It is likely that a counselor-in-training (CITs) will counsel a lesbian, gay, or bisexual (LGB) client in practice. The American Counseling Association (ACA, 2014) and the Council for Accreditation of Counseling and Related Educational Programs (CACREP, 2009) address ethical and training standards about counseling clients from diverse populations and multicultural counselor competence. The purpose of this dissertation was to examine master's-level CITs' perceived LGB counselor competence and potentially related and predictive factors including age, gender, religiosity, spirituality, personal relationships with LGB individuals, and having an "out" faculty or peer in the training program. This quantitative study included 105 CITs from CACREP accredited counselor education programs, surveyed from a national stratified sample, based on CACREP regions. The survey included the Sexual Orientation Counselor Competence Scale (Bidell, 2005), the Toronto Empathy Questionnaire (Spreng, McKinnon, Mar, & Levine, 2009); a Religiosity Index (Statistics Canada, 2006), Spiritual Transcendence Index-Modified (Seidlitz et al., 2002), the Marlowe-Crowne Social Desirability Scale-Sort Form C (Reynolds, 1982), and additional survey items to collect information on the number of personal relationships with LGB individuals (i.e., family, friends, faculty, and peers), age, gender and demographic information. Results indicated that CITs felt least competent in their skills to work with LGB clients and most competent in their attitudes towards LGB individuals. A regression analysis revealed that religiosity negatively predicts perceived LGB counselor competence and personal relationships with LGB individuals positively predicted LGB competence. Additionally, there was a significant positive relationship between having an "out" LGB peer in the training program and perceived LGB counselor competence. Implications suggest that counselor training programs increase the use of LGB themed case studies, role plays, and other classroom initiatives to help CITs meet the needs of their future LGB clients. An interesting finding in this study was the positive relationship between having an "out" LGB peer in the training program and increased LGB competence. Future research should investigate the dynamics of this relationship.

Capacitation of spermatozoa is essential for fertilization. Rabbit spermatozoa are particularly difficult to capacitate in vitro and require treatment with high-ionic-strength Brackett's defined medium. Spermatozoa treated with platelet activating factor had significantly higher fertilization rates when compared with nontreated (fresh, twice washed) spermatozoa (63% vs 34%). Fertilization rates of spermatozoa treated with platelet activating factor, although higher than those of high-ionic-strength capacitated spermatozoa, were not significantly different (63% vs 57%). Spermatozoa treated with lyso-platelet activating factor, the biologically inactive form of platelet activating factor, were noted to have fertilization rates similar to those of the untreated (noncapacitated) group. These data show that synthetic platelet activating factor treatment of uncapacitatedspermatozoa induces fertilization of rabbit oocytes in vitro in a manner similar to that for spermatozoa capacitated by high-ionic-strength media and significantly higher than that for untreated spermatozoa or after treatment with the biologically inactive form of platelet activating factor (lyso-platelet activating factor).

Previous studies have suggested that T cell contact‐dependent signaling of macrophages (MΦ) is mediated by membrane tumor necrosis factor‐α (memTNF‐α), based on the observation that anti‐TNF‐α could inhibit T cell‐mediated MΦ activation. The current report confirms that anti‐TNF‐α does inhibit activation of interferon‐γ (IFN‐γ)‐primed MΦ by paraformaldehyde‐fixed activated T cells. However, the involvement of membrane molecules other than memTNF‐α in the contact‐dependent signaling is suggested by two lines of evidence. First, the TH2 clone, AK8, displayed neither secreted TNF‐α/β nor memTNF‐α/β detectable by bioassay or immunofluorescence. Nonetheless, AK8 cells were equally effective, on a per cell basis, in contact‐dependent signaling of MΦ activation as TH2 and TH1 cells which do express memTNF‐α. Second, the expression of memTNF‐α by the TH clone, D10.G4, is maximal 24 h after activation, whereas the ability of this clone to activate MΦ is maximal at 6–8 h of activation and declines thereafter. Since TNF‐α is known to play a critical role in activation of MΦ effector function, it was hypothesized that T cell membrane components other than memTNF‐α might signal MΦ production of TNF‐α, thus allowing autocrine TNF‐α stimulation of MΦ effector function. In support of this, it is demonstrated that paraformaldehyde‐fixed activated TH2 cells can induce de novo production and release of TNF‐α by MΦ. This effect was not an artifactual result of paraformaldehyde fixation since paraformaldehyde‐fixed resting T cells did not induce TNF‐α gene expression. Previous studies have demonstrated a role for autocrine TNF‐α stimulation in LPS induction of effector function in recombinant IFN‐γ‐primed MΦ. The current study confirms that TNF‐α plays a critical role in T cell contact‐dependent signaling of MΦ but indicates that memTNF on the T cells may not be a sine qua non factor for contact‐dependent signaling. The data suggest that other T cell membrane molecules contribute to activation of MΦ effector function by stimulation of MΦ TNF‐α production.

This thesis work focuses on the study and improvement of single-stage power factor correction techniques. The generalized structures of the present pulse-width-modulation (PWM) integrated single-stage power factor correction (PFC) converters are presented. The typical PFC cells in the single-stage PFC converter are identified. After that, the necessary PFC condition is derived and verified to understand the principle of the single-stage PFC converters. As an example, the continuous current mode (CCM) current source single-stage PFC converter is studied. The circuit intuitions and design consideration of this converter are presented. Also, an improved current source single-stage PFC converter with a low-frequency auxiliary switch is proposed to overcome the problem of the previous converter. Experimental verification shows the improvement is effective. To evaluate single-stage PFC technique, a comparison study between the current source single-stage and the boost two-stage PFC converters is done in this thesis. It shows that for universal line application, due to the wide bus-capacitor voltage range, single-stage PFC converters have higher component ratings than two-stage PFC converters. This limits the application of single-stage PFC converter. Therefore, an interesting future work will be how to reduce the bus voltage range of single-stage PFC converters. / Master of Science

Many attempts have been made to improve the in vitro production of cattle embryos by refining in vitro maturation (IVM) and culture systems. Cysteine supplementation to IVM media of bovine oocytes increases cellular glutathione production, which reduces reactive oxygen species (ROS). Similarly, beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of the antioxidant cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on subsequent embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mM cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/mL); EGF (10 ng/mL); and IGF-I+EGF (100 ng/mL+10 ng/mL) for all IVM treatments. Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the IVM media (12 h C IGF-I+EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I+EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD; SOD1) and manganese (Mn) SOD (SOD2) in embryos was assessed. No significant treatment effect was observed on the expression of apoptotic and oxidative stress genes. Bax was expressed strongly (4-fold) in morulae with the addition of IGF-I, but was less prevalent in all other morula and blastocyst groups relative to FCS. There was slightly less expression of both SOD1 and SOD2 with treatments compared to FCS in morulae and blastocysts, indicative of low mitochondrial activity and/or a low level of oxidative stress in treatments. There was no significant treatment effect on total cell number, apoptotic nuclei, or apoptotic index. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS. / Master of Science

Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, Factor VII and in small amounts in its activated form, Factor VIIa. However, the mechanism of initial generation of Factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases Factor VII activating protease, and hepsin play a role in activating Factor VII, however, it has remained controversial. In this work I estimated the levels of Factor VIIa and Factor VII for the first time in adult zebrafish plasma and also reevaluated the role of the above two serine proteases in activating Factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease did not reduce Factor VIIa levels while hepsin knockdown reduced Factor VIIa levels. After identifying role of hepsin in Factor VII activation in zebrafish, I wanted to identify novel serine proteases playing a role in Factor VII activation. However, a large scale knockdown of all serine proteases in zebrafish genome using available knockdown techniques is prohibitively expensive. Hence, I developed an inexpensive gene knockdown method which was validated with IIb gene knockdown, and knockdown all serine proteases in zebrafish genome. On performing the genetic screen I identified 2 novel genes, hepatocytes growth factor like and prostasin involved in Factor VII activation.

PhD Thesis / Morphogens (often acting as transcription activators or repressors) govern pattern formation and cell differentiation during early embryogenesis. Abnormal distributions of morphogens can result in developmental defects or even death. Oftentimes, thresholds of concentrations of morphogens behave like an ON/OFF switch for the activation or repression of downstream genes. Accurate measurements of morphogen concentration and mobility in space and time can help tackle the puzzle of how exactly cascades of hundreds of morphogens coordinate their targets precisely and promptly amidst crowded and complicated cellular environments. The research question at the centre of my thesis is that of the concentration and dynamics of two morphogens with opposite functions in the early fly embryo. In the work presented in this thesis, we use Fluorescence Correlation Spectroscopy (FCS) and confocal imaging to achieve extremely low ($\sim$ nM) concentration measurements in live \textit{Drosophila} embryos expressing recombinant fluorescent morphogens, by carefully taking into account background noise and photobleaching effects. The dynamics of both Bicoid (Bcd) and Capicua (Cic), an activator and a repressor morphogens, were further studied using FCS, Fluorescence Recovery After Photobleaching (FRAP) and Monte Carlo simulation. We found that both types of morphogens are very mobile in nuclei, explaining how they are able to turn on or off gene expression in only a few minutes. However, these two morphogens with opposite functions have drastically different nucleo-cytoplasmic transport behaviours, where the activator can pass through the nuclear envelop (NE) relatively freely while the repressor is jailed inside nuclei during interphase. These findings can provide clues to distinguish between several hypothetical models (including the newly proposed hub hypothesis) trying to explain the mechanisms of target gene search and transcription regulation. In this thesis, a background introduction on transcription factors and morphogens is given in Chapter 1, with a focus on the two transcription factors (the activator Bicoid and the repressor Capicua) studied in this thesis. Next, experimental details such as fruit fly maintenance, and fluorescent techniques used to measure concentration and mobility are described in Chapter 2. From Chapter 3 to Chapter 5, three manuscripts from the thesis author, either published or in preparation for submission are presented in sequence. Chapter 3 introduces a new method to accurately measure protein concentration in the presence of noise and photobleahing in early \textit{Drosophila} embryos using FCS. Chapter 4 contains the results of concentration and mobility measurements for Cic which contribute to the finding that Cic acts like a fast brake in transcription repression. Chapter 5 compares the similarities and differences of the dynamics of Bcd and Cic through multiple lenses. Finally, a conclusion and future outlook are given in Chapter 6. / Thesis / Doctor of Philosophy (PhD) / Have you ever wondered how a single fertilized egg turns miraculouly into a beautifully organized living being, be it an insect, a cat, or a human? It turns out that an important group of molecules called morphogens govern the formation of body pattern. These molecules (usually proteins) form concentration gradients along the different body axes of that organism and influence gene expression. Abnormal distribution of morphogen can result in defects in embryo development and even death. Thus knowing how much morphogen is present in the early developing embryo, as well as how it forms gradients and how the morphogen concentration is translated into a pattern can help us better understand early embryo development. My thesis focuses on accurate measurements of morphogen concentrations and dynamics using fluorescence techniques. We were able to obtain concentration maps for two morphogens, the activator Bicoid and the repressor Capicua, in early developing fruit fly embryos. We also found that despite having opposite functions, the activator and the repressor have similar intranuclear dynamics, but drastically different internuclear mobility. Our findings provide clues to distinguish between multiple hypothetical models scientists have put forward to explain the mechanisms of transcription regulation.

Traditionally, pre-cracking has been performed under tension-tension loading, followed by a load reduction scheme to obtain fatigue crack growth rate data in the near threshold regime. These data have been shown to exhibit load history effects due to remote crack closure. An alternative test method has been developed to minimize these load history effects. This test procedure uses compression pre-cracking to initiate a crack, followed by constant amplitude loading to grow the crack to failure. Compression-compression (C-C) loading as a means of forming a starter crack for fatigue crack growth is a relatively new concept. Cracks grown under C-C loading emanate from the notch tip due to a tensile residual stress field formed during the unloading cycle. The subsequent constant amplitude steady-state crack growth is free of load history effects, after crack growth beyond several compressive plastic zone sizes, and therefore will give a better steady-state representation of the near-threshold regime. A more in-depth examination at this phenomenon is performed herein.

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