No description available.

ABSTRACT This study describes selected aspects of the phonetic and phonological structure of the G|ui language, a poorly documented endangered Khoe (Central Khoisan) language spoken in Botwana. It conducts instrumental phonetic investigations, namely analyses of palatograms, linguograms, aerodynamic recordings, sound spectrograms, spectra, waveforms, and pitch measurements, in order to provide an objective basis for the detailed description of phonetic features of consonants, vowels, and tones. The description includes phonetic and phonological topics, involving consonants, vowels, and tones, and in addition, it deals with relevant morphological phenomena, such as the compound verb, verbal reduplication and verbal suffixes. This research also explores some theoretical issues, such as the unitary nature of clicks and their accompaniments, the integration of the clicks and non-clicks within a single set of features, the correct interpretation of tonal structure. Two types of historical sound shifts are also dealt with: namely, palatalization which is involved in the nonclick consonant system, and the click replacement which is involved in the click consonant system. In addition to the phonetic and phonological topics, selected aspects of the sociolinguistic profile of this endangered language are also documented.

The bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction exhibits complex but well-defined kinetics in aqueous and organic solution for soluble azides, alkynes, and ligand-bound copper(I). The kinetic profile in two dimensions, however, for CuAAC systems within a lipid bilayer membrane, has yet to be defined. The effect of triazole formation with lipid membrane-bound components on membrane properties such as fluidity and permeability is also of interest. Azide- and alkyne-functionalized lysolipids were synthesized and incorporated into non-fluid vesicles, which were then subject to CuAAC. The rate order for membrane-bound lipid substrates in non-fluid vesicles was observed to be comperable to that of the reaction in solution. Reactions between vesicles showed evidence of lipid transfer between non-fluid membranes, which has not been previously reported. For intervesicular and intravesicular reactions in non-fluid membranes, the observed reactivity was found to be opposite that of previously published reactions between nucleophiles and electrophiles in fluid lipid systems. Applications of this work include the potential for novel symmetric membrane leaflet labeling, bioorthogonal manipulation of cell and tissue function, and the creation of membranes with precisely controlled properties that may not be available in naturally-occurring membranes.

Bayreuth, Univ., Diss, 2009/2010.

Despite their ubiquitous use, Pt(II) anti-cancer drugs still suffer from many issues such as off-drug target effects, renal and nephrotoxicity as well as acquired and intrinsic drug resistance. To obtain a better understanding of how to mitigate these deleterious effects can be mitigated we first must know all the targets of these drugs. Highlighted in this dissertation is previous work performed by groups exploring the localization of Pt in cells using fluorescence microscopy. While Pt drugs such as cisplatin contain no native fluorescence, a great deal of work has been done to covalently modify complexes with fluorescent tags. From studies using this technique, it been reported that Pt can target a number of compartments within the cell ranging from the nucleus to the cytoplasm. With each different derivative being observed in varied cell lines it becomes difficult to deconvolute a universal pattern to where Pt localizes, furthermore, the connected fluorophore could also bias Pt localization. To add general functionality and eliminate the bias of a pre-tethered fluorophore our lab has developed a number of different azide and alkyne-modified complexes that append a “reactive handle” to Pt compounds. This modification allows for use of the bioorthogonal azide-alkyne click reaction we are able to observe Pt localization after treatment. The focus of this work includes method development to conjugate a fluorophore to our Pt complexes in vitro and in cell cultures. We examined a number of different cell lines and observed frequent localization in the nucleolus of the cell. Also in this work is the development of methods to append multiple fluorophores to each Pt site to increase our ability to visualize these complexes in cells. Finally, we have also constructed a new Pt-azide that exhibits slower exchange kinetics due to a chelating exchangeable group. The use of this new complex will enable studies to determine whether changing the leaving group results in differential localization of Pt drugs in cells.

Methods to map small-molecule interactions with cellular RNAs are important for understanding endogenous activation, such as in riboswitches, as well as the potential for exogenous compounds to target RNA. Cisplatin is one of the most widely used of the platinum anticancer drugs that are prescribed in approximately 40-50% of all chemotherapy treatments (Dyson and Sava, 2006; Harper et al., 2010). Despite nearly 40 years of experience with this class of drugs, we still lack a comprehensive understanding of the targets of Pt compounds and their effects on cells. Pt(II) compounds are well-known DNA and RNA crosslinking agents, but the latter area is under-studied. In order to better understand the impacts of cisplatin and other platinum(II)-derived small molecules on cellular RNA, we have developed a technique we call “Platinum-seq,” which couples reverse transcription mapping of platinated RNAs to high-throughput sequencing. Chapter 1 is a study of cisplatin and a novel click-functionalized platinum compound (2-ADAP Pt) binding to the HDV ribozyme, a small catalytic RNA. Chapter 2 moves our platinum mapping approaches from low-throughput, sequencing gel based methods into next-generation sequencing for high-throughput analysis of all platinum sites in cellular RNA, a method we have named “Platinum-seq.” Chapter 3 is a study of differential gene expression of Saccharomyces cerevisiae treated with cisplatin and a second novel platinum(II) compound (azaplatin), using data acquired from the work in Chapter 2. Chapter 4 describes recent efforts to implement pre-enrichment of sequencing targets using click chemistry followed by DNA hybridization, in order to enrich for platinated fragments before sequencing library construction. Together, this work represents a significant step forward in advancing analysis of Pt(II) binding to cellular RNA, a potentially important target for this widely used class of anticancer compounds. Methods developed here are broadly applicable to genome-wide identification of platinum accumulation on DNA as well, which has not been pursued despite the extensive use of these compounds.

La synthèse de nanoparticules (NPs) d’or fonctionnalisées en phase aqueuse est encore aujourd’hui un enjeu majeur de la recherche dans le domaine des nanomatériaux. Depuis les travaux de J. Turkevich de 1951, la synthèse utilisant le citrate comme ligand et agent réducteur est la méthode de choix pour obtenir des NPs d'or. Cependant cette synthèse nécessite une étape supplémentaire de modification de surface par échange de ligand, pour pouvoir accrocher des molécules d’intérêt. Afin de simplifier la procédure, notre projet propose de synthétiser en une seule étape des NPs qui possèdent un groupement permettant une post-fonctionnalisation. La nouvelle voie de synthèse fait intervenir des composés bifonctionnels de la famille des 1-hydroxy-1,1-méthylène bisphosphonates (HMBP). Ainsi la base conjuguée de l'acide (1-hydroxy-1-phosphonopent-4-ènyl) phosphonique (HMBPène), qui possède une fonction éthylénique terminale nous a permis d'obtenir des dispersions de nanosphères de tailles contrôlées et nous avons pu rationaliser le mécanisme de synthèse utilisant ce type de molécules. Nous avons ensuite évalué plusieurs modalités de post-fonctionnalisation de notre nanoplateforme et validé une approche par chimie « Click » la via cycloaddition de composés tétrazine. En utilisant une nouvelle classe de HMBP couplés à une chaine polyéthylène glycol, des NPs stables en milieu physiologique ont pu être synthétisées selon le même modèle. Elles offrent également des possibilités de post-fonctionnalisation par couplage carbodiimide, que nous avons illustré par le couplage d'un fluorophore. Nous développons en dernière partie les résultats préliminaires sur deux types NPs d'or synthétisées à l'aide des HMBP pour des applications thérapeutiques. / In the ever growing fields of nanoscience the control of the synthesis of gold nanoparticles (GNPs) owing to their large variety of applications has emerged as an important domain. Among all methodologies Turkevich-Frens synthesis using citrates that act as ligand and reducing agent remains a method of choice for the obtaining of water soluble GNPs. Nevertheless, in post-synthesis, citrates are often exchanged with other ligands to enhanced stabilization and allow further functionalisation. In our work we present a new class of bi-functional molecules (1-hydroxy-1,1-methylene bisphosphonates HMBP) that can both reduce Au(III) and act as an efficient stabilizer of the formed GNPs in water. The first size controlled GNPs “one pot” synthesis was achieved by using an alkene conjugated HMBP, the (1-hydroxy-1-phosphonopent-4-enyl)phosphonic acid (HMBPene). We moreover, rationalized the mechanism of the GNPs synthesis using this type of molecule. We then, evaluated several methodologies for the post-functionalization of our nanoplateform and developed a « Click » chemistry approach to nanoparticle coating by tetrazine cycloaddition. Other nanoplatforms were synthesized using pegylated hydroxyl methylene bisphosphonates. This new class of bisphosphonate coated GNPs showed an improved stability in biological media and brought reactive groups available for post-functionalization as well, illustrated by the coupling of a fluorescent dye. The last part of this was dedicated to our latest results on GNPs synthesis for biomedical applications with HMBP compounds.

No description available.

This study illustrates the utility of click chemistry in functionalizing triazabutadienes by allowing access to various applications both biological and material based. Triazabutadienes have been shown to trigger the release of highly reactive diazonium species in a pH dependent way when placed in acidic conditions. Electron-rich phenyl systems such as tyrosine residues have been shown to react with diazonium compounds to form stable azo bonds. Modification of these triazabutadiene motifs can functionalize them as linkers or impact solubility; which can allow for target specificity and mild cleavage of linker in order to liberate diazonium near site of interest. Incorporation of azide-alkyne cycloadditions onto these molecules will allow chemical functionalization and cross-linking properties. The 1,2,3-triazole triazabutadiene derivatives are synthesized via Huisgen 1,3-dipolar cycloaddition from alkynyl modifications on the triazabutadiene that are reacted with various azides that show substrate diversity.

The isoprenoid biosynthetic pathway (IBP) plays an important role in cellular metabolism. Currently there are drugs, including lovastatin and the nitrogenous bisphosphonates risedronate and zoledronate, that are used clinically to lower cholesterol levels and treat bone disease, respectively. These drugs work by inhibition of the upstream enzymes, HMG-CoA reductase and farnesyl diphosphate synthase (FDPS), respectively. The enzyme FDPS catalyzes the formation of farnesyl pyrophosphate (FPP), an important intermediate that represents a branch point in the pathway. The post-translational modification known as protein prenylation is mediated by the three prenyltransferase enzymes. Even though compounds like lovastatin, risedronate, and zoledronate indirectly disrupt protein prenylation, they also impair processes downstream from the point of inhibition. Therefore a direct approach would be desirable where downstream enzymes are targeted so that the rest of the cellular processes can continue to function. One such downstream enzyme is geranylgeranyl transferase II (GGTase II). This enzyme and it catalyzes the transfer of two hydrophobic geranylgeranyl chains from geranylgeranyl pyrophosphate (GGPP) to Rab proteins, which are essential for intracellular membrane trafficking. Inhibition of GGTase II may be a good therapeutic target for diseases such as multiple myeloma characterized by an over secretion of proteins. A known GGTase II inhibitor is the carboxy phosphonate 3-PEHPC, however millimolar concentrations are necessary to observe cellular effects with this compound. In an effort to develop more potent inhibitors of this enzyme, a family of isoprenoid triazole bisphosphonates was initially prepared by click chemistry, first as a mixture of olefin isomers due to an allylic azide rearrangement. These compounds were tested by our collaborators to determine the compounds’ activity as GGTase II inhibitors. Because some triazole bisphosphonates showed good activity as a mixture of isomers, a family of isoprenoid triazole bisphosphonates as single olefin isomers now has been prepared through the use of epoxy azides to avoid the azide rearrangement. The biological activity of these compounds has been studied and some of these triazole bisphosphonates were found to be potent and selective inhibitors of geranylgeranyl diphosphate synthase (GGDPS). While the enzyme GGDPS is upstream of the geranylgeranyltransferases, it is still downstream of the pathway’s primary branch point and provides GGPP for Rab geranylgeranylation. Two other families of triazole bisphosphonate analogues, homo- and bishomoisoprenoid triazole bisphosphonates, also have been prepared and tested by our collaborators to explore the compounds’ activity as GGDPS inhibitors, as well as the structure-activity-relationship. Previous research has shown digeranyl bisphosphonate (DGBP) and the bisphosphonate ether C-prenyl-O-geranyl bisphosphonate to be inhibitors of GGDPS. Two C-alkyl-C-homoalkyl DGBP analogues have been synthesized in order to study further the binding of these compounds to GGDPS, and dialkylated triazole bisphosphonates have been prepared to explore the effect of a triazole moiety on the analogue’s ability to inhibit GGDPS. The activity uncovered through these studies encourages further research on inhibitors of GGDPS.