The localization, trafficking and regulation of messenger ribonucleic acids (RNA) and viral RNA play crucial roles in cellular homeostasis and disease pathogenesis. In recent years biochemical and molecular biology methods used to study RNA function have made several important advances in the areas of RNA interference, expression of transgenes, and the sequencing of transcriptomes. In contrast, current technologies for imaging RNA in live cells remain in limited use. Previous studies of RNA localization and dynamics have relied primarily on the expression of a reporter RNA and a fluorescent protein fusion that binds to aptamer sequences in the expressed RNA. While these plasmid based systems offer methodological flexibility, there remains a need to develop methods to image native, non-engineered RNA as plasmid derived RNAs may not have the same regulatory elements (3'UTR and introns) or copy number as the native RNA. Additionally, viral pathogenesis is often sensitive to the size and sequence of their genomic RNA and may not be suitable for study using engineered systems. We sought to develop and validate a new method for imaging native, non-engineered RNA with single molecule-sensitivity. These probes have four important properties. They are modular, compatible with fixation and immunostaining, bind quickly and specifically to targets, and do not interfere with RNA function. We built upon the technique of delivering exogenous, linear probes that bind to their target by Watson-Crick base pairing. The probes are multiply labeled and tetramerized to increase their brightness. To validate the probes, targeting and utility was demonstrated in two model systems: beta-actin mRNA to show targeting of an endogenous target and the genomic RNA of human respiratory syncytial virus to show targeting of a viral RNA target. All video files are in QuickTime format.

Combinations of sterically encumbered Lewis acids and Lewis bases are precluded from dative bond formation, failing to yield classical Lewis acid-base adducts. These unique systems are referred to as frustrated Lewis pairs (FLPs) and demonstrate a plethora of unique small molecule activations.Herein, the syntheses of phosphonium alkoxy- and aryloxyborate salts in addition to phosphonium thioborate salts are detailed. The scope of Lewis acid and base, alcohol and thiol, that are effective in this chemistry, is also detailed. The syntheses of novel borate and boronate esters of the form B(OR)3 and (C6R4O2)B(C6F5) are detailed and applied in metal-free heterolytic dihydrogen activation with phosphines. The further study of a variety of perfluoroboranes in the activation of H2 with tertiary phosphines is also detailed. Derivatization of triarylboranes, boronate esters, borinic esters and chloroboranes by reaction with one or two equivalents of alky- or aryldiazomethane is achieved yielding the corresponding RR’C insertion products. The solid-state structures of the free boranes, in addition to Lewis base adducts of a sampling of these species, are detailed. Reactivity of the resulting sterically encumbered boranes in imine hydrogenations, H2 and XeF2 activation are also detailed. Combinations of intermolecular frustrated Lewis pairs are found to react collaboratively to activate greenhouse gases such as carbon dioxide (CO2) and nitrous oxide (N2O), yielding the corresponding zwitterionic compounds R3P(CO2)BR2R’ and R3P(N2O)BR’3. Atom connectivity has been established via X-ray crystallographic studies and molecular structures and parameters are discussed. Subsequent exchange chemistry of both CO2 and N2O species with transition metal and other d-block Lewis acids are investigated and yield zwitterionic compounds and ion pairs which are otherwise unobtainable employing more conventional methods. Transition metal containing Lewis acids are employed in conjunction with tri(tert-butyl)phosphine for the FLP-mediated direct activation of N2O.

Combinations of sterically encumbered Lewis acids and Lewis bases are precluded from dative bond formation, failing to yield classical Lewis acid-base adducts. These unique systems are referred to as frustrated Lewis pairs (FLPs) and demonstrate a plethora of unique small molecule activations.Herein, the syntheses of phosphonium alkoxy- and aryloxyborate salts in addition to phosphonium thioborate salts are detailed. The scope of Lewis acid and base, alcohol and thiol, that are effective in this chemistry, is also detailed. The syntheses of novel borate and boronate esters of the form B(OR)3 and (C6R4O2)B(C6F5) are detailed and applied in metal-free heterolytic dihydrogen activation with phosphines. The further study of a variety of perfluoroboranes in the activation of H2 with tertiary phosphines is also detailed. Derivatization of triarylboranes, boronate esters, borinic esters and chloroboranes by reaction with one or two equivalents of alky- or aryldiazomethane is achieved yielding the corresponding RR’C insertion products. The solid-state structures of the free boranes, in addition to Lewis base adducts of a sampling of these species, are detailed. Reactivity of the resulting sterically encumbered boranes in imine hydrogenations, H2 and XeF2 activation are also detailed. Combinations of intermolecular frustrated Lewis pairs are found to react collaboratively to activate greenhouse gases such as carbon dioxide (CO2) and nitrous oxide (N2O), yielding the corresponding zwitterionic compounds R3P(CO2)BR2R’ and R3P(N2O)BR’3. Atom connectivity has been established via X-ray crystallographic studies and molecular structures and parameters are discussed. Subsequent exchange chemistry of both CO2 and N2O species with transition metal and other d-block Lewis acids are investigated and yield zwitterionic compounds and ion pairs which are otherwise unobtainable employing more conventional methods. Transition metal containing Lewis acids are employed in conjunction with tri(tert-butyl)phosphine for the FLP-mediated direct activation of N2O.

The field of molecular magnetism has expanded rapidly since the discovery of single molecule magnets (SMMs) in the 1990’s and has witnessed extraordinary advances in the last several decades. One of the current trends in molecular magnetic research is to incorporate metal ions that have pronounced single-ion anisotropy in an effort to improve magnetic exchange interactions. The 4d and 5d transition metal ions have large spin-orbit coupling parameters which contribute to the orbital angular momentum effects that lead to anisotropic behavior. The work herein describes efforts to synthesize and characterize new cyanide-bridged molecular materials incorporating 4d and 5d transition metal ions, specifically the [Os(CN)_(6)]^(3-), [Mo(CN)_(6)]^(3-) and [W(CN)_(8)]^(3-) ions. The 5d hexacyanometallate [Os(CN)_(6)]^(3-) was incorporated into a trinuclear cyanide bridged molecule and the [Fe(CN)_(6)]^(3-) analog was prepared as a reference compound for assessing the effect of the 5d versus 3d metal ion on the magnetic properties. Both molecules exhibit SMM bistability with a pronounced increase (~90 %) in the blocking temperature (TB) of the OsIII analogue. In addition to typical SMM behavior, both compounds exhibit exchange-biased SMM behavior, a shift in the quantum tunneling of the magnetization (QTM) from zero field. This exchange-bias can be turned “on” or “off” depending on the presence of interstitial methanol molecules. New trigonal bipyramidal (TBP) molecules incorporating the rarely studies hexacyanomolybdate(III) ion are presented in chapter III of this dissertation. The molecules of general formula [M(tmphen)_(2)]_(3)[Mo(CN)_(6)]_(2) (M = V^(II), Mn^(II) and Fe^(II); tmphen = 3,4,7,8-tetramethyl-1,10-phenanthroline), represent additions to a large homologous family of TBP molecules reported by the Dunbar group over the years. The [Mo(CN)_(6)]^(3-) ion was prepared in situ by loss of one cyanide ligand from [Mo(CN)_(7)]^(4-). Of particular interest among the compounds reported is the V_(3)Mo_(2) analog which exhibits extraordinarily strong antiferromagnetic coupling (estimated J = -134 cm^(-1). The observed exchange coupling parameter is more than twice the current record for the antiferromagnetic coupling parameter for a cyanide-bridged magnetic molecule. Another set of results were obtained using the octacyanometallate anion [WV(CN)_(8)]^(3-) as a building block for the synthesis and magnetic studies of a family of new cyanide-bridged magnetic materials. The compounds exhibit several different structural motifs including three 0-D molecular compounds (two pentanuclear molecules and a linear trinuclear molecule) and a 1-D chain, findings that illustrate the structural versatility of the octacyanotungstate(V) ions. The TBP molecule, [Mn(tmphen)_(2)]_(3)[W(CN)_(8)]_(2), exhibits evidence for an out-of-phase signal when subjected to ac measurements in zero applied field. The 1-D chain also reveals evidence for the beginning of an out-of-phase signal under zero applied field which hints at single chain magnet behavior.

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In vitro analysis of osteoblasts deficient in endothelial NO synthase (eNOS) or inducible NOS (iNOS) enzymes, indicated that NO produced via eNOS is required for normal osteoblast growth and differentiation. The transcriptional regulation of two genes, the extracellular matrix protein Tenascin-Cytotactin (Tn-C) and eNOS, which contain strain-related regulatory sites and are known to respond to mechanical stimuli, was investigated in osteoblasts and osteocytes (ob-mix). Stretch was applied via 2 different systems and the transcription level of the gene promoters assayed by dual luciferase reporter assay. It was demonstrated that transcription of Tn-C and eNOS is upregulated in response to physiological levels of tensile and/or shear stress. This signal is mediated via the putative StRE and SSRE in Tn-C promoter and at the proximal end of the eNOS promoter is indicated. A system was built to study calcium (Ca²⁺) fluxes in response to shear stress in primary ob-mix and an immortalised simian virus human foetal osteoblast cell-line (SV-HFO). Studies have shown that pulsating fluid flow (PFF) stimulates a rapid [Ca²⁺]_i increase in primary ob-mix. Rapid transient increases in [Ca²⁺]_i were also recorded in a synchronised culture of SV-HFO when subjected to PFF. Both these observations are consistent with the hypothesis that Ca²⁺ increases contribute to the osteoblastic NO response to shear stress. Taken together, these results confirm the important role NO plays in bone physiology and elucidate aspects of transcriptional regulation and activation of the eNOS enzyme.

Throughout history, cancer has been severely plaguing mankind; the search for a cure to cancer had long been sought by scientists and still poses as one of the greatest challenges scientists have yet to overcome. Hypoxia in cells is a condition where there is little to no oxygen availability in its environment. In general, this event is detrimental since this can lead to cell necrosis or reoxygenation injuries. However, hypoxia, a prominent property of most solid tumors, activates the hypoxia-inducible factor (HIF-1) family of transcription factors that promotes angiogenesis. In this study, we describe the design and synthesis of small-molecule inhibitors of the HIF-1 pathway.

During development, Dictyostelium discoideum cells assume muticellularity via their collective aggregation. Cell-cell adhesion is required for morphogenesis, cell differentiation, cell sorting and gene expression during development. TgrC1 is a heterophilic cell adhesion molecule which is indispendable for complete development. TgrC1 can be considered as the most important cell adhesion molecule for D. discoideum development because deletion of the tgrC1 gene completely arrests development at the loose aggregate stage and inhibits fruiting body formation. In order to investigate the biological role of TgrC1 during development, I have chosen to identify and charactize the extracellular heterophilic partner and the cytoplasmic binding partner(s) of TgrC1. Using different biochemical approaches, we identified TgrB1 as the heterophilic binding partner of TgrC1 and demonstrated that their association is mediated through IPT/TIG domains in the extracellular region of both proteins. Both tgrB1 and tgrC1 share the same transcriptional promoter and their spatiotemporal expression pattern is identical during development. We also examined the assembly of TgrC1-TgrB1 complexes via the split green fluorescence protein complementation assay and the fluorescence resonance energy transfer approach. Whereas TgrC1 is capable of forming cis-homodimers spontaneously, cis-homodimerization of TgrB1 depends on its trans-interaction with TgrC1. A model of the assembly process has been proposed. To investigate signalling events initiated by the interaction between TgrB1 and TgrC1, pull-down assays were employed and led to the identification of myosin heavy chain kinase C as the cytoplamic partner of TgrC1. Mutational analysis showed that the basic residues in the short cytoplasmic domain of TgrC1 are critical to the binding with MHCK-C. Disruption of the interation between MHCK-C and TgrC1 results in an alteration of cell motility at the aggregation stage and aberrant cell sorting in slugs. These studies have highlighted the role of TgrB1-TgrC1 complexes in the regulation of morphogenesis during Dictyostelium development.

The repression of the E. coli lac operon seems to be more efficient than the current theoretical model allows for. Specifically, it is more quiet than expected during the replication of the chromosome. I have induced cells during short periods and counted the number of protein products from the operon to determine if there is a delay in activation of transcription that could account for the discrepancy. The results are compatible with a delay of 10-20 s, but the delay could not be conclusively proven. Furthermore, it has been investigated if the mechanism behind the delay might be differential localization of the lac operon with and without induction. It is shown that the lac operon is more often located in the periphery of the cell and in the internucleoid region when induced. These might be regions where genes are higher expressed, giving a delay in expression after de-repression before the gene is transported there.

The binding between the 45 kDa N-terminal domain of the a subunit of the GPIb-IX-V complex (GPIbαN) on the platelet membrane and the A1 domain of von Willebrand factor (VWF-A1), a multimeric protein circulating in the plasma, plays a key role in platelet adhesion and thrombus initiation at sites of cut-injury and atherosclerotic plaque rupture where blood vessels are subjected to high haemodynamic shear. A fundamental yet unresolved issue is how haemodynamic force upregulates this interaction (binding kinetics) and how a mechanical stimulus is translated into a biochemical signal (mechanotransduction). In order to address above issues, we setup a new biomembrane force probe (BFP) with the drifting reduction, temperature control and concurrent fluorescence imaging. My research findings are summarized into three aims: 1. VWF regions surrounding A1 hinder A1-GPIbα interaction at zero force, which is relieved by increasing force that stabilizes the interaction, giving rise to a VWF-GPIbα catch bond. 2. Three transport-related physical factors: receptor-ligand separation distance, Brownian motion and diffusivity govern the VWF-GPIbα association. 3. Mechanical force and structural variation regulate platelet signaling via the engagement duration of GPIbα mechanosensor. My thesis study advances our understanding of the biophysical and structural basis of how the VWF activation, its interaction with GPIbα and signal transduction are regulated by force when platelets' haemostatic functions are most needed.