Little is known about the outward trafficking of receptor dimers from the endoplasmic reticulum to the plasma membrane, or the role that trafficking plays in assembly, targeting and specificity of receptor signalling. Bimolecular fluorescence complementation was used to follow prescribed receptor homo/heterodimers in Jurkat cells and clarify the trafficking itineraries those receptors follow to reach the plasma membrane. Chemokine receptors CXCR4 and CCR5 were chosen due to their implication in numerous pathologies including, HIV and cancer, and their ability to form homo and hetero-oligomers. This study demonstrates that although the individual receptors composing heterodimeric complexes are the same as in homodimeric complexes, the heterodimer traffics and signals independently of its constituent homodimers. The presence of CD4 affects the trafficking of CCR5 containing dimers but not the CXCR4 homodimer. These observations demonstrate the importance of considering receptor heterodimers as distinct signalling entities that should be more carefully and individually characterized.

Oxabicyclic alkenes have been the focus of many synthetic studies as they are versatile compounds which act as synthetic intermediates to produce a variety of useful heterocyclic, carbocyclic and acyclic products. The nucleophilic ring opening reaction of oxabenzonorbornadiene was studied. Methanol was the primary nucleophile used throughout the investigation, however various other alcohol nucleophiles were also tested for their efficacy. The effects of substitution were explored, providing ring opened products in yields of up to 81%. The [2+2] cyclodimerization reaction of oxanorbornadienes was also examined providing the first examples of dimers of this kind. The scope was expanded to include other 2,3-diester oxanorbornadienes as well as various C1 substitutions. Changing the ester moiety did not affect the reaction, however the addition of a C1 alkyl substituent did result in lower yields. Moderate yields of up to 66% were obtained. Additionally, a new ruthenium complex was discovered in the process.

In the past three decades, various steps of the human immunodeficiency virus type 1 (HIV-1) life cycle have been thoroughly studied. Many of these steps, such as viral entry, reverse transcription and proteolysis have been targets of antiretroviral therapy. Retroviral genomic RNA (gRNA) dimerization appears essential for viral infectivity and this process appears to be chaperoned by the nucleocapsid (NC) protein of HIV-1. In this dissertation, the role of NC in genome dimerization and other aspects of the viral life cycle have been thoroughly studied. Various positions of the NC protein have been mutated through site-directed mutagenesis and relevant and dispensable positions of NC have been identified through this method. 34 of its 55 residues were mutated, individually or in small groups, in a panel of 40 HIV-1 mutants. It was found that the amino-terminus, the proximal zinc finger, the linker, and the distal zinc finger of NC each contributed roughly equally to efficient HIV-1 gRNA dimerization. The various mutations introduced into NC show the first evidence that gRNA dimerization can be inhibited by: 1) mutations in the N-terminus or the linker of retroviral NC; 2) mutations in the proximal or distal zinc finger of lentiviral NC; 3) mutations in the hydrophobic patch (plateau) or the conserved glycines of the proximal or the distal retroviral zinc finger. Some NC mutations impaired gRNA dimerization more than mutations inactivating the viral protease, indicating that gRNA dimerization may be stimulated by the NC component of the Gag polyprotein (Pr55gag). In the second section of my work, I studied the effect of Pr55gag processing on gRNA dimerization by introducing rate alternating mutants into Pr55gag protein cleavage sites. I showed that Maturation ofNCp15 into NCp9 is essential for fast rates of genomic RNA dimerization and maturation of NCp9 into NCp7 has no incidence on genomic RNA dimerization but is essential for viral replication. In order to delineate the amount of viral protease activity needed to produce mature virus 48 hours post transfection, we also studied, by cotransfection studies, the effect of various ratios of wild-type (BH10) and protease-inactive (PR- ) plasmids and found that HIV-1 reaches its full genomic RNA dimerization despite 75% unprocessed Pr55gag polyproteins. We have also shown that wild type BH10 plasmid can rescue those mutations in NCp7 protein that have an effect on gRNA dimerization through rescue experiments. Overall, this thesis sheds light on the role of NC in HIV-1 genome dimerization and other aspects of the viral life cycle and identifies the importance of each component of NC during these processes.

Abnormal expression and signaling of the ErbB receptors is associated with the development and progression of several forms of cancer. In this thesis, new ErbB-targeting affibody molecules are evaluated regarding their cellular effects in vitro. Since ligand binding to an ErbB receptor might have an impact on the cell it is important to be aware of these effects as they may have consequences for the continued growth of the tumor when used in vivo. The affibody molecules are intended for tumor targeting with the prospect of clinical use in imaging or therapy. Three types of affibody molecules were studied, HER2-binding, EGFR-binding and bispecific binders that target both EGFR and HER2. The HER2-targeting (ZHER2:342)2 showed promising characteristics. It sensitized SKBR-3 cells to irradiation and decreased cell growth to the same extent as the clinically approved antibody Herceptin. The monomeric version, ZHER2:342, did not induce any large effects on intracellular signaling or biological outcome. This makes (ZHER2:342)2 interesting for therapy purposes, while ZHER2:342 may be better suited for imaging. The bispecific affibody molecules were all able to simultaneously bind to both EGFR and HER2, but none of the six constructs resulted in any large effects on cellular outcome. Interestingly, all three monovalent binders are more functional when positioned at the N-terminal part of the construct and the (S4G)3 linker renders higher affinity of the bispecific binders compared to (G4S)3. Tumors that co-express several ErbB receptors are often more aggressive and associated with a worse prognosis, suggesting that the total ErbB expression pattern might be more informative than the expression level of one receptor regarding cancer prognosis and prediction of response to targeted therapies. Bispecific ligands could thus be used as imaging agents with prognostic value. Another aspect of dual targeting is the possibility of increased tumor specificity since tumors are more likely than healthy tissue to express high amounts of two receptors.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Campus access only. Vita. Bibliography: 176-188.

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. Their extracellular structure is composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of short consensus repeats SCR. The adhesion of cells (expressing ligands) to the endothelium (expressing the selectin i.e., E-selectin) occurs through spatio-temporally regulated interactions that are mediated by multiple intra- and inter-cellular components. Furthermore, selectins play a role beyond fixing cells to a specific location by regulating important signaling pathways in the migrating cell during physiological and pathological processes. These interactions start mainly with the binding of the lectin domain of selectins and ligand on cells. Therefore, structural/functional studies to date have mainly focused on the direct interactions of the lectin domain of E-selectin with its ligands while other domains and conformational dynamics received less attention. For this purpose, we produced a number of different recombinant E-selectin proteins with and without artificial oligomerization and with changes in the SCR units in addition to proteins where strategic residues will be mutated to change the conformation of the selectin to an extended conformer. Moreover, double cysteine mutant candidates were produced for maleimide labeling for the real-time SM-FRET (single molecule fluorescence resonance energy transfer) studies to assess conformational dynamics of E-selectin. Using a comprehensive set of static- and flow-based assays, we concluded that SCR domains play a role by enhancing the interaction of recombinant E-selectin proteins with E-selectin ligand, while dimerization and extension of the lectin domain improve the binding. However, our double cysteine mutants purification and labeling requires further optimization to be utilized to study the conformational dynamics of E-selectin binding to its ligands using SM-FRET and force microscopy. Furthermore, our experiments extend to highlight the importance of phosphatases in regulating signaling pathways that are affected by E-selectin binding to migrating cells. Collectively, these studies are beneficial to understand the mechanistic details of cell adhesion and migration of cells using the established model system of hematopoietic stem cells (HSCs) adhesion to the selectin expressing endothelial cells.

Klotho (KL), an anti-aging protein, attracted interest in the aging field because of the dramatic phenotype of KL deficient mice and its connection to signaling pathways implicated in aging. The KLVS variant consists of the F352V (KLV) and C370S (KLS) substitutions. It was detected in genome wide association studies (GWAS) that linked it to alterations in longevity and disease risk. The molecular mechanism(s) underlying these associations are unknown. To understand how KL increases the risk of age-related diseases, the studies in this dissertation investigated whether expression of the KLVS variant, when compared to wildtype (KLWT), displays differences in processing, protein-protein interactions and enzymatic activity. Differences in processing were evaluated by studying changes in shedding, half-life and plasma membrane localization of KL variants. The decrease in KLV shedding, as measured by the intracellular: extracellular ratio, were explained by a decreased half-life. This decreased half-life is potentially due to decreased KLV plasma membrane localization, which is attenuated by co-expression of dominant negative dynamin, suggesting a role of endocytosis in these differences. To assess whether there are changes in KLVS protein-protein interactions, differences in dimerization were measured by Blue Native gel electrophoresis and cross-linking. KLV dimerization was increased while KLS and KLVS variants decreased dimerization. Co-immunoprecipitation of tagged KL assessed whether these changes were due to alterations in homodimerization. The presence of KLVS in dimers decreased the levels of immunoprecipitated KL suggesting KLVS decreases homodimerization. Changes in heterodimerization of KLVS with fibroblast growth factor receptor (FGFR) 1c were also investigated through co-immunoprecipitation. KLVS increased heterodimerization with FGFR1c. Addition of FGF23, for which KL is a co-receptor, showed that KLVS increases FGF signaling downstream of FGFR1c. To determine differences in enzymatic activity of KLVS, 4-metylumbelliferyl-beta-D-glucuronide was used to measure alterations in glucuronidase activity. Results showed that KLVS had decreased enzymatic activity compared to KLWT. These findings are the first to show that KLVS leads to differences in function as demonstrated by decreased homodimerization and enzymatic activity and increased heterodimerization with FGFR1c. Given the association of KLVS with disease and longevity, these results suggest that these functions are integral in KL's anti-aging role in humans.

Soot formation mechanisms have been a target of intense research for decades. The various stages in the soot formation mechanism have been accepted and recognized, nevertheless, the nucleation stage, which corresponds to the transition from gas phase polycyclic aromatic hydrocarbons (PAH) to condensed particles, is controversial. Pyrene dimerization is considered by many models to be the first step in soot nucleation. In this work, a jet-stirred reactor (JSR) in the temperature range of 700 – 1200 K was used to perform pyrene pyrolysis and to study the various dimerization Nascent particles were chemically analyzed using Fourier-transform ion cyclotron resonance high resolution mass spectrometry (FT-ICR MS) with a laser desorption ionization (LDI) source. Simulations were realized based on a simple kinetic model using CHEMKIN-PRO, which addressed three different dimerization pathways: 1) physical dimerization of two pyrene molecules (P-DIM), 2) physical dimerization between a pyrene molecule and a pyrenyl radical (PR-DIM), and/or 3) chemical dimerization between two pyrenyl radicals (C-DIM). The detected species presented 202 and 402 Da masses in the mass spectra with different intensities. At higher temperatures, the formation rate was enhanced due to the sensibility of particle formation to the reaction temperature. The first temperature regime was identified at 700 – 900 K, where the detected species contained only pyrene molecules, stacked by Van der Waals forces (P-DIM). In the 900–1100 K range, the formation of pyrenyl radicals was considered, and the production of PR-DIM was favored. In the higher temperature range (1100–1200 K), the greater species' mass were located and related to the dimerization of two pyrenyl radicals (C-DIM). The temperature increase was reflected in the production of higher concentrations of the pyrenyl radical, resulting in the dominance of the chemical dimerization pathway at 1200 K. The use of different initial concentrations of pyrene in the simulations did not significantly affect the outcome. Results of the experiment were reflected in the simulations, based on the model used, revealing the tendency of the three dimerization pathways, the decreased survival rate of physically-formed dimers, and the enhanced production of chemically-linked dimers at high temperatures.

The focal adhesion kinase (FAK) and the protein tyrosine kinase 2-beta (PYK2) are two closely related non-receptor tyrosine kinases that link cell adhesion, migration and proliferation, and thus also promote cancer cell invasiveness. FAK and PYK2 have the same domain structure (comprising the FERM, kinase and FAT domains) and possess several overlapping functions, however their cellular roles can be different or even opposing. In particular, PYK2 can be activated by calcium, and has important functions in the brain and neurodegenerative disease. The molecular basis for calcium-based activation of PYK2 is unclear and controversial. In this work we combined biophysical and structural methods to determine the molecular basis for calcium-sensing in PYK2. For this, we investigated the least-studied region of these kinases, namely the long linker (KFL) region between the kinase and FAT domains. This linker is only ~20% conserved between FAK and PYK2, and, therefore, is a prime candidate for causing their differential properties. We find that the linker harbors a helical segment, which is conserved in both FAK and PYK2, and contributes to their dimerization (an important step in their activation). Helix-flanking regions differ between both proteins, and we show that these of PYK2 create a non-canonical dimeric binding site for calcium-bound calmodulin. Calmodulin-binding is synergistic with linker dimerization in PYK2, explaining how calcium influx can be translated into activation of PYK2. Collectively, our work clarifies the capacities for FAK and PYK2 to receive, process and transduce cellular signals, and may provide new opportunities for targeted therapeutic intervention.

Benzothiazole dibenzoic acid derivative (BTDB) is well-known organic linkers utilized for the syntheses of various metal organic frameworks, and demonstrates interesting photophysical properties upon concentration variations in solution. The presence of two carboxylic acid functional groups at each side of the rod-like molecule, facilitates dimerization and oligomerization equilibria. Interestingly, dimers and oligomers have completely different emission behaviors from the monomer of the same species. At a low range of concentration, 0.1 – 64 μM, dimerization process is dominant, and that the equilibrium constant of dimer formation found to be 18,000 M-1. On the other hand, in the 64 – 1000 μM concentration range, oligomerization takes over, and that it results in the formation of a small linear chain of 8 molecules, or 4 dimers, with a high equilibrium constant of 1.2 × 1013 M-3. Various experimental measurements and theoretical calculations have suggested hydrogen-bond formation is the main driving force for the dimerization and oligomerization in the nano- and micro- molar regime, and that structure rigidity of a species is a key factor in controlling its photophysical properties, such as emission quantum yield and excited state lifetime.