does not colocalize with AR binding sites, indicating that the mechanism by which PBAF regulates AR signaling is indirect. This observation was further supported by the fact that knocking down BRD7 prevents expression of genes related to adaptive processes, but not AR target genes, in response to androgen treatment. Further mechanistic studies will aid in understanding the function of PBAF in PCa. However, overall, our results indicate that PBAF is a promising therapeutic target in PCa models expressing AR, including CRPC systems.</p>

No description available.

In this research, a proton exchange membrane fuel cell (PEMFC) sensor was investigated for specific detection of volatile organic compounds (VOCs) for point-of-care (POC) diagnosis of the physiological conditions of humans. A PEMFC is an electrochemical transducer that converts chemical energy into electrical energy. A Redox reaction takes place at its electrodes whereas the volatile biomolecules (e.g. ethanol) are oxidized at the anode and ambient oxygen is reduced at the cathode. The compounds which were the focus of this investigation were ethanol (C2H5OH) and isoflurane (C3H2ClF5O), but theoretically, the sensor is not limited to only those VOCs given proper calibration. Detection in biosensing, which needs to be carried out in a controlled system, becomes complex in a multivariate environment. Major limitations of all types of biosensors would include poor selectivity, drifting, overlapping, and degradation of signals. Specific detection of VOCs in multi-dimensional environments is also a challenge in fuel cell sensing. Humidity, temperature, and the presence of other analytes interfere with the functionality of the fuel cell and provide false readings. Hence, accurate and precise quantification of VOC(s) and calibration are the major challenges when using PEMFC biosensor. To resolve this problem, a statistical model was derived for the calibration of PEMFC employing multivariate analysis, such as the “Principal Component Regression (PCR)” method for the sensing of VOC(s). PCR can correlate larger data sets and provides an accurate fitting between a known and an unknown data set. PCR improves calibration for multivariate conditions as compared to the overlapping signals obtained when using linear (univariate) regression models. Results show that this biosensor investigated has a 75% accuracy improvement over the commercial alcohol breathalyzer used in this study when detecting ethanol. When detecting isoflurane, this sensor has an average deviation in the steady-state response of ~14.29% from the gold-standard infrared spectroscopy system used in hospital operating theaters. The significance of this research lies in its versatility in dealing with the existing challenge of the accuracy and precision of the calibration of the PEMFC sensor. Also, this research may improve the diagnosis of several diseases through the detection of concerned biomarkers.