Bioluminescence is the chemical production of light that results when a luciferase enzyme catalyzes the luminogenic oxidation of a small-molecule luciferin substrate. The numerous luciferases and luciferins nature has evolved can be used to illuminate biological processes, from in vitro assays to imaging processes in live animals. However, we can improve the utility of bioluminescence through modification of these enzymes and substrates. My thesis work focuses on developing reporters that expand the bioluminescent toolkit and improving our understanding of how bioluminescence works on a molecular level. The first part of my thesis focuses on characterizing luciferases and luciferins that improve bioluminescence imaging in vivo. Some of our luciferins can outperform the natural D-luciferin substrate in live mouse imaging, while others are selectively utilized by mutant luciferases in live mouse brain. We also engineered luciferins that can selectively report on endogenous enzymatic activity in live mice. The second part of my thesis focuses on determining the molecular details of how enzymes related to firefly luciferase, long-chain fatty acyl-CoA synthetases (ACSLs), can function as latent luciferases. I have determined the structure for one of these enzymes and improved its bioluminescent activity with synthetic luciferins enough to image in live mouse brain. I also characterized the selectivity in chimerized enzymes that combine firefly luciferase and ACSLs. In summary, my work improves the utility of bioluminescence for in vivo use and informs us about how evolutionarily-related enzymes function as luciferases on a molecular level.

An adult female sample of childhood sexual abuse survivors (N=467) were surveyed to determine whether internalized shame moderated the effects of sexual inhibited difficulties. Other variables such as severity, duration, and frequency of sexual abuse, as well as whether physical abuse was also part of their experience, were examined to determine their role in later sexual inhibition. It was predicted that there would be a significant positive relationship between 1) Scores on variables of physical abuse, severity of abuse, frequency of abuse, duration of abuse, identity of the perpetrator and scores on the variable of inhibited sexual difficulties; 2) Scores of internalized shame and scores of inhibited sexual difficulties and 3) Scores on variable of physical abuse, severity of abuse, frequency of abuse, duration of abuse, identity of the perpetrator and scores on the variables of internalized shame. Through Structural Equation Modeling using AMOS, the results indicated a statistically significant positive relationship between severity, frequency and inhibited sexual disturbances but found no direct relationship between physical abuse, the identity of the perpetrator, the duration of the abuse and inhibited sexual disturbances. Results also indicated that shame had a direct positive relationship to inhibited sexual disturbances. The third finding was that physical abuse and severity of abuse had a significant positive relationship with shame which implies that shame is a moderating variable for inhibited sexual disturbances in adult women survivors of childhood sexual abuse. Internalized shame may be an important factor for therapists to consider in helping survivors overcome sexual inhibition as a result of childhood sexual abuse. If a woman develops a shame-prone identity she may be at risk for experiencing inhibition in her sexuality.

SH3-Domänen sind Proteinmodule, die über die Erkennung prolinreicher Peptidsequenzen Protein-Protein-Interaktionen in einer Vielzahl von zellulären Prozessen vermitteln. In der vorliegenden Arbeit wurde die Erkennung von Peptidliganden durch ausgewählte SH3–Domänen hinsichtlich Selektivität versus Promiskuität untersucht. Diese Arbeiten dienten als Vorversuche für einen kompletten proteomischen Ansatz aller SH3-Domänen aus S. cerevisiae. Für die vier ausgewählten SH3-Domänen, nämlich Myo5, Abp1, Yhr016 und Rvs167, wurden die Ligandenpräferenzen durch Interaktionsanalysen mit einem vollständigen Satz prolinreichen Peptidliganden, abgeleitet aus dem Hefeproteom ermittelt. Hierzu wurde ein Peptid–Array Ansatz gewählt. Mit der SPOT-Technologie wurden 2953 15mere Peptide als Array auf einem Celluloseträger Schritt für Schritt synthetisiert. Hierfür wurde zuerst die Art des Celluloseträgers festgelegt und anschließend überprüft, ob sich ein Array mehrmals in Interaktionsexperimenten verwenden lässt. Es zeigte sich, dass eine Regeneration der Arrays nicht möglich war, so dass für jedes Interaktionsexperiment ein neues Peptidarray synthetisiert werden musste. Die Interaktionsstudien bestätigten die Bindung prolinreicher Sequenzmotive als eine universelle Eigenschaft der vier SH3-Domänen, wobei die einzelnen SH3–Domänen-Peptid-Interaktionen von zusätzlichen, spezifitätsdetermierenden Resten im Liganden abhängig waren. Darüber hinaus zeigten die Experimente deutliche Unterschiede hinsichtlich Selektivität und Promiskuität im Erkennungsverhalten der vier Domänen. Die Domänen konnten nach steigender Selektivität an Hand der Bestimmung von Grenzwerten und Integralen geordnet werden, nämlich Myo5 < Abp1 < Yhr016 < Rvs167. Ein gemeinsamer Ligand für alle vier Domänen konnte nicht identifiziert werden. Dagegen konnten gemeinsame Liganden für die Domänenpaare Myo5/Abp1 und Yhr016/Rvs167 bestimmt werden. Die Bindungspräferenzen zweier ausgewählter Liganden, dem Myo5/Abp1 Liganden ERPKRRAPPPAPKKP und dem Yhr016/Rvs167 Liganden VQQDSLPKLPFRSWG, wurden mit Hilfe von Substitutions- und Längenanalysen detailliert charakterisiert. Es zeigte sich deutlich, dass die selektiveren SH3-Domänen Yhr016 und Rvs167 einen klar definierten und kurzen Konsensus im Liganden binden, hingegen die mehr promiskuitiven Domänen Myo5 und Abp1 ein stärker variables und längeres Motiv im Liganden erkennen. Die Ergebnisse dieser Arbeit zeigten, dass die hier gewählte Methode geeignet ist, die proteomische Charakterisierung aller dreißig Hefe SH3-Domänen in Angriff zu nehmen. / SH3-Domains are protein modules, which recognize polyproline sequences. The Domains are involved in a variety of cellular processes. The recognition rules between peptide ligands and selected SH3-Domains were analysed in respect of selectivity versus promiscuity. This dissertation is a preliminary test for subsequent scanning the whole number of yeast SH3-Domains. The SH3-Domains Myo5, Abp1, Yhr016 and Rvs167 were selected and chosen for interaction with all yeast polyproline ligands. A peptide array was chosen and built up with SPOT-Synthesis. 2953 15-mere peptides were spotted as an array on a cellulose membrane and inspected for regeneration method. The regeneration failed and therefore four arrays were synthesised. The scanning experiments revealed an interaction between Ligand and Domain with a common polyproline core. However, these experiments demonstrated as well that specific residues were needed for operative ligand domain interaction. In addition, these four SH3-Domains exhibited clear differences in selectivity and promiscuity of recognition profiles. The integrals and the results after the setting of the threshold showed a distinct increase in selectivity. The arrangement was based on the increasing selectivity: 1. Myo5 2. Abp1 3. but cases of consistency were found between Abp1- and Myo5-SH3, as well as ligands between Yhr16- and Rvs167-SH3. The Myo5-/Abp1-Ligand ERPKRRAPPPAPKKP and the Yhr016-/Rvs167 ligand VQQDSLPKLPFRSWG were selected for characterisation of the peptide domain interaction through the analysis of substitution and length. The data showed that more selective Domains like Yhr016 and Rvs167 had a well-defined and short consensus, whereas more promiscuous Domains like Yhr016 and Rvs167 showed a mutable and longer consensus. In conclusion, the data showed that the SPOT-Synthesis is a suitable technique for a proteomic characterisation of the whole 30 yeast SH3-Domains.

The purpose of this study was to investigate the perceptions of young people regarding the O Icheke MCP campaign who are members of Selebi Phikwe District Youth Council, in a mining town in central Botswana. The main objective of the study was to explore young people‘s views about MCP, to solicit their views on the key strength and weaknesses of the campaign, and to make recommendations to further strengthen the campaign. Within the framework of the Health Belief Model, a qualitative research approach was employed and data was collected using four focus group discussions categorised into the following strata  Males aged 18-24 years  Females aged 18-24 years  Males aged 25-35 years  Females aged 25-35 years The overall study finding was that financial issues, the apparent need for sexual variety, quest for material possession, and the impact of unemployment, migration and alcohol abuse are seen as some of the major factors underlying MCP in Botswana. The O Icheke Multiple Concurrent Partnership campaign was positively viewed as playing a key role in addressing the apparent knowledge gap regarding the impacts of these partnerships and in changing people‘s attitudes towards MCP and discouraging wide sexual networks. The campaign‘s target population, funding, stakeholders and delivery mode of the program were identified as its pillars and an effective vehicle to achieve its goals. The consultation between funding office and implementing organisations, coverage of the programme in surrounding areas and growing non-profit organisation were identified to affect the MCP campaign to achieve its goals. The recommendation is that more has to be done to improve the implementation of O Icheke programme through more funding, consultation with programme implementers, tailor made for people with disability and covering surrounding areas. The recommendation is that more has to be done to improve the implementation of O Icheke programme through more funding, consultation and capacity building of volunteers. / Health Studies / MA (Social Behaviour Studies in HIV and AIDS)

The purpose of this study was to investigate the perceptions of young people regarding the O Icheke MCP campaign who are members of Selebi Phikwe District Youth Council, in a mining town in central Botswana. The main objective of the study was to explore young people‘s views about MCP, to solicit their views on the key strength and weaknesses of the campaign, and to make recommendations to further strengthen the campaign. Within the framework of the Health Belief Model, a qualitative research approach was employed and data was collected using four focus group discussions categorised into the following strata  Males aged 18-24 years  Females aged 18-24 years  Males aged 25-35 years  Females aged 25-35 years The overall study finding was that financial issues, the apparent need for sexual variety, quest for material possession, and the impact of unemployment, migration and alcohol abuse are seen as some of the major factors underlying MCP in Botswana. The O Icheke Multiple Concurrent Partnership campaign was positively viewed as playing a key role in addressing the apparent knowledge gap regarding the impacts of these partnerships and in changing people‘s attitudes towards MCP and discouraging wide sexual networks. The campaign‘s target population, funding, stakeholders and delivery mode of the program were identified as its pillars and an effective vehicle to achieve its goals. The consultation between funding office and implementing organisations, coverage of the programme in surrounding areas and growing non-profit organisation were identified to affect the MCP campaign to achieve its goals. The recommendation is that more has to be done to improve the implementation of O Icheke programme through more funding, consultation with programme implementers, tailor made for people with disability and covering surrounding areas. The recommendation is that more has to be done to improve the implementation of O Icheke programme through more funding, consultation and capacity building of volunteers. / Health Studies / M.A. (Social Behaviour Studies in HIV and AIDS)