Aryl Hydrocarbon Receptor (AhR) mediates drug and toxin action. The AhR proteins have been characterised in several mammalian species, and are soluble proteins found in various tissues. The AhR is normally found in the cytoplasm in a complex with 90 KDa heat shock protein (hsp90) and cellular chaperones such as ARA9 (AIP or XAP2) and p23. However, there has not been a systematic analysis of the proteins which chaperone the AhR ligand-binding domain (LBD). This work investigates the interaction between ligands and the AhR, the protein composition of the AhR ligand-binding domain (LBD) complex, by establishing translation of AhR LBD in reticulocyte lysate, which contains molecular chaperones such as hsp90, and p23 that stabilise the ligand binding form of AhR. EGFP (Enhanced Green Fluorescent Protein) has been coupled to the mouse AhR b-1 LBD, to enable fluorescence analytical techniques of ligand-binding to the AhR. The Glutathione S-Transferase (GST) affinity tag was fused to EGFP (GST-EGFP), then fused to AhR.LBD with one or two EGFP (GST-EGFP-AhR.LBD and GST-EGFP-AhR.LBD-EGFP) to enable rapid one-step purification of AhR fusion proteins, and associated chaperone proteins. Proteins were expressed in E.coli (BL21(DE3)plysS). The GST protein is soluble, and not fluorescent, and GST-EGFP and GST-EGFP-AhR.LBD-EGFP was soluble and fluoresecent. The GST-EGFP-AhR.LBD was an insoluble fluorescent protein. Thus, the AhR proteins were purified from bacteria to test the specificity of the pulldown system under conditions which do not yield functional Ah Receptor. The tagged AhR constructs were translated with [35S]-methionine in reticulocyte lysate and translation products were ~36, 66, 84 and 109 kDa on SDS-PAGE. Reticulocyte lysate programmed with GST-EGFP-AhR.LBD and GST-EGFP-AhR.LBD-EGFP both bound ~800 d.p.m 2,3,7,8-[1,6-3H]tetrachlorodibenzo-p-dioxin (a ligand for the AhR), while lysate programmed with GST.EGFP showed binding of ~15 d.p.m, indistinguishable from unprogrammed lysate. The AhR proteins were purified from reticulocyte lysate and subsequent pulldown experiments will enable proteomic analysis of the proteins associated with the AhR LBD.

Variation in the calpain-10 gene has been linked to a three-fold increased risk for type 2 diabetes in Pima Indian and some European populations. Furthermore, reduced skeletal muscle expression of calpain-10 is associated with reduced insulin mediated glucose disposal and carbohydrate oxidation. The skeletal muscle specific calpain-3 plays a key role in skeletal muscle integrity and has also been linked to insulin resistance in humans and rodents. The major aims of this thesis were to 1) investigate the hypothesis that alterations in insulin sensitivity in healthy humans would lead to significant changes in the mRNA and protein expression of calpain-10 and -3, 2) investigate the effect of hyperinsulinaemia and lipid availability on calpain-10 and -3 expression, 3) further address the role of genetic variation in the calpain-10 gene on glucose utilisation in humans and finally 4) investigate the expression of calpain-10 in skeletal muscle of type 2 diabetic patients. The studies in this thesis show for the first time that insulin resistance as a result of short term fasting or high fat availability is not associated with changes in calpain-10 and -3 mRNA and protein expression, providing evidence against an adaptive role for these genes in the development of fasting- and lipid-induced insulin resistance.

Human embryonic stem cells (hESCs) are thought to have enormous potential for use in regenerative medicine, whilst simultaneously allowing us insights into human embryonic development, disease modelling and drug discovery. Differentiation to mesodermal lineages, such as cardiomyocytes and blood, may allow for improved treatment of cardiac and haematopoietic diseases. hESC-derived immune cell types may also allow the circumnavigation of the immune barrier. This thesis aims to test the hypothesis that formation of hESC derivatives is regulated by the same mechanisms and ontology as in vivo embryo development. Therefore, by identifying and facilitating the mechanisms of mesoderm induction, hESC differentiation can be optimised to maximise the production of mesoderm, and, ultimately, mesoderm derivatives. Using a Xenopus laevis animal cap model with simultaneous treatment with activin B or fgf4, together with tall, Im02 and gatal mRNA, resulted in substantial increases in mesodermal, haemangioblast and erythropoietic cell markers. One of the most successful methods for hESC differentiation is by the formation of human embryoid bodies (hEBs). To reduce first the number of variables in current mass culture protocols for hEB formation, such as hEB size, a forced aggregation system was established that produced homogeneous hEBs from defined numbers of cells. This system was then optimised to enhance production beating cardiomyocytes by varying the number of hESCs used for hEB formation and also the number of days in culture. This system was assessed in four hESC lines and demonstrated substantial inter-line variability in cardiomyocyte production (1.6± 1.0% to 9.5±0.9°0). Differentiation was also performed using chemically defined media (CDM) with the addition of actiyin A and FGF2 and resulted in 23.6±3.6% of hESs producing beating cardiomyocytcs. In addition immunohistochemistry was performed to assess the relationship of cells expressing markers for mesoderm, pluripotency, ectoderm, and endoderm to establish a standard spatial and temporal map of hEB differentiation.

Thesis--University of Michigan. / Includes bibliographical references.

Thesis (Ph. D)--Guo li zheng zhi da xue, 1977. / Reproduced from ms. copy. Bibliography: p. 305-337.

Thesis (Ph. D.)--Yale University, 1981. / Typescript. Includes bibliographical references (v. 1, p. 294-324).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1973. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

This thesis comprises of the study performed on the extraction, isolation and structural characterisation of macromolecular components from the three members of the family Cucurbitaceae. In particular, the polysaccharides from C.moschata, C. maxima and C. pepo (butternut squash, zucchini and pumpkin, respectively) and oil bodies from the seeds of C. pepo are selected on the basis of their antidiabetic potential. The study centred around structural characterisation of the polysaccharides using hydrodynamical methods such as analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium), viscometry and dynamic light scattering followed by the use of gas chromatography and gas chromatography coupled with mass spectrophotometry for the assessment of monosaccharide composition. Bioactivity of these polysaccharides was also examined using complement fixation assay. Pumpkin seed oil bodies were extracted, isolated and characterised under various laboratory conditions to establish the zeta potential and size distribution of oil bodies in the solvent provided. Although the selection of the biomaterial for this study from the three species was based on their antidiabetic potential, other health benefits and practical applications are also associated with them. For example, the characterisation of these macromolecules could act as a stepping stone for the future investigation in therapeutics. These biomaterials can potentially be used in the pharmaceutical industry to act as a drug themselves or can be used as a part of any formulation or otherwise can be used as a nutraceutical compound.

The global cost of heart failure is USD$45 billion and set to double in the next 15 years. The only method of treatment is heart transplant but demand far exceeds supply and is projected to increase. Meanwhile, global pharmaceutical development has been hindered by poor drug development success rates. Of the drugs that make it to phase I clinical trials, only 8 % pass phase III and existing drug screens do not always accurately predict or detect adverse cardiac events. Cardiotoxicity is the underlying reason for 26 % of safety related drug withdrawals between 1990-2006. Therefore, a source of human cardiomyocytes (CMs) is required to fill the need for regenerative medicine and drug screening applications. Differentiation of human pluripotent stem cells (hPSCs) to CMs is a viable solution to this bottleneck but the number of cells required is staggering; up to 5 million novel compounds are registered annually by pharmaceutical and academic institutions, while cell replacement studies in primates suggest that 10 billion CMs will be required per patient to repair the damaged myocardium post infarction. The objective of this thesis was to evaluate whether automated high throughput manufacture of hPSCs and CMs was possible, and to demonstrate that hPSC-CMs could be used in automated high throughput drug screening by carrying out assays in 384-well plates. This thesis started by carrying out three manual differentiation methods; an embryoid body (EB) based method and two monolayer methods. Batch variability in mouse embryonic fibroblast conditioned medium (MEF-CM) led to erratic and variable differentiation outcomes (as high as 94+/-0.3 % to as low as 25.6+/-39.7 % beating EBs per 96 well plate). Two monolayer methods, using defined media (mTeSR and E8) increased cell yields by up to 12-fold and 65-fold respectively and simplified the process technically. When these methods were automated, EB differentiation failed to generate spontaneously beating EBs, whereas both monolayer methods succeeded in generating spontaneously beating cardiomyocytes of purities >90 %. Finally, cryopreserved stocks of hiPSC-CMs produced by automation were used to evaluate whether cardiotoxicity from the anticancer drug doxorubicin could be decreased by co-treating with dexrazoxane (an existing doxorubicin cardio-protectant), carvedilol (a β-blocker), sildenafil (a vasoactive agent) and isoprenaline (a β-adrenoreceptor agonist). This was carried out in a real-time, fully automated assay setup to monitor induction of apoptosis by the marker propidium iodide using the Operetta confocal plate reader. The concentration of doxorubicin that led to 50 % hiPSC-CM death (TD50) was significantly reduced by co-treatment with dexrazoxane, carvedilol and sildenafil. Carvedilol showed the highest level of cardioprotection by increasing TD50 of doxorubicin by 7.5-fold. In contrast, isoprenaline reduced TD50 of doxorubicin, suggesting that isoprenaline would be contraindicated in patients undergoing doxorubicin treatment. Thus, this thesis demonstrated that automated differentiation of cardiomyocytes was technically feasible with capability of generating high yields (up to 39 million cells per flask) and high purity (>90 %) cardiomyocytes. Furthermore, this system was compatible with high content assays in 384-well plates for evaluating drug toxicity.

Introduction: The emergence of antibiotic resistance has led to the continuing search for discovery of effective antibiotics. Host defence peptides (HDPs) confer defence mechanisms against infection and investigation of their specific roles and interplays are ongoing. Among the HDPs, defensins are a group of effector molecules which plays important roles in humans. Although several stereotypes of human beta defensins (hBDs) such as the hBD1-3 are well studied, other members including the human beta defensin 9 (hBD9), are not entirely known. Understanding the properties of these HDPs will enable us to discover a safe and efficacious, broad-spectrum and resistance-free antibiotic for therapeutic application in the future. Purpose: The purpose of this study is to clone the DEFB109 gene, express and purify the hBD9 propeptide, before determining the hBD9 propeptide antimicrobial property using a recombinant system in Escherichia coli. Methods: The second exon of the DEFB109 was amplified through reverse transcription polymerase chain reaction (RT-PCR) and inserted into selected plasmid vectors. The recombinant plasmid construct was cloned, and transformed into E coli expression host. The correctly transformed colonies were selected before the plasmid constructs were purified and verified through nucleotide sequencing. Expression and purification of the hBD9 propeptide were carried out and antimicrobial property of the peptide was investigated. Result: HBD9 fusion protein was successfully expressed and purified. It was shown to have antimicrobial efficacy against Staphylococcus aureus and Pseudomonas aeruginosa. The effect of the free hBD9 propeptide against wider spectrum of organisms needs to be studied in the future.