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Neurotrophic factors are proteins involved in the maturation, differentiation and survival of neurons. Due to their neuroprotective properties, they have been regarded as potent candidates for the treatment of neurodegenerative diseases. Recently, a novel family of neurotrophic factors was discovered comprising mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF). These factors have been shown to protect against the degeneration of nigrostriatal dopaminergic neurons in mammalian models of Parkinson's disease, however their neuroprotective mechanisms of action are not yet understood. Although distinct in vertebrates, MANF and CDNF constitute a single homolog in invertebrates. In the present study, we have characterized the in vivo expression and function of the C. elegans homolog manf-1. We have shown that manf-1 is not essential for neuronal development, however when knocked down, mutants exhibit enhanced age-related dopaminergic neuronal degeneration accompanied by an increase in the endogenous ER stress response. Loss of manf-1 function also results in enhanced alpha-synuclein expression and aggregation, a pathological hallmark of Parkinson’s disease. / Thesis / Master of Science (MSc)

An alternative gene therapy method, non-autologous somatic-gene therapy, is the use of a genetically modified universal cultured cell line that can be implanted into different allogeneic recipients. When used as recombinant cells in microcapsules, myoblasts possess several advantages over other cell types, namely their ability to terminally differentiate thus preventing overcrowding within the capsular space. However, encapsulated myoblasts demonstrate decreased proliferation and myogenic differentiation when compared to unencapsulated myoblasts due to the unnatural capsule environment. This study aims to improve the microcapsule environment by incorporating basic fibroblast growth factor (bFGF) and insulin-like growth factor-11 (IGF-11) and the extracellular matrix materials, collagen, laminin-1 and merosin (laminin-2) within the microcapsules in an attempt to mimic the natural surrounding required for myoblast growth and differentiation. While bFGF lead to significant increases in encapsulated myoblast proliferation, it did not appear to be an ideal choice for optimizing the microcapsule environment due to its inhibitory effect on differentiation and the relative cost in therapeutic delivery of proteins. Both merosin and the combination of laminin and merosin together provide a better alternative for increasing myoblast growth and survival within microcapsules since they have no apparent inhibitory effect on myogenic differentiation, and produce similar proliferative results seen when using bFGF. In terms of differentiation, the addition of IGF-11 to the microcapsules or the use of a myoblast cell line overexpressing IGF-11, aid in increasing the myogenic differentiation of encapsulated myoblasts, however, differentiation levels still do not approach those seen in unencapsulated myoblasts. The positive results obtained with the growth factors and matrix materials employed in this study are important steps towards the optimization of microcapsules by improving both the proliferation and differentiation of encapsulated myoblasts. However, more study is needed to elucidate possible solutions to the continued problem of decreased differentiation of myoblasts within APA microcapsules in order to achieve myogenic differentiation that is comparable to what is seen in unencapsulated myoblasts. / Thesis / Master of Science (MS)

Improved corneal epithelial cell growth over artificial cornea materials is required to improve device retention within the eye. In this work, varying concentrations of epidermal growth factor (EGF), a potent mitogen for epithelial cells, were immobilized to polydimethylsiloxane (PDMS) substrates, and the cellular response was analyzed. Three methods were developed to bind EGF to PDMS via polyethylene glycol (PEG) tethers. 1) Plasma Modification: EGF was first reacted with homobifunctional NHS2PEG and then bound to allylamine plasma-modified PDMS. 2) Hydrosilylation: PDMS was modified with heterobifunctional allyl-PEG-NBS and then EGF was attached to the surface-bound PEG. 3) Thiol Modification: EGF was first reacted with heterobifunctional NHS-PEG-maleimide and then bound to thiol-modified PDMS. Using Method 1 (Plasma Modification), 40 to 90 ng/cm2 of EGF was bound, however 70% of this was adsorbed even under optimized EGF-PEG reaction conditions. Cells rapidly grew to confluence on these surfaces, and cell counts increased significantly compared to control surfaces. Extracellular matrix protein production was also increased on the EGF-modified surfaces, corresponding to significantly higher levels of cell adhesion observed under a detachment force. Modification by Method 2 (Hydrosilylation) resulted in 10 to 300 ng/cm2 of bound EGF, of which 20% was adsorbed. However, despite increased EGF binding homogeneity, the cell growth was slower on these surfaces than on those prepared by Method 1, and coverage was non-uniform at all EGF concentrations. This is likely due to a higher underlying PEG density, and binding of the PEG and EGF in clusters on the surface. Simultaneous tethering of the cell adhesion peptide YIGSR had no further effect on cell coverage. Using Method 3 (Thiol Modification), 24 to 65 ng/cm2 of EGF was bound, of which 22% was adsorbed. This method enables more homogeneous EGF surface binding than Method 1, with a lower PEG density than Method 2. However, free thiol groups were inhibitory to corneal epithelial cell growth, even in the presence of bound EGF. Defunctionalization of free thiols by reaction with 3-maleimidopropionic acid restored cell growth and morphology on the PDMS, and may hence allow for retention of the proliferative effect of the EGF. These results indicate that while tethering of EGF to PDMS can improve the coverage by corneal epithelial cells, and presents a promising strategy for modification of polymeric artificial cornea materials, the effects are highly dependent on the underlying surface chemistry. / Thesis / Doctor of Philosophy (PhD)

Microarray gene expression data for thousands of genes in many organisms is quickly becoming available. The information this data can provide the experimental biologist is powerful. This data may provide information clarifying the regulatory linkages between genes within a single metabolic pathway, or alternative pathway routes under different environmental conditions, or provide information leading to the identification of genes for selection in animal and plant genetic improvement programs or targets for drug therapy. Many analysis methods to unlock this information have been both proposed and utilized, but not evaluated under known conditions (e.g. simulations). Within this dissertation, an analysis method is proposed and evaluated for identifying independent and linked metabolic pathways and compared to a popular analysis method. Also, this same analysis method is investigated for its ability to identify regulatory linkages within a single metabolic pathway. Lastly, a variant of this same method is used to analyze time series microarray data. In Chapter 2, Factor Analysis is shown to identify and group genes according to membership within independent metabolic pathways for steady state microarray gene expression data. There were cases, however, where the allocation of all genes to a pathway was not complete. A competing analysis method, Hierarchical Clustering, was shown to perform poorly when negatively correlated genes are assumed unrelated, but performance improved when the sign of the correlation coefficient was ignored. In Chapter 3, Factor Analysis is shown to identify regulatory relationships between genes within a single metabolic pathway. These relationships can be explained using metabolic control analysis, along with external knowledge of the pathway structure and activation and inhibition of transcription regulation. In this chapter, it is also shown why factor analysis can group genes by metabolic pathway using metabolic control analysis. In Chapter 4, a Bayesian exploratory factor analysis is developed and used to analyze microarray gene expression data. This Bayesian model differs from a previous implementation in that it is purely exploratory and can be used with vague or uninformative priors. Additionally, 95% highest posterior density regions can be calculated for each factor loading to aid in interpretation of factor loadings. A correlated Bayesian exploratory factor analysis model is also developed in this chapter for application to time series microarray gene expression data. While this method is appropriate for the analysis of correlated observation vectors, it fails to group genes by metabolic pathway for simulated time series data. / Ph. D.

Five new single-stage power factor correction (PFC) techniques are developed for single-phase applications. These converters are: Integrated single-stage PFC converters, voltage source charge pump power factor correction (VS-CPPFC) converters, current source CPPFC converters, combined voltage source current source (VSCS) CPPFC converters, and continuous input current (CIC) CPPFC converters. Integrated single-stage PFC converters are first developed, which combine the PFC converter with a DC/DC converter into a single-stage converter. DC bus voltage stress at light load for the single-stage PFC converters are analyzed. DC bus voltage feedback concept is proposed to reduce the DC bus voltage stress at light load. The principle of operations of proposed converters are presented, implemented and evaluated. The experimental results verify the theoretical analysis. VS-CPPFC technique use a capacitor in series with a high frequency voltage source to achieve the PFC function. In this way, the input inductor is eliminated. VS-CPPFC AC/DC converters are developed, and their performance is evaluated. VS-CPPFC electronic ballasts with and without dimming function are also presented. The average lamp current control with duty ratio modulation is developed so that the lamp operates in constant power with a low crest factor over the line variation. The experimental results verify the CPPFC concept. CS-CPPFC technique employs a capacitor in parallel with a high frequency current source to obtain the PFC function. The unity power factor condition and principle of operation are analyzed. By doing so, the switch has less switching current stress, and deals only with the resonant inductor current. Design considerations and experimental results of the CS-CPPFC electronic ballast are presented. VSCS-CPPFC technique integrates the VS-CPPFC with the CS-CPPFC converters. The circuit derivation, unity power factor condition and design considerations are presented. The developed VSCS-CPPFC converters has constant lamp operation, low crest factor with a high power factor even without any feedback control. CIC-CPPFC technique is developed by inserting a small inductor in series with the line rectifier for the conceptual VS-CPPFC, CS-CPPFC and VSCS-CPPFC circuits. The circuit derivation and its unity power factor condition are discussed. The input current can be designed to be continuous, and a small line input filter can be used. The circulating current in the resonant tank and the switching current stress are minimized. The average lamp current control with switching frequency modulation is developed, so the developed electronic ballast operates in constant power, low crest factor. The developed CIC-CPPFC electronic ballast has features of low line input current harmonics, constant lamp power, low crest factor, continuous input current, low DC bus voltage stress, small circulating current and switching current stress over a wide range of line input voltage. / Ph. D.

This study examined the relationship between self-leadership and personality through an analysis and comparison of hierarchical factor structures. More specifically, this study examined the relationships between the self-leadership dimensions of behavior-focused strategies, natural reward strategies, and constructive thought strategies, and the personality dimensions of extraversion, emotional stability, and conscientiousness. The results of the study provide evidence that the self-leadership dimensions are distinct from, yet related to, the specified personality traits. The hypothesis that self-leadership strategies are distinct from the selected personality traits was supported through structural equations modeling analyses examining competing models combining the hierarchical factor structures of self-leadership and personality. Model fit increased significantly through a progression of models that showed increasingly greater distinction between self-leadership dimensions and personality traits. The best fitting model in the progression, in harmony with both self-leadership and trait personality theory, consisted of a hierarchical factor structure with three first order self-leadership factors, three first order personality factors, and two correlated second order factors (i.e., self-leadership and personality). Furthermore, intercorrelations were greater within the self-leadership dimensions than between the self-leadership dimensions and the personality traits, thus providing additional evidence of differentiation. Although the evidence indicates that self-leadership skill dimensions are unique with respect to personality traits, these results also suggest that self-leadership and personality factors are nevertheless significantly related. Specifically, both extraversion and conscientiousness were significantly related to all three self-leadership dimensions, while emotional stability was significantly related only to the natural rewards strategies dimension. In summation, the results of this study suggest that self-leadership represents a distinct constellation of strategies that are significantly related to certain key personality traits. The implications of these results for future self-leadership research and practice are discussed. / Ph. D.

Tumor necrosis factor-a (TNFa) is primarily a macrophagederived cytokine. TNFa, in vitro, kills or inhibits growth of approximately one third of surveyed transformed cell lines dincluding the L929 and WEHI 164 murine fibrosarcoma cell lines. Very little is known about the mechanisms of TNFa action. However, recently, it has been theorized that TNFa has no activity of its own and that the receptor for TNFa on the cell surface, when properly triggered, activates the cellular mechanisms which may result in the cell's death. The objective of this study was to produce an antiidiotypic antibody to TNFa to be used as a tool to study the mechanisms of TNFa action. A hybridoma that secretes an antiidiotypic antibody to TNFa (Ab2J1) has been produced and isolated following standard procedures. This antibody was found to be of isotype IgG2a as determined by an indirect ELISA test. The Ab2J1 exhibited TNFa target cell-killing capabilities in vitro. The TNFa-resistant cell lines, SP2jO and NS-1 were resistant to Ab2J1 and TNFa sensitive cells, L929 and WEHI 164, were sensitive to Ab2J1. The cell killing activity of both TNFa and Ab2f3 could be neutralized by a monoclonal anti-TNFa antibody. Both TN Fa and Ab2f3 acted in parallel having an effect on the killing of Brucella abortus strain RB51 by peritoneal macrophages, whereas neither TNFa nor Ab2f3 had an effect on the killing of strain 2308 by macrophages. These results, again indicate that TNFa and Ab2f3 have parallel dbactericidal effects and that Ab2f3 is capable of mimicking TNFa activity. The Ab2J1 was further characterized by gel electrophoresis and Western blot and was found to have two subunits of 25 and 50 kDa molecular weights similar to IgG. This anti-idiotypic antibody to TNFa may help in understanding the mechanisms of the cytotoxic activity of TNFa. / Master of Science