Doctor of Philosophy

dissertationTransition metal catalyzed [2+2+2] cycloaddition reactions are an important class of reactions that provide the means for the rapid construction of various carbocyclic and heterocyclic compounds. Over the past several years, our efforts have been focused on exploring metal catalyzed cycloaddition reactions to find new methods and improve existing methods to access heterocyclic core structures such as pyridines, pyridones and pyrimidones. Enynes and isocyanates in the presence of a nickel-based catalyst system undergoes cycloaddition affording E- and the Z-dienamides in moderate to good yields with the Edienamide being the major product. The substrate scope with respect to isocyanate and enyne structures was also determined. It was observed that aryl as well as alkyl isocyanates undergo this cycloaddition reaction. Internal enynes afforded the dienamide products while terminal enynes afforded lactams. A new catalytic system involving iron acetate and a sterically hindered bis(aldimino)pyridyl ligand was also developed. This Fe-complex catalyzed the cycloaddition reaction of alkynenitriles and alkynes to afford pyridines in moderate to good yields. Symmetrical and unsymmetrical exogenous alkynes can be used in this cycloaddition reaction. Alkyl, aryl, and terminal alkynenitrile afford good yields of the pyridine products. Five- and six-membered fused pyridines can be synthesized in good yields by this methodology. The synthesis of 2-aminopyridines by the cycloaddition reaction of diynes and cyanamides in the presence of an iron catalyst system has also been studied. The Fecatalytic system is a combination of iron chloride and a bis(aldimino)pyridyl ligand and it leads to good to excellent yields of desired product. Five- and six-membered fused 2-aminopyridines were prepared in good yields by utilizing this methodology. Various Nalkyl- alkyl, N-aryl-aryl, and N-alkyl-aryl cyanamides undergo this cycloaddition reaction with diynes to afford 2-aminopyridines in good yields

Regulation of apoptosis by the Epstein-Barr virus latent membrane protein LMP1

The Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is implicated in the aetiology of African Burkitt’s lymphoma (BL) and several other cancers of lymphoid/epithelial origin. In vitro, expression of all of the eleven EBV latent proteins or just the oncogenic Latent Membrane Protein 1 (LMP1) can protect BL cells from apoptosis by several stimuli including growth factor deprivation and this is related to the ability to upregulate the expression of several anti-apoptotic 6c/-2-related genes. In this study, it is shown that EBV latent gene expression and in particular LMP1 can induce the upregulation of expression of the anti-apoptotic gene bfl-1 in addition to bcl-2 in BL cells and also that Bfl- 1 expression can not onlt protect BL cells from serum-deprivation-induced apoptosis but also exert a proliferative effect under these culture conditions Bfl-1 has been shown by others to suppress apoptosis and exhibits proliferative and potent cooperative transforming properties. Both increased mRNA stability and increased promoter activity were found to contribute to the effect of LMP1 on bfl-1 expression. The transcription factor NF-kB was shown to mediate the effect of LMP1 on bfl-1 promoter activity in both BL and T cells however, celltype- dependent differences in the regions of the promoter targeted by LMP1 were observed CD40, which engages similar signaling proteins (TRAFs) as LMP1, also stimulates bfl-1 mRNA expression, however, mechanistic similarities and differences were found between the two TNFR family members in this effect LMP1 could also be demonstrated to contribute to activation of the bfl-1 promoter in EBV-immortahsed B lymphoblastoid cell lines (LCLs), however, NF-kB activation may not be the only mechanism regulating bfl-1 promoter activity in different LCLs. A minor but significant finding in this study is that both transcriptional and translational mechanisms are involved in the LMPl-mediated upregulation of bcl-2 expression, with the latter exerting a stronger effect. The upregulation of bfl-1 expression by LMP1 represents the first example of regulation of expression of this anti-apoptotic gene by a viral protein and extends the list of anti-apoptotic proteins whose expression is controlled by LMP1. Protection against apoptosis constitutes an important part of the latent phase of the EBV life cycle, in that it provides a mechanism to ensure life-long viral persistence in the host by mimicking the natural process of selection of B cells into the memory compartment. The regulation of expression of bcl-2-related genes by LMP1 provides an important link between EBV and its cellular survival and growth transforming properties

DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur

Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation

Improved Outcomes After Autologous Hematopoietic Cell Transplantation for Light Chain Amyloidosis: A Center for International Blood and Marrow Transplant Research Study

Autologous hematopoietic cell transplantation, or autotransplantation, is effective in light-chain amyloidosis (AL), but it is associated with a high risk of early mortality (EM). In a multicenter randomized comparison against oral chemotherapy, autotransplantation was associated with 24% EM. We analyzed trends in outcomes after autologous hematopoietic cell transplantation for AL in North America

Indoor position tracking using received signal strength-based fingerprint context aware partitioning

Mobile indoor localisation has numerous uses for logistics and health applications. Current wireless localisation systems experience reliability difficulties in indoor environments due to interference and also require a large number of wireless access points to ensure position accuracy and resolution. Localisation using wireless channel propagation characteristics, such as radio-frequency (RF) receives signal strength are subject to wireless interference. The Fingerprint Context Aware Partitioning (FCAP) tracking model used received RF signal strength fingerprinting, combined with context aware information about the user's indoor environment. The authors show the use of context aware information in the FCAP model, reduces the effect of wireless interference and lowers the spatial density of access points required. The wireless localisation network consisted of reference nodes placed at locations in a building. Reference nodes are used by mobile nodes, to localise a user's position. The authors tested the FCAP model in a typical indoor environment and compared the performance and accuracy to other received signal strength indicator fingerprint localisation methods. They found the FCAP model had improved performance and was able to achieve a similar accuracy to other protocols, with fewer reference nodes

Gold-catalyzed oxidation of propargylic ethers with internal C–C triple bonds: Impressive regioselectivity enabled by inductive effect

Inductive perturbations of C-C triple bonds are shown to dictate the regiochemistry of gold-catalyzed oxidation of internal C-C triple bonds in the cases of propargylic ethers, resulting in highly regioselective formation of β-alkoxy-α,β-unsaturated ketones (up to >50/1 selectivity) via α-oxo gold carbene intermediates. Ethers derived from primary propargylic alcohols can be reliably transformed in good yields, and various functional groups are tolerated. With substrates derived from secondary propargylic alcohols, the development of a new P,N-bidentate ligand enables the minimization of competing alkyl group migration to the gold carbene center over the desired hydride migration; the preferred migration of a phenyl group, however, results in efficient formation of a α-phenyl-β-alkoxy-α,β-unsaturated ketone. These results further advance the surrogacy of a propargyl moiety to synthetically versatile enone function with reliable and readily predictable regioselectivity

The sigmoid take-off: An anatomical imaging definition of the rectum validated on specimen analysis

Background A pre-operative imaging landmark to define the rectum would optimise clinical care of rectal cancer patients and research efforts to improve outcomes. The sigmoid take-off has been suggested as an imaging landmark for the rectosigmoid junction (RSJ). This study aimed to investigate whether this imaging definition of the rectum was validated by surgical specimen analysis. Methods This prospective study recruited 20 patients undergoing surgery and undertook radiological and pathological analysis of their rectal specimens. The radiological landmark of the sigmoid take-off was identified on pre-operative magnetic resonance imaging (MRI), and the distance to the anterior peritoneal reflection was measured by two readers. After surgery, the distance from the beginning of the sigmoid mesocolon to the anterior peritoneal reflection to the beginning of the sigmoid mesocolon on the specimen was measured, and compared to the distance on MRI using Pearson's Correlation Coefficient and Bland-Altman plots. Results In 17 patients, the mean distance from the anterior peritoneal reflection to the RSJ on MRI was 20.3 mm and 23.1 mm for two readers, and on pathology was 20.6 mm. The mean differences between MRI and specimen measurements were −0.31 mm (−2.83 to 2.20 mm), and 2.51 mm (95% confidence interval −0.31 to 5.33 mm) for each reader, with correlation coefficients of 0.77 and 0.81. Conclusion The sigmoid take-off has been validated on specimen analysis to be an imaging landmark that defines the termination of the rectum. This anatomical landmark can be used to classify tumours and guide treatment and research of sigmoid colon and rectal cancer

Nuclear Factor κB-Dependent Activation of the Antiapoptotic bfl-1 Gene by the Epstein-Barr Virus Latent Membrane Protein 1 and Activated CD40 Receptor

Suppression of the cellular apoptotic program by the oncogenic herpesvirus Epstein-Barr virus (EBV) is central to both the establishment of latent infection and the development of EBV-associated malignancies. We have previously shown that expression of the EBV latent membrane protein 1 (LMP1) in Burkitt's lymphoma cell lines leads to increased mRNA levels from the cellular antiapoptotic bfl-1 gene (also known as A1). Furthermore, ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type 1 infection protects against apoptosis induced by growth factor deprivation (B. N. D'Souza, M. Rowe, and D. Walls, J. Virol. 74:6652-6658, 2000). We now report that LMP1 drives bfl-1 promoter activity through interactions with components of the tumor necrosis factor receptor (TNFR)/CD40 signaling pathway. We present evidence that this process is NF-κB dependent, involves the recruitment of TNFR-associated factor 2, and is mediated to a greater extent by the carboxyl-terminal activating region 2 (CTAR2) relative to the CTAR1 domain of LMP1. Activation of CD40 receptor also led to increased bfl-1 mRNA levels and an NF-κB-dependent increase in bfl-1 promoter activity in Burkitt's lymphoma-derived cell lines. We have delineated a 95-bp region of the promoter that functions as an LMP1-dependent transcriptional enhancer in this cellular context. This sequence contains a novel NF-κB-like binding motif that is essential for transactivation of bfl-1 by LMP1, CD40, and the NF-κB subunit protein p65. These findings highlight the role of LMP1 as a mediator of EBV-host cell interactions and may indicate an important route by which it exerts its cellular growth transforming properties