Melanocytes are pigment-producing cells that colour our hair, skin and eyes. Melanocytes are evolutionary conserved in vertebrates, and in addition to contributing to pigmentation and pattern formation, can contribute to background adaptation (zebrafish) and protection against harmful UV irradiation (humans). Many of the processes involved in melanocyte development – such as migration, proliferation and differentiation - are misregulated in melanoma. Here, I use chemical biology in zebrafish to identify targetable pathways in melanocyte development and regeneration, with a view to how these processes may be misregulated in melanoma and other pigmentation syndromes. We first wanted to address the potential for small molecules to regulate multiple stages of melanocyte development and differentiation. In Chapter 3, I describe my work involved in a small molecule screen for clinically active compounds that alter melanocyte biology (Colanesi et al., 2012). In this work we have identified small-molecules that affect melanocyte migration, differentiation, survival, morphology and number. This is important as it highlights new pathways essential for normal melanocyte development and consequently provides further tools in which to study melanocytes. Identifying the target of small molecules in vivo is a challenge in chemical biology. In Chapter 4, I describe my contributions to understanding how 5-nitrofuran compounds act in zebrafish (Zhou et al., 2012). My work has contributed to understanding the activity of 5-nitrofurans is dependent upon its nitrofuran ring structure. I have also helped confirm a conserved interaction between 5-nitrofurans and ALDH2, which may contribute to the off-target effects observed in the clinic. These results are important as they aid further understand of the 5-nitrofuran class of drugs and give evidence to support combination therapy of 5-nitrofurans with ALDH2 inhibitors as a way to overcome clinical side effects. Additionally I show that NFN1 treatment limits ensuing melanocyte regeneration thereby suggesting a role at the Melanocyte Stem Cell (MSC), which provides me with a key tool to study melanocyte regeneration in zebrafish. How tissue specific cell numbers are specified and maintained is a key question in developmental biology. In Chapter 5, I describe the identification of the MITF gene in the maintenance of cell cycle arrest in differentiated melanocytes (Taylor et al., 2011). We show that the human melanoma mutation MITF4TΔ2B promotes melanocyte division, thereby suggesting a role for melanocyte division in the pathogenesis of melanoma. This work is valuable because it highlights Mitf as a molecular rheostat that controls melanocyte proliferation and differentiation in living vertebrates, and helps us to understand the role of MITF in melanoma progression. Little is known about the pathways that control melanocyte stem cells in animals. To identify new melanocyte stem cell pathways, I used NFN1 as the basis for a small molecule screen for enhancers of melanocyte regeneration (Chapter 6). I find that chemical inhibition of Phosphatase of Regenerating Liver-3 (Prl-3) in zebrafish can enhance melanocyte regeneration. Importantly, I have found that there are an increased number of melanocyte progenitor cells in PRL3-inhibitor treated zebrafish. I propose that PRL-3 may control progenitor cell number in melanocyte regeneration. This is significant because it identifies PRL-3 as a novel molecular target controlling melanocyte progenitor cells, and identifies a new chemical tool with which to study melanocyte differentiation from a progenitor population. In the final chapter, I discuss how this work relates to the larger field of melanocyte developmental biology, and the new insight it provides into the fundamental processes of how organisms control cell number and pattern formation. In addition, I discuss how this work may have implications for understanding and treating melanocyte diseases, such as vitiligo (loss of melanocytes) and melanoma (cancer of the melanocyte).

Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy, characterized by its hallmark symptom myotonia. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded non-coding CTG/CCTG repeats, and these CUG/CCUG repeats sequester a family of RNA binding proteins. The Muscleblind-like (MBNL) family of RNA binding proteins are sequestered to the expanded CUG/CCUG repeats. The MBNL proteins are regulators of alternative splicing, and their sequestration to the toxic RNA leads to mis-splicing events, which are believed to cause the symptoms observed in DM patients. A previously reported screen for small molecules used to identify compounds that could disrupt MBNL from binding the toxic CUG repeats found that pentamidine was able to rescue splicing defects associated with DM. Herein, we present a new class of molecules (phenolsulphonphthaleins) that inhibited MBNL1/CUG repeat complex formation in a competitive electrophoretic mobility shift assay (EMSA). Additionally, one of these molecules, bromophenol blue (BPB), acted in a synergistic manner with the previously described inhibitor pentamidine. We also demonstrated that the halogenation of the phenolsulphonphthalein dyes is an important factor for activity. Moreover, we presentant analysis of a series of methylene linker variants of pentamidine that revealed heptamidine (an analog of pentamidine) could reverse splicing defects in a DM1 tissue culture model and rescue myotonia in a DM1 mouse model. Finally, we report on a new crystal structure of CUG repeats, crystallized in the context of a GAAA tetraloop/receptor which facilitated ordered packing within the crystal. This structure was consistent with previous structures showing that the repeats are essentially A-form RNA, despite having a U-U mismatch every third base pair. We also identified six types of U-U mismatch in the context of the 5'CUG/3'GUC motif, suggesting that the interactions between the uridines are dynamic. This structure also contains the highest resolution GAAA tetraloop/receptor structure (1.95 Å) reported to date. This dissertation includes previously unpublished co-authored material.

Une transduction efficace des cellules souches hematopoïetiques est un préalable pour la thérapie génique des maladies génétiques comme la β‐thalassemie, l’Adrenoleucodystrophie et le Déficit Immunitaire Combiné Sévère. La petite molécule UM171 à été décrite comme étant une molécule capable de stimuler l’expansion in vitro des cellules souches hématopoïétiques humaines, permettant ainsi une plus large application des thérapies basées sur les cellules souches. Nous avons aussi conduit des études supplémetaires pour confirmer la capacité de UM171 à expandre les souches hématopoïétiques. Durant ce travail, nous avons découvert que UM171 pouvait aussi augmenter de maniére significative, l’efficience de la transduction lentivirale des cellules hématopoïetiques primitives dérivées de sang de cordon. En plus, nous avons montré que UM171 augmentait la transduction des cellules hématopoïeques ayant les phénotypes les plus immatures. Des études plus approfondies ont aussi révélé que UM171 pouvait aussi augmenter la transduction des cellules souches hématopoïétiques avec des lentivirus ayant diffèrent pseudotypes. Au total ces découvertes ont pour conséquence, une nette amélioration des protocoles d’expansion et de transduction des cellules souches hématopoïétiques à travers un meilleur rendement en cellules souches et des taux élevés de transfert de gène en utilisant des quantités réduites de particules virales / Efficient lentiviral gene transfer to hematopoietic stem cells is a prerequisite for theultimate goal of gene therapy for a range of major genetic diseases such as β‐thalassemia, Adrenoleucodystrophy and severe combined immnodeficiency. The small molecule UM171 was recently described as having potent ability to stimulate ex vivo expansion of human hematopoietic stem cells, another key to safer and wider application of stem cell mediated therapies. Here we have conducted additional studies to confirm the stem cell expansion properties of UM171 and in the course of this work discovered that it also has the ability to significantly enhance the efficiency of the lentiviral transduction of primitive hematopietic cells in human cord blood. Subsequent work confirmed that this enhancing effect extends importantly to the most primitive hematopoietic subset as assessed phenotypically and by functional readout in immunodeficient mouse xenografts. Further detailed characterization ofthis phenomenom revealed that UM171’s effects are manifest rapidly and extend to a range of lentiviral pseudotypes. Together these findingsprovide an avenue for improved protocols for hematopoietic stem cell transduction that achieve higher gene efficiency and stem cell recovery coupled with the potential for reduced viral titer requirements.

E2A-PBX1 is expressed as a consequence of a recurring chromosomal translocation seen in 5% of acute lymphoblastic leukemia cases. We recently reported that substitution of a leucine residue (L20A) within the N-terminal transcriptional activation domain (AD1) of E2A-PBX1 markedly impairs binding to the KIX domain of CBP/p300 and, importantly, leukemia induction in a mouse bone marrow transplantation model. Since both the protein-protein interaction and consequent leukemogenesis rely on a focal contact point and might therefore be susceptible to antagonism by small molecules, we devised a cell-free assay based on fluorescence anisotropy (FA) to detect binding of a fluorescently labeled peptide derived from AD1 of E2A-PBX1 (FITC-E2A) with recombinantly expressed KIX domain. The optimized FA assay reveals a dissociation constant of 2 µM for the wild-type interaction and correctly detects disruption of the complex by naphthol AS-E phosphate, a compound previously shown to antagonize KIX binding. The optimized FA assay was used to screen the Prestwick, Spectrum and Chembridge libraries containing 12400 compounds in total. Of the initial 43 positive hits from the libraries, 10 caused a reproducible decrease in FA. Since intrinsic small molecule fluorescence can produce false positive results in the FA-based screen, intrinsically fluorescent compounds were excluded from further analysis unless they could be shown to bind to KIX. Two hits, L1 and C2, were intrinsically fluorescent but demonstrated KIX interactions and one hit, P9, was not intrinsically fluorescent. These three compounds were tested for their ability to inhibit binding of a larger portion of E2A (residues 1 to 483) to full length CBP in a pull down assay with only compound P9 demonstrating efficacy. Further characterization of P9 by NMR showed no binding to KIX, however evaluation by FA showed binding to FITC-E2A with a 20 µM affinity. A cell-based cytotoxicity assay demonstrated that compound P9 was slightly more toxic on leukemic cells that express E2A-PBX1, compared to leukemic cells lacking E2A-PBX1 expression. Mammalian two-hybrid analysis did not provide details of the effects of P9 on the E2A:KIX interaction. We expect the identification of a novel compound, P9, capable of disrupting the oncogenic E2A-PBX1:CBP interaction, to guide the development of effective, less toxic leukemia drugs and provide new tools for elucidating the molecular mechanisms of leukemia induction by E2A-PBX1. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-08-31 11:13:19.517

No description available.

With the incidence of Alzheimer’s disease (AD) growing worldwide and in Canada along with the growing economic and social burdens, the need for more effective therapies becomes of great importance. Since the discovery of AD, a number of proposed theories have arisen to explain the pathophysiology including the i) cholinergic theory, ii) oxidative stress pathways, and iii) metal ion imbalance. The major class of drug therapies to treat AD are cholinesterase inhibitors; however, the “one drug, one target” approach has not proven fruitful and generally becomes ineffective in later stages of disease progression. In this project, we synthesized a library of 1,2,3,4-tetrahydroacridine derivatives (10a-d, 11a-e, 12a-e, and 13a-f) as potential agents to target the cholinergic and oxidative stress pathways of AD. Chapter I provides background information on the role of AChE and BuChE enzymes in AD. Furthermore, this chapter describes the neurotoxicity of reactive oxygen species (ROS) and metals in AD. Chapter II provides a summary of project hypothesis and rationale. Chapter III describes the synthetic details regarding the synthesis of target small molecules. It further describes the principles involved in carrying out biological evaluation such as AChE and BuChE inhibition, antioxidant properties via DPPH stable radical scavenging, iron chelation capacity using ferrozine and in vitro cell viability data in neuroblastoma cells. Chapter IV describes the SAR details on ChE inhibition, antioxidant activities, iron chelation and cell viability profiles and molecular modeling details. A brief conclusion and future directions are included in Chapter V and the final section, Chapter VI provides experimental details for synthetic chemistry including analytical data of synthesized compounds and protocols for biological evaluations. This study identified novel tetrahydroacridine derivatives with nanomolar inhibition of both human AChE and human BuChE enzymes that were more potent relative to the reference agent tacrine. Compound 10d[N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified as a potent inhibitor of BuChE (IC50 = 24.0 nM) and compound 13c [6-chloro-N-(pyridine- 2-ylmethyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified as a potent inhibitor of AChE (IC50 = 95.0 nM) with good inhibition of BuChE (IC50 = 1.61 μM) whereas compound 11e [6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine] was identified with an optimum combination of dual AChE and BuChE inhibition (AChE IC50 = 0.9 μM; BuChE IC50= 1.4 μM). In conclusion, our studies provide new insight into the design and development of novel tetrahydroacridine derivatives to target multiple pathological routes of AD.

Ce travail de thèse est axé sur la synthèse et la réactivité d'un nouveau germylène P,N-hétérocyclique stabilisé par un fragment phosphanylidène-phosphorane. Le premier chapitre constitue un état de l'art des études portant sur la réactivité des espèces dérivées du groupe principal vis-à-vis de petites molécules (H2, NH3, éthylène, etc.). Une emphase particulière a été apportée à la description des différents mécanismes impliqués dans l'activation des liaisons de hautes énergies et à leur comparaison avec ceux rencontrés dans le cas des métaux de transitions. Le deuxième chapitre présente la synthèse et la caractérisation complète de germylene-phosphacétènes stabilisés par différents ligands phosphine fonctionnalisés. Ces dérivés sont thermiquement labiles et leur décarbonylation à diverse températures conduit au germylène-phosphinidène transitoire correspondant. L'évolution de ces intermédiaires est étroitement liée à la nature du ligand phosphine coordinné au Ge(II) central et, dans le cas du germylène stabilisé par un ligand diaminophosphine à quatre chaînons très volumineux, une migration intramoléculaire conduisant à un nouveau germylène cyclique à six chaînons est observée. L'étude de ce germylène P,N-hétérocyclique fait l'objet du troisième chapitre de ce manuscrit. Ce germylène présente un écart énergétique HOMO-LUMO faible du fait de sa stabilisation par le fragment phosphanilidène-phosphorane. En conséquence, sa réactivité est accrue en comparaison des germylènes N-hétérocycliques classiques pour qui l'écart d'énergie singulet-triplet est supérieur. La réactivité de ce nouveau germylène, incluant une étude en chimie de coordination vers acides de Lewis, est présentée. Dans le quatrième chapitre, un adduit réactif du germylène P,N-hétérocyclique avec un borane a été étudié expérimental et théoriquement. Du fait de l'existence de plusieurs sites réactifs de différentes natures au sein de ce composé, il se comporte comme un système "multi-activation" vis-à-vis de petites molécules. Son utilisation en tant que catalyseur dans des transformations organiques sera également présentée. / The synthesis and reactivity of a new P,N-heterocyclic germylene, stabilized by a phosphanylidene-phosphorane moiety, are the subjects of this work. In the first chapter, a bibliographic study resumes the state of the art of principal main-group systems capable to activate small molecules, such as H2, NH3, ethylene, etc. following similar activation mechanism that those of transition metal complexes. The second chapter present the synthesis and full characterization of stable phosphaketenes functionalized germylenes supported by different phosphine ligands. These derivatives are thermally labile and easily undergo thermal decarbonylation, affording the corresponding transient germylene-phosphinidene. The evolution of these intermediates is strongly related to the nature of the phosphine ligand coordinated to the Ge(II) centre and, in the case of the germylene stabilized by the bulkier four-membered cyclic diamino-phosphine, the phosphine ligand migrates from the germanium to the phosphorus atom to form a new six-membered cyclic germylene. The study of this P,N-heterocyclic germylene is the main subject in the thirds chapter. This germylene presents a small HOMO-LUMO energy gap due to the stabilization provided by the phosphanilidene-phosphorane fragment. As a consequence, it presents an enhanced reactivity compared to that of classical N-heterocyclic germylenes, where the singlet-triplet energy gap is larger. The reactivity of this new germylene, as well as its coordination ability towards Lewis acids, will be discussed. In the fourth chapter, a stable adduct of the P,N-heterocyclic germylene with a bulky borane will be experimental and theoretically studied. Due to the presence of multiple reactive sites in this germylene-borane complex, it behaves as a multiple-activation system towards small molecules. Its use as catalyst in some organic transformations will be also presented.

Abstract: With the worldwide dependence on non-renewable fossil fuels and increasing concerns over their impact on our planet through greenhouse gas emissions finding an alternative source of clean energy is a global imperative. The solar energy is one source of renewable energy resources, and It has the highest potential to contribute substantially to the future of carbon-free power needs. Solar to hydrogen has attracted much attention in the past decade due to its abundance and the spotlessness of hydrogen as fuel for energy usage. However, practically the requirements to convert solar energy to hydrogen, require a stable photocatalyst that’s able to operate efficiently over a wide range of the UV-VIS spectrum. Organic semiconductors have been widely used in hydrogen evolution due to their earth abundance, aqueous stability, and optical absorption that can be tuned to the UV-VIS spectrum. In chapter 3, The effect of different sacrificial regents on hydrogen evolution activity was systemically investigated by using poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) nanoparticles dispersion large and small diameter with Sodium dodecyl sulfate (SDS) as stabilizer. Ascorbic acid (AA), diethylamine (DEA), triethanolamine (TEOA), and triethylamine mixed with methanol (TEA/MeOH) were chosen as sacrificial reagents. The results indicate that the large diameter give improved efficiency with ascorbic acid, and the small diameter improved activity in the presence of diethylamine. The results indicated that the comparison between different sacrificial reagents is difficult because, the conditions of every experiment is different to another, depending on (the type of photocatalyst used, solubility, activity..) so to date, there is no clear concurrence in which sacrificial reagent is better than others. Photocatalysts formed from a single organic semiconductor typically suffer from inefficient intrinsic charge generation, which leads to low photocatalytic activities. In chapter 4, To overcome this limitation, we have used BTR, O-IDTBR, and PC71BM in binary and tertiary heterojunction nanoparticles between non fullerene donors’ small molecules and fullerene acceptor. The resulting photocatalyst display unprecedentedly a high hydrogen evolution rate over 12000 μmolh-1g-1 under AM 1.5g illumination.

Influenza A viruses (IAV) are negative sense RNA viruses that naturally infect a wide variety of animals. Different subtypes of the virus infect waterfowl, poultry, pigs, horses, ferrets, bats, dogs, cats and humans, causing zoonotic outbreaks and pandemics. In humans, IAV strains cause seasonal epidemics that can result in up to 50000 deaths and 700000 hospitalizations each year, and ranks in the top ten causes of death in the United States. In addition, virulent strains of IAV have caused pandemic outbreaks triggering numerous fatalities. While tropism to the upper respiratory tract is important for virus transmission, infection of the lower lung is most correlated with pathogenesis. The mammalian lung has multiple structural cell types, of which two classes are considered most important for IAV pathogenesis. These are (1) alveolar unit cells and (2) cells of the conducting airways, primarily those of the bronchi and bronchioles. Alveolar unit cells, or pneumocytes, chiefly comprise type I and type II alveolar epithelial cells, and are involved in gas exchange and surfactant production. Cells of the bronchi and bronchioles (e.g., basal, secretory, ciliated, club, goblet and neuroendocrine cells) perform numerous functions related to tissue repair/renewal, and mucous production. Upon IAV infection the regulated (or programmed) death of the infected cell represents an important pathogen clearance mechanism. Programmed cell death can be largely non-inflammatory (e.g., apoptosis) or pro-inflammatory (e.g., necroptosis). In this dissertation, I outline experiments carried out to identify the role of pro-inflammatory programmed cell death in influenza A virus clearance and pathogenesis both in vitro and in vivo. My work outlines the role of necroptosis in IAV clearance, and how this controlled form of cell death, particularly in alveolar unit cells, can be exploited as a potential new therapeutic avenue for severe influenza disease. / Cancer Biology & Genetics

Small-molecule libraries with enhanced structural diversity are of value in drug discovery campaigns where novel biologically active hits are desired. As such, multicomponent reactions (MCRs) have proven fruitful to enhance the molecular diversity of chemical collections and expedite forward progression of the drug discovery chain. Bicalutamide (Casodex), an anticancer drug, and Telaprevir (Incivek), an antiviral, are two examples of marketed drugs that can be synthesized using an MCR. The research topic of this dissertation involves the design, discovery, and development of novel MCRs and new combinations of MCRs with post-condensation modifications to generate over twenty-five new drug-like scaffolds in an operationally friendly, atom-economical, time- and cost-effective fashion. The developed chemical methodologies possess inherent 'iterative efficiency','high exploratory power', and 'bond forming efficiency' that allow them to quickly explore chemical space and navigate the 'hypothesis-synthesis-screening' loop that is key for a medicinal chemistry project. The prepared molecules were submitted to the Community for Open Antimicrobial Drug Discovery (CO-ADD) for antimicrobial screening against pathogens that are known to cause drug-resistance infections.