published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

The main focus of this thesis is to study the physical characteristics of the N-terminus of human XAF1 protein, a 17kD protein named NTA1, by biophysical methods. Structural studies of the N-terminus of XAF1 serves as a base for the studies of the structure and function relationship of the N-terminus, and the same maybe true for the full length XAF1. Bioinformatics analysis shows that NTA1 shares high sequence identity with the TRAF-type zinc finger domain-containing protein 1 (TRAFD1) and FLN29. Protein structure prediction has been performed on NTA1 by the I-TASSER web server. The prediction result suggests that NTA1 is a structure that consists of α-helices which are joined by flexible linkers. The loose structure shown by I-TASSER is expected to have high solvent accessibility. This coincides with the deuterium exchange data. In addition, by the CD approach, NTA1 was estimated to contain high α-helix content. This result is consistent with the bioinformatics prediction and the secondary structure obtained from the chemical shift index method as well. The physical characterizations of NTA1 showed that NTA1 is a loosely packed protein; and the five zinc ions are bound in the protein structure. Based on the chemical shifts of β-carbons, the Cysteine residues Cys8, Cys11, Cys34, Cys37, Cys50, Cys59, Cys62, Cys86, Cys89 and Cys115 showed a significantly downfield shift, they are probably involved in the zinc ions coordination. The dynamic property of NTA1 was investigated by NMR techniques. Backbone dynamics of NTA1 reveal that NTA1 does not have a typical spherical structure, it is anisotropic. Residues corresponding to the zinc finger regions in the predicted structure show large R2/R1 and S2 values, while regions shown to be flexible linkers in the 3D structure prediction show small R2/R1 and S2 values. Thus, the protein structure homology modeling data are supported by the backbone dynamics data. / published_or_final_version / Chemistry / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

Previous in vitro studies showed that exchange protein directly activated by cyclic AMP 1 (Epac1), which is a cAMP mediator, plays an important role in maintenance of endothelial barrier function. Diabetic retinopathy is characterized by impairment of retinal blood vessel integrity leading to breakdown of blood retinal barrier, retinal hypoxia, and neuronal damage. Here, we hypothesize that Epac1 regulates endothelial permeability and protects retina from the retinal damage associated with diabetes. To test such hypothesis, we first demonstrated that human retinal microvascular endothelial cells (HRMECs) exposed to high glucose concentration at 25 mM or 35 mM showed the decreased Epac1 expression level. Our preliminary data also showed that Epac1-downstream activator, Rap1, a member of Ras GTPase, was also altered by different glucose levels. In addition, retina from type 2 diabetic, db/db, mice also showed the decreased Epac1 expression compared to that of non-diabetic, db/m, mice. To further determine the role of Epac1 in diabetic retinopathy, we made use of Epac1-deficient mice. The pathogenesis of diabetic retinopathy share similar characteristics to that of ischemic retinopathy, such as neuronal cell death, glial reactivity, and glutamate toxicity. Therefore, we used our previous retinal ischemic model, i.e., transient middle cerebral artery occlusion (tMCAO). Firstly, we determined the retinal morphology of Epac1-/- mice under normal condition at 3wks. At 3 wks old, the Epac1-/- retinae showed a significantly decreased thickness of outer plexiform layer (OPL) with a trend of increase in inner nuclear layer (INL) thickness. Interestingly, there were obviously more glutamine synthetase (GS)-positive M?ller cells and protein kinase C (PKC)-α positive rod bipolar cells in INL. In addition, there were more IgG-positive blood vessels in OPL. To further determine whether these phenotypes will lead to more severe retinal damage, Epac1-/- mice were exposed to 2 hours of MCAO followed by 22 hours of reperfusion, which we have previously shown to induce retinal ischemia. There was no obvious difference in retinal thickness and expressions of glial fibrillary acidic protein (GFAP) and GS in the contralateral sides of Epac1+/+ and Epac1-/- retina after tMCAO suggesting that the Epac1-deficiency may be compensated by either protein kinase A (PKA) or Epac2. However, Epac2 level was not altered by Epac1-deficiency by Western blot analysis. The ipsilateral sides of the retina of Epac1+/+ and Epac1-/- after tMCAO also did not show obvious difference in swelling and cell death in inner retina, GFAP, glutamate, GS, nitrotyrosine (NT), and peroxiredoxin 6 (Prx6), suggesting that Epac1-deficiency may have been compensated by other cAMP mediators, such as Epac2. However, Epac2 expression in the ipsilateral side of Epac1+/+ and Epac1-/- retinae was not significantly different, although the activities of Epac and PKA were not determined. Taken together, the Epac1-deficient mice would serve as a useful model to determine the role of Epac1 in retinal development, and to determine the detail mechanisms of pathogenesis of diabetic and ischemic retinopathy. / published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

As a ubiquitous protein-folding helper in bacterial cytosol, SlyD is a peptidylprolyl isomerase (PPIase) of the FK506-binding protein (FKBP) family. It has two important functional domains, the IF (insert-in-flap) domain with chaperone activity and the FKBP domain with PPIase activity. It also possesses a histidine- and cysteine-rich C-terminal metal-binding domain, which binds to selected divalent metal ions (e.g. Ni2+, Zn2+) and is critical for participation in metal trafficking for metalloenzymes. SlyD from Helicobacter pylori was investigated both structurally and functionally by a variety of biophysical, biochemical and molecular biology techniques. HpSlyD was cloned, expressed and purified. It binds to Ni2+ and Zn2+ with dissociation constants (Kd) of 2.74 and 3.79 μM, respectively. Both Ni2+ and Zn2+ can competitively bind to HpSlyD. The C-terminus was demonstrated to convey nickel resistance in vivo. It also binds to Bi3+ with Kd of 4.4 × 10-24 M. Furthermore, Zn2+, Cu2+ and Bi3+ can induce the dimerization or oligomerization of HpSlyD. The solution structure of the C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC) was determined by NMR, which demonstrates that HpSlyDΔC folds into two well-separated, orientation-independent domains. Both the FKBP and IF domains fold into a structure consisting of a four-stranded antiparallel β-sheet and an α-helix. Binding of Ni2+ instead of Zn2+ induced the conformational changes in FKBP domain, where the active sites are positioned, suggesting a regulatory role of nickel on the function of HpSlyD. It was also confirmed that HpSlyD can associate with the Tat (twin-arginine translocation) signal peptide from small subunit of [NiFe] hydrogenase (HydA), an accessory protein HpHypB for [NiFe] hydrogenase mainly by the IF domain. Surprisingly HpSlyD was found to form a complex with HpUreE, a urease chaperone, indicative of the “cross-talk” between [NiFe] hydrogenase and urease. The possible mechanism of HpSlyD for the cooperation with HpHypB was also explored. In the presence of different metal ions, HpSlyD was shown to regulate the GTPase activity of HpHypB, implicating the possible metal transfer induced by HpSlyD. It was suggested that HpSlyD modulates the nickel insertion of [NiFe] hydrogenase by controlling the GTPase activity of HpHypB. In this thesis, the SlyD protein from H. pylori was shown as an important regulator for the activation of both [NiFe] hydrogenase and urease. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

In flowering plants, pollen grains are surrounded by extremely strong outer walls providing solid and firm structure for protecting pollen and species-specific interactions with female stigma. The outer wall of pollen, referred to as exine, is composed of sporopollenin polymer, but the composition and synthesis of sporopollenin remains poorly understood. Previous studies have indicated that several genes such as Fatty Acyl-CoA Synthetase (ACOS5), Polyketide Synthases (PKSA and PKSB), and Tetraketide α-Pyrone Reductase (TKPR1) take part in the biosynthesis of sporopollenin in Arabidopsis thaliana. The existence of ancient biochemical pathways for sporopollenin biosynthesis has been widely proposed but experimental evidence from plant species other than Arabidopsis is not extensively available. In this study, two homologous PKS genes, NtPKS1 and NtPKS2, were found in tobacco (Nicotiana tabacum). Results of RT-PCR and in situ hybridization revealed that NtPKS1 and NtPKS2 are specifically and transiently expressed in tapetal cells during microspore development in tobacco anthers. RNAi plants of NtACOS1 and NtPKS1 were investigated. Comparing with wild-type tobacco (SR1), abnormal pollens, defect exine structure, and male sterility were found in the RNAi lines. Enzymatic assays show that NtPKS1 and NtPKS2 encode anther-specific enzymes using fatty acyl-coenzyme A and p-coumaroyl coenzyme A as substrates to yield tri- and tetra- ketide α-pyrone and bisnoryangonin respectively. In this study, the metabolic steps catalyzed by the anther-specific acyl- CoA synthetase (ACOS), polyketide synthase (PKS), and tetraketide α-pyrone reductase (TKPR) were investigated. Using fatty acids as starting substrates, sequential activities of heterologously-expressed tobacco enzymes NtACOS1, NtPKS1, and NtTKPR1 resulted in the production of reduced tetraketide α- pyrones which propose to contribute to the biosynthesis of sporopollenin precursors in tobacco. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

The ability to sense and rapidly respond to oxygen availability is crucial to the survival and physiology of facultative anaerobes. In many gram negative bacteria such as Escherichia coli, this process is primarily controlled by the dimeric, [4Fe〖-4S]〗^(2+) containing global transcription factor FNR, which regulates transcription of genes necessary for the anaerobic metabolism. Activity of FNR is directly regulated by the presence of oxygen, which inactivates FNR by oxidizing the [4Fe〖-4S]〗^(2+) cluster and causing the dissociation of the FNR dimer. Although the biological function of FNR has been well established, structural and biochemical characterization of the FNR dimer has been limited due to its extreme lability to oxygen. In the current study, I conduct molecular engineering on FNR protein and obtain oxygen stable variants that are suitable for in vitro biochemical studies. By combining several approaches including covalently linking two FNR monomers using a flexible peptide linker, amino acid substitutions to promote dimerization, and removal of protease recognition sites to prevent proteolysis, a series of FNR variants which are potentially active in the presence of oxygen are constructed. Various in vivo and in vitro assays led to the identification of the construct (FNRD154A)2 which covalently links two copies of FNRD154A, an FNR variant that has greater dimerization capability, in tandem displays significantly improved transcription regulation and DNA binding to various FNR regulated promoters in the presence of O2. Circular Dichroism analysis showed that this variant maintains a similar secondary structure as that of native FNRD154A and in vivo transcription assay demonstrated that this protein retains other properties of the native FNR dimer including [4Fe〖-4S]〗^(2+) cluster binding, oxygen sensing, and capability to support the anaerobic growth of E. coli. All these together led the conclusion that an FNR variant that retains structural and functional properties of native FNR has been constructed, but with significantly improved O2 stability. Thus, it has the potential to be widely used in various biochemical and structural studies of FNR in the presence of oxygen. In addition to the major project of molecular engineering of FNR protein, in this thesis, I also initiated the study of using metabolomics approaches to identify the cellular substrates of the multidrug efflux pump MdtEF. MdtEF is an important efflux pump in E. coli and its expression has been shown to be induced under a number of stressed conditions. It is thus proposed to have a general detoxification function in E. coli, but the cellular substrates it expels have not been identified. In this study we established and applied metabolite profiling on the wild type and ΔmdtEF E. coli strains and confirmed that indole red, a metabolic by-product formed during anaerobic respiration of nitrate, is one of the cellular substrates of MdtEF under anaerobic conditions. This study provides a general methodology to identify endogenous substrates of efflux pumps and contributes to the understanding of the physiological roles of multidrug efflux pumps in bacteria. / published_or_final_version / Biological Sciences / Master / Master of Philosophy