Alpha7 Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in essential physiological functions and play a role in disorders such as Alzheimer’s disease. MD-354 (3-chlorophenylguanidine; 21), the first small–molecule negative allosteric modulator (NAM) at alpha7 nAChRs, served as a lead in developing structure–activity relationships for NAMs at a7 nAChRs. MD-354 (21) also binds at 5-HT3 receptors. Analogs of MD-354 with structural features detrimental to 5-HT3 receptor affinity were evaluated in patch-clamp recordings and an aniline N-methyl analog resulted in a more potent and selective NAM than MD-354. A new N-methyl series of compounds was synthesized in which the 3-position was replaced with different substituents considering their electronic, lipophilic, and steric nature. Comparative studies were initiated to investigate whether or not the MD-354 series and the N-methyl series bind in the same manner; 3D models of the extracellular domain of human alpha7 nAChRs were developed, allosteric sites identified, and docking studies conducted.

Neste trabalho, analisamos a interação de pequenas moléculas, marcadores hidrofóbicos, com hemoglobina, em diferentes estados: monocristal, pó e solução aquosa. Os métodos de análise empregados, são baseados nas teorias de relaxação em líquidos e técnicas não lineares de ressonância ST-RPE (transferência de saturação), fornecendo informações sobre mudanças locais nas vizinhanças desses marcadores. Um dos marcadores hidrofóbicos, o TEMPO, mostrou uma anisotropia considerável nos espectros de RPE do monocristal de hemoglobina que está relacionado com o empacotamento molecular da proteína do cristal. A associação desses métodos de análise conduziu a importantes informações sobre mudanças na camada de hidratação em várias proteínas: hemoglobina, mioglobina e lisozima, monitoradas pelo marcador covalente derivado da maleimida, e induzidas pela temperatura, sob diferentes condições de hidratação. Desta forma o uso da espectroscopia de RPE especialmente com simulação espectral e DT-RPE mostro ser um método potente e ainda não explorado no estado de hidratação de proteínas / In this work, the interactions of small molecules, hydrophobic spin labels, with hemoglobin, under different states was analysed: single crystal, powder and aqueous solution. The methods of analysis employed, are based in relaxation theory in liquids and non-linear techniques, saturation transfer (ST-EPR), giving informations on the local changes in neighbourhood of the labels. One of the hydrophobic labels, TEMPO, showed a considerable anisotropy in the hemoglobin single crystal spectra, a result related to the molecular packing in the protein single crystal. The association of the techniques of analysis all together lead to important informations an temperature induced changes in the hydration layer in several proteins: hemoglobin, myoglobin, NEM*, a maleimide derivated, in different conditions of hydration. In this way the use of EPR spectroscopy and particularly with spectral simulations and ST-EPR, proved to be a powerful and not yet very much explored method to study the problem of protein hydration

Myotonic dystrophy (DM) is an inherited disease characterized by myotonia, insulin resistance, cardiomyopathy, and cognitive deficiencies. DM is a triplet repeat disorder, meaning that affected individuals carry anywhere between 50 and thousands of CTG/CCTG repeats in their genetic makeup. When transcribed into RNA, these repeats become “toxic” in the sense that they serve to bind and sequester important RNA binding proteins. One such family of proteins, the Muscleblind-like (MBNL) family, is important in the regulation of alternative mRNA splicing, and thus the sequestration of MBNL proteins leads to a number of mis-splicing events. Many of these events are directly correlated to DM symptoms. While there is no known cure for DM, the use of small molecules to treat symptoms is a well-characterized therapeutic tactic with immense promise. Pentamidine is a small molecule that was found to reverse mis-splicing in both DM cell and mouse models. Mechanistically, this molecule is particularly unique because unlike many small molecules, which physically displace MBNL from the toxic CUG RNA, pentamidine reduces CUG RNA levels, possibly through inhibition of CTG transcription. Chapter I summarizes alternative splicing mechanisms and regulation, defines MBNL protein structure and function, describes DM pathophysiology and molecular mechanism, and finally provides an overview of pentamidine characterization as a small molecule therapeutic. Chapter II reports the development of an in vitro T7 transcription assay, which allowed us to compare the relative efficacy by which pentamidine is able to inhibit the transcription of various repeat and non-repeat DNA sequences. This chapter further reports the characterization of a series of methylene linker analogues of pentamidine, which were also characterized through the T7 transcription assay. Chapter III details our thorough structure-activity relationship investigation of bisbenzamidine analogues of pentamidine, both in in vivo and in vitro models. Chapter IV describes our characterization of actinomycin D, a known transcription inhibitor and chemotherapeutic, within the DM disease framework. Chapter V summarizes these data, which ultimately serve as a proof of concept for the potential of CTG transcription inhibition in therapeutic contexts and broadly describe their application in other repeat diseases. This dissertation contains previously published and unpublished co-authored material. / 10000-01-01

Foram determinadas quatro estruturas de complexos de Lantanóides utilizando a difração de raios-X. As intensidades dos feixes difratados foram medidas com um difratômetro automático de 4 ciclos CAD-4. As estruturas foram resolvidas utilizando o método de Patterson, sínteses de Fourier e Fourier Diferença e refinadas por métodos de mínimos quadrados com matriz bloqueada. Os poliedros de coordenação foram obtidos considerando as distâncias quadráticas médias mínimas entre os coordenantes e poliedros ideais. Y(C6H2N3O)3(6H18N3OP)2 e Ho(C6H2N3O)3(C6H18N3OP)2 são complexos isomorfos pertencentes ao sistema monoclínico, P21/n, tendo como parâmetros de rede para o complexo de Y: a = 17.104(2), b = 16.328(1), c = 17.671(6) &#197, &#946 = 95.40(1)&#176, Z = 4, Dc = 1.53 gcm-3, V = 4913(4) ޵ e para o complexo de Ho: a = 17.097(2), b = 16.299(1), c = 17.685(3) &#197, &#946 = 95.41(1)&#176, V = 4907(2) &#1973, Dc = 1.63 gcm-3. O poliedro de coordenação para esses complexos é o dodecaedro, simetria D2d (42m). Ce(C6H2N3O)3(C5H12N2O)3, é uma estrutura pertencente ao sistema triclínico, P-1, a = 1.495(5), b = 11.815(5), c = 18.40(1)&#197, &#945 = 83.09(5)&#176, &#946 = 76.7995)&#176, &#947 = 77.00(3)&#176, Z = 2, V = 4800(2)&#1973, Dc = 1.63 gcm-3. Existem 8 coordenantes ao redor do átomo pesado formando um Prisma Trigonal Biencapuzado. Nd(C6H2N3O)3(C5H12N2O)3, pertence ao sistema monoclínico, P21/c, a = 18.913(5), b = 12.386(5), c = 22.134(5)&#197, &#946 = 120.20(5)&#176, V = 4800(2)&#1973, Dc = 1.63 gcm-3. O número de coordenação é igual a 9 e o poliedro de coordenação é uma forma intermediária entre o antiprisma quadrado monoencapuzado e o prisma trigonal Trigonal Triencapuzado. / It were determinated four small molecules structures by X-Ray diffraction. The intensities of the reflections were measured with an automatic four-cicle difractometer CAD-4. The structures were solved by Patterson, Fourier Synthesis and refined by least squares methods. The coordination polyedras were obtained considering the minimal root mean distances between the polyedras founded and the ideal polyedras. Y(C6H2N3O)3(6H18N3OP)2 e Ho(C6H2N3O)3(C6H18N3OP)2 are isomorphos strutures belong to the monoclinic system, P21/n; Y: a = 17.104(2), b = 16.328(1), c = 17.671(6) &#197, &#946 = 95.40(1)&#176, Z = 4, Dc = 1.53 gcm-3, V = 4913(4) ޵ Ho: a = 17.097(2), b = 16.299(1), c = 17.685(3)&#197, &#946 = 95.41(1)&#176, V = 4907(2)&#1973, Dc = 1.63 gcm-3. The coordination number of these structures is 8 and the coordination polyedra is dodecahedro, with simetry D2d (42m). Ce(C6H2N3O)3(C5H12N2O)3, is a structure belongs to the triclinic system. P-1, a = 1.495(5), b = 11.815(5), c = 18.40(1)&#197, &#945 = 83.09(5)&#176, &#946 = 76.7995)&#176, &#947 = 77.00(3)&#176, Z = 2, V = 4800(2)&#1973, Dc = 1.63 gcm-3. There are 8 coordinates around the heavy metal forming a Two-couped Trigonal Prism. Nd(C6H2N3O)3(C5H12N2O)3, belongs to the monoclinic system, P21/c, a = 18.913(5), b = 12.386(5), c = 22.134(5)&#197, &#946 = 120.20(5)&#176, V = 4800(2)&#1973, Dc = 1.63 gcm-3. The coordination number is a 9 and the coordination polyedra is intermediate between mono-couped square antiprism and three-couped trigonal.

The budding yeast S. cerevisiae is widely used as a model organism to study biological processes that are conserved among eukaryotes. Di fferent genomic approaches have been applied successfully to interrogate the mode of action of small molecules and their combinations. In this thesis, these technologies were applied to di fferent sets of chemical compounds in the context of two collaborative projects. In addition to insight into the mode of action of these molecules, novel approaches for analysis of chemical-genetic pro files to integrate GO annotation, genetic interactions and protein complex data have been developed. The fi rst project was motivated by a pressing need to design novel therapeutic strategies to combat infections caused by opportunistic fungal pathogens. Systematic screens of 1180 FDA approved drugs identifi ed 148 small molecules that exhibit synergy in combination with uconcazole, a widely used anti-fungal drug (Wright lab, McMaster University, Canada). Genome-wide chemical-genetic profiles for 6 of these drugs revealed two di fferent modes of action of synergy. Five of the compounds a ffected membrane integrity; these chemical-genetic interactions were supported by microscopy analysis and sorbitol rescue assays. The sixth compound targets a distinct membrane-associated pathway, sphingolipid biosynthesis. These results not only give insight into the mechanism of the synergistic interactions, they also provide starting points for the prediction of synergistic anti-fungal combinations with potential clinical applications. The second project characterised compounds that aff ected melanocytes in a chemical screen in zebra fish (Patton lab, Edinburgh). Chemical-genetic screens in S.cerevisiae enabled us to show that melanocyte pigmentation reducing compounds do so by interfering with copper metabolism. Further, we found that defects in intracellular AP1 and AP3 trafficking pathways cause sensitivity to low copper conditions. Surprisingly, we observed that the widely-used MAP-kinase inhibitor U0126 a ffects copper metabolism. A nitrofuran compound was found to speci fically promote melanocyte cell death in zebrafi sh. This enabled us to study off -target eff ects of these compounds that are used to treat trypanosome infections. Nifurtimox is a nitrofuran prodrug that is activated by pathogen-specifi c nitroreductases. Using yeast and zebra fish we were able to show that nitrofurans are also bioactivated by host-specifi c aldehyde dehydrogenases suggesting that a combination therapy with an aldehyde dehydrogenase inhibitor might reduce side e ffects associated with nifurtimox.

Pluripotent embryonic stem cells (ESCs) possess an unlimited capacity for self-renewal. This property of ES cells is both defining and unique. Harnessing this potential of ESCs would provide tremendous opportunity in the field of regenerative medicine and its attempts to combat degenerative diseases such as Parkinson’s, muscular dystrophy, etc. In 2006, Shinya Yamanaka was able to demonstrate that the ectopic expression of four proteins could reverse the process of differentiation and provide somatic cells with the characteristics ESCs. One year later, James Thompson’s group proved the same feat could be accomplished in human somatic cells using a different set of four proteins, including Nanog. The prospect of converting one’s own cells into a stem cell which could subsequently differentiate and repopulate an area of the body afflicted by gross degeneration was revolutionary. In the years following Yamanaka’s and Thompson’s discoveries, however, there has been little insight gained into how these proteins are regulated post-translationally. In this study, four proteins which had previously been identified by Yamanaka as being ‘pluripotency factors’ were used as baits in order to ascertain a protein-protein interaction network. This network was subsequently interrogated using various chemical compounds and small molecules in order to dissect the signal transduction pathways feeding into pluripotency, as well as, post-translational modifications regulating the factors themselves. In this way, the chemical inhibitor H89 was found to decrease the presence of Nanog phosphorylation and possibly its dimerization resulting in the Nanog protein being destabilized and targeted for degradation. Inversely, the pan-cullin inhibitor MLN4924 was identified to increase the abundance of both phosphorylated Nanog and total Nanog protein. In an attempt to identify the Cullin Ring Ligase (CRL) responsible for the degradation of Nanog protein in ESCs, each cullin identified in the protein interaction network was inhibited using specific shRNAs. Quantitative fluorescence microscopy was performed and identified that inhibition of CUL3 increases Nanog protein levels, suggesting that a CUL3-based CRL may be responsible for the post-translation regulation of Nanog. Additionally, the quantitation of Sox2 protein levels in CUL4B shRNA cell line demonstrates that Sox2 protein levels may be regulated by a CUL4B-based CRL. Further studies will reveal whether or not CUL4A depletion also results in elevated Sox2 protein levels. If not, this would include the pluripotency factor Sox2 among the recently identified CUL4B-isoform-specific substrates for degradation and possibly provide the basis for a hypothesis of developmentally regulated substrate specificity. In addition to MLN4924, several other small molecules were identified as being able to increase phospho-Nanog protein levels in this study. Among them were the cell permeable peptides Ht-31 and PKI (14-22) amide. These peptides were found to both stabilize phospho-Nanog and produce ES cell colonies that uniformly express the Nanog protein. The development of a growth medium containing these peptides in order to maintain homogeneous pluripotent ES cells is currently in progress and received backing for a patent application by the University of Edinburgh on February 23, 2012.

Face à la croissance de la demande énergétique, les énergies alternatives, telles que l’énergie photovoltaïque, représentent des solutions réalistes. Cette dernière nécessite des matériaux efficaces pour la capture des photons et leur conversion en électricité.Les cellules solaires organiques (CSOs) sont basées sur les propriétés semiconductrices de certaines molécules ou de certains polymères π-conjugués. Dans le domaine des CSOs, les efforts de recherche actuels se concentrent selon trois axes : la réduction des coûts, l’augmentation de la durée de vie des cellules solaires et l’augmentation des rendements de conversion photovoltaïque. Les récentsdéveloppements ont conduit à une complexification des architectures des CSOs ainsi que des semi-conducteurs organiques utilisés, induisant une augmentation des coûts de fabrication. Dans une logique de développement économiquement efficace et écologiquement soutenable, il est nécessaire aujourd’hui de se concentrer sur des semi-conducteurs organiques viables économiquement et dont la synthèse est respectueuse de l’environnement. Ce travail doctoral a pour but de développer de nouveaux matériaux semi-conducteurs organiques bio-inspirés et bas coût. Les molécules étudiées présentent une structure donneur-accepteur-donneur. Leur squelette est celui de la curcumine, molécule qui donne sa couleur au curcuma. Le groupement accepteur est un difluorure de bore. Les groupements donneurs quant à eux varient suivant les semi-conducteurs. Les propriétés optoélectroniques de dix-sept dérivés curcuminoïdes ont été étudiées. Plusieurs d’entre eux se sont démarqués : ceux avec des groupements anthracène, ceux avec des dérivés thiophènes, enfin et impact sur les performances photovoltaïques de la formulation de l’encre utilisée pour le dépôt de la couche a été étudié en détail. Différents matériaux accepteurs ont été testés, de même que l’utilisation de mélanges ternaires. Pour l’un de dérivés curcuminoïde en combinaison avec du PC61BM, des rendements supérieurs à 4 % ont été obtenus avec des tensions de circuit ouvert supérieures à 1,0 V. Au regard de la simplicité structurale de ce semi-conducteur, ces résultats figurent à notre connaissance parmi les meilleurs reportés dans la littérature. Les phénomènes photophysiques ont également été étudiés par spectroscopie d’absorption des espèces transitoires. Enfin, le procédé de fabrication a été rapproché des conditions industrielles en éliminant les solvants halogénés utilisés et en travaillant à l’air ambiant. Finalement, bien qu’intéressantes, les propriétés photovoltaïques restent limitées pour une application industrielle du fait de la faible mobilité des trous de ces matériaux. / To face the growing needs in energy, renewable energies like solar photovoltaic represent realistic solutions. Photovoltaic energy requires efficient materials to absorb photons and to convert them into electricity. Organic solar cells (OSCs) are based on semiconducting π-conjugated polymer or small molecules. Current research in this field focuses on three main topics: the reduction of costs, the increase of device lifetime and the increase of power conversion efficiency. This last issue led to an increase in the complexity of OSCs architecture as well as organic semi-conductors, leading to anincrease in manufacturing costs. In order to develop sustainable and eco-friendly processes, it is now important to work on cost effective semi-conductors obtained fromgreen synthetic methodology. The aim of this thesis was to develop new bio-inspired organic semi-conductors. These materials are potentially low cost. Molecules studied present donor-acceptor-donor structure. They have the skeleton of curcumine. Curcumine is a natural yellow dye present in curcuma. Acceptor group is boron difluoride. Donor groups vary depending on the curcuminoid derivative. Optoelectronicproperties of seventeen semi-conductors were studied. Several of them stood out: those with anthracene groups, those with thiophene derivatives, finally and especially, those with triphenylamine groups. For this last family, the impact on the photovoltaic performances of the ink formulation used for deposition has been deeply studied. Several acceptor materials were tested, as well as ternary blend. For one curcuminoid derivative combined with PC61BM, efficiency above 4% has been achieved with open circuit voltage up to 1.0 V. Due to the very simple chemical structure of the donor, this represents one of the best result reported in literature to our knowledge. Transient species were also studied by ultrafast spectroscopy. The fabrication process was also changed to eliminate halogenated solvent and to enable processing in ambient air like in industrial process. Finally, photovoltaic properties observed are interesting. Nevertheless, they are not sufficient for industrial application due to low hole mobility in these materials.

Aptamers are short oligonucleotide sequences (DNA or RNA) capable of high affinity and specific binding to a molecule or a family of molecules. Aptamers are lower in cost and exhibit higher reproducibility when compared to antibodies and thus are well-suited for recognition and detection of small molecular targets such as drugs of abuse and small medical biomarkers. While aptamers have been extensively utilized for development of small molecule sensors, several limitations prevent measurements of complex or real-world samples. This dissertation describes methods, technologies, and assays that were developed with the goal of producing and/or improving aptamer-based sensors for target detection in complex samples. Aptamer engineering is detailed as an important facet of maximizing aptamer-sensor sensitivity and specificity, along with adaptation to various read-out mechanisms for improved selectivity. In chapter 3, an aptamer vii sensor for cocaine is developed based on binding between the fluorophore ATMND to the cocaine aptamer which results in quenching (i.e., ‘turn-off’) of the fluorescence of ATMND. Cocaine binding results in displacement of the ATMND and recovery of the fluorescence signal. Detection of cocaine is demonstrated with an engineered cocaine aptamer with higher affinity for cocaine, permitting over a 50-fold increase in sensitivity over other aptamer-based sensors. The method can be used in dilute biological fluids (e.g., saliva) with a single step reaction (seconds) and robust signal output. In chapter 4, a new adenosine specific aptamer is identified through rational engineering of a previously reported ATP-binding aptamer. The new adenosine aptamer is utilized to develop an electrochemical sensor for detection of adenosine in undiluted serum. The method displays 40-fold higher sensitivity in undiluted serum measurements over previously reported aptamer-based sensors for adenosine but also demonstrates specificity for adenosine over ATP, ADP and AMP that has not been previously reported. In chapter 5, a nuclease-guided truncation method is developed to yield optimal structure-switching aptamer sequences for the emergent illicit drug methylenedioxypyrovalerone (MDPV) and medical biomarkers ATP and deoxycorticosterone 21-glucoside (DOG). The method intelligently removes unessential nucleotides, producing truncated aptamer sequences with structure-switching functionality. This technique will be immediately useful for simple and low-cost development of aptamer-based electrochemical sensors.

No description available.

Organic solar cells have caught considerable attention in the past few years due to their potential for providing environmentally safe, flexible, lightweight, inexpensive, and roll-to-roll feasible production solar cells. However, the efficiency achieved in current organic solar cells is quite low, yet quick and successive improvements render it as a promising alternative. A hopeful approach to improve the efficiency is by exploiting the tandem concept which consists of stacking two or more organic solar cells in series. One important constituent in tandem solar cells is the middle electrode layer which is transparent and functions as a cathode for the first cell and an anode for the second cell. Most studies done so far have employed noble metals such as gold or silver as the middle electrode layer; however, they suffered from several shortcomings especially with respect to reproducibility. This thesis focuses on studying a new trend which employs an oxide material based on nano-particles as a transparent cathode (such as Zinc-oxide-nano-particles) along with a transparent anode so as to replace the middle electrode. Thus, this work presents a study on solution processable zinc oxide (ZnO) nanostructures, their proper handling techniques, and their potential as a middle electrode material in Tandem solar cells in many different configurations involving both polymer and small molecule materials. Moreover, the ZnO-np potential as a candidate for acceptor material is also investigated.