The illegal bushmeat harvest has been identified as a reason for declining wildlife populations throughout much of Africa. For many years the trade was thought to exist primarily in Western Africa. The illegal use of bushmeat in Eastern Africa, including Kenya, went undocumented and unstudied. In 2004, the Born Free Foundation published a study which claimed illegal sale of wild game was substantial in butcheries throughout Nairobi, Kenya. In an effort to determine other markets of the commercial trade, the goal of this study was to analyze local restaurants in Voi, Kenya for illegal bushmeat sale. The town of Voi was selected due to recent published reports in the popular press, its proximity to Tsavo National Park, and its access to a major highway. Samples were collected and analyzed using mDNA sequencing analysis of the cytochrome B gene. None of the collected samples were identified as illegal game meat. The restaurants in Voi, Kenya were not a commercial outlet for illegal bushmeat trading in the local economy during the period of this study. The results from this study provide valuable baseline data which can be used in future research to help determine possible vectors of the bushmeat trade.

Chlamydomonas reinhardtii, a unicellular eukaryotic green alga, serves as a model organism to study the circadian clock in plants and animals. Rhodopsins are blue/green-light photoreceptors also found in C. reinhardtii. Chlamyrhodopsin (COP), the most abundant eyespot protein, was reported to have no role in the phototactic and photophobic responses in C. reinhardtii. Its function is yet unknown. In the present study, we hypothesized that the function of COP is to mediate entrainment of the circadian clock by light. In order to test this hypothesis, a C. reinhardtii selection marker conferring resistance to the antibiotic paromomycin was cloned into a COP RNAi construct obtained from another lab. Firstly, the COP RNAi construct was restriction digested to linearize it. The linearized plasmid was then blunt ended with T4 DNA polymerase and dephosphorylated with phosphatase. The linearized fragment was ligated with the paromomycin resistance marker obtained by restriction digestion of the plasmid containing it and transformed into E.coli. The recombinant clones obtained were confirmed by restriction digests. Fusion regions and the orientation of the insert in the recombinant plasmid were confirmed by sequencing. An attempt was made to transform C. reinhardtii with the construct, but this was not successful. Future studies will be required to optimize the C. reinhardtii transformation method. Transformants with reduced COP amounts can then be tested for defects in resetting the clock after light pulses. This will determine whether chlamyrhodopsin is involved in the circadian input pathway or not. The results of the complete project are expected to contribute to our understanding of the circadian clock of many other organisms including humans.

Diet-induced obesity promotes type 2 diabetes (T2D). Drugs that inhibit the renin-angiotensin system (RAS) have been demonstrated in clinical trials to decrease the onset of T2D. Previously, we demonstrated that mice made obese from chronic consumption of a high-fat (HF) diet have marked elevations in systemic concentrations of angiotensin II (AngII). Pancreatic islets have been reported to possess components of the renin-angiotensin system (RAS), including angiotensin type 1a receptors (AT1aR), the primary receptor for AngII, and angiotensin converting-enzyme 2 (ACE2), which negatively regulates the RAS by catabolizing AngII to angiotensin-(1-7) (Ang-(1-7)). These two opposing proteins have been implicated in the regulation of β-cell function. We hypothesized that the RAS contributes to the decline of β-cell function during the development of T2D with obesity. To test this hypothesis we first examined the effects of whole-body deficiency of ACE2 in mice on β-cell function in vivo and in vitro during the development of T2D. Whole-body deficiency of ACE2 resulted in impaired β-cell adaptation to insulin resistance with HF-feeding and a reduction of in vivo glucose-stimulated insulin secretion (GSIS) associated with reduced β- cell mass and proliferation. These results demonstrate that ACE2 plays a role in the adaptive response to hyperinsulinemia with obesity. In islets from HF-fed mice, AngII inhibited GSIS. In mice with pancreatic-specific deletion of AT1aR, AngII-induced inhibition of GSIS in vitro from islets of HF-fed mice was abolished. However, there was no effect of pancreatic AT1aR-deficiency on glucose homeostasis in vivo in HF-fed mice exhibiting pronounced hyperinsulinemia. Notably, pancreatic weight, insulin content and basal and glucose-stimulated insulin secretion from islets were decreased in mice with pancreatic AT1aR deficiency. These results suggest that AT1aR may contribute to pancreatic cell development, and also contribute to AngII-induced reductions in GSIS from islets of HF-fed mice. Overall, these studies suggest a role for the RAS in the regulation of β-cell function in T2D.

An estimated 12% of couples worldwide are infertile. The contributing factor is approximately equal between men and women with nearly 25% diagnosed as idiopathic. Despite the increasing numbers of couples seeking assistance from infertility clinics, few molecular mechanisms have been identified for treatment. Autophagy is an evolutionarily conserved cellular process for bulk degradation and recycling of cytosolic components through the lysosome to maintain homeostasis. Several studies have observed increased levels of autophagy during ovarian folliculogenesis and gonadal steroidogenesis; however, no genetic studies to determine the significance of autophagy exist. To investigate the function of autophagy in the ovary and testis, a directed genetic knockout approach was used to independently knockout two key autophagy genes, Becn1 and Atg7. Chapter 2 reports that deficiency of Becn1 results in 56% fewer primordial follicles at postnatal day 1. In addition, Atg7 knockout mice do not have identifiable primordial follicles, suggesting that autophagy is necessary for survival of female germ cells during embryogenesis. Chapter 3 presents that Becn1 is necessary to sustain pregnancy and the deficiency of Becn1 in granulosa cells is a novel genetic model to study preterm labor due to impaired corpora lutea function. The results indicate that Becn1 is necessary for lipid droplet formation and subsequent progesterone production in luteal cells. In contrast, Atg7 is not necessary and deficiency results in overproduction of progesterone throughout pregnancy, suggesting that the defect in Becn1 conditional knockout mice is additional to autophagy. Chapter 4 presents that Sertoli cell expression of Becn1 is required for spermatogenesis after 8 weeks of age. Beyond 9-weeks-old, Becn1 conditional knockout mice are unable to sire a litter due to a failure of spermatogenesis and a Sertoli-cell-only phenotype in a majority of the seminiferous tubules. Atg7 was also identified as a necessary factor for spermatogenesis beyond 26-weeks-old. Together the data presented in Chapter 4 suggests that autophagy is necessary for adult Sertoli cell function. Primarily, this dissertation presents data from the first functional studies on autophagy in the reproductive tract. The results demonstrate an understanding of the functional significance for Becn1 and Atg7 in both the ovary and testis.

A primary goal of climate change research is to determine if species will be able to persist in a warmer environment. Most studies predict climate change will cause many species to become extinct. However, these predictions are based on experiments where only a single life stage or generation of a species was exposed to predicted future conditions (i.e. shock treatments), and thus overlook the possibility of species adapting or acclimatizing to new environmental conditions over multiple generations. As a result, current projections of species persistence through climate change are likely to overestimate species extinction. In this study, the rate and extent to which adaptation and transgenerational acclimation may allow species to persist through climate change was measured. Marine rotifers, Brachionus plicatilis, were reared for ~75 generations at: i) Optimal temperature (25°C), ii) Optimal temperature (25°C) with weekly sub-lethal shocks (35°C), iii) Maximum temperature (33°C), and iv) Maximum temperature (33°C) with weekly sub-lethal shocks (35°C). Changes in population growth rates and fitness were assessed weekly through rotifer density, adult size and aerobic performance (respiration rate). There was no adaptation observed, but there was evidence of transgenerational acclimation. However, populations were unable to acclimate when exposed to high temperature shocks. This study shows that acclimation through the selection of thermally tolerant individuals can occur over multiple generations in a thermally stable environment, as seen by a reversible increase in aerobic performance, and thus species with short life cycles may be better able to keep up with the pace of climate change. This multi-generational study can enhance our understanding of the rate and extent in which transgenerational acclimation may allow species to persist through climate change. These estimates can then be incorporated into models to improve projections of survival through climate change of species with longer lifespans.

Please read the abstract in the section, 00front, of this document / Thesis (DPhil)--University of Pretoria, 2006. / Humanities Education / DPhil / Unrestricted

Metastasis is characterized pathologically by uncontrolled cell invasion, proliferation, migration and angiogenesis. Steroid hormones, such as estrogen, and growth factors, which include insulin growth factor I/II (IGF-1/IGF-2) therapy has been associated with most if not all of the features of metastasis. It has been determined that IGF-1 increases cell survival of cancer cells and potentiate the effect of E2 and other ligand growth factors on breast cancer cells. However not much information is available that comprehensively expounds on the roles of insulin growth factor receptor (IGFR) and Rab GTPases may play in breast cancer. The latter, Rab GTPases, are small signaling molecules and critical in the regulation of many cellular processes including cell migration, growth via the endocytic pathway. This research involves the role of Rab GTPases, specifically Rab5 and its guanine exchange factors (GEFs), in the promotion of cancer cell migration and invasion. Two important questions abound: Are IGFR stimulation and downstream effect involved the endocytic pathway in carcinogenesis? What role does Rab5 play in cell migration and invasion of cancer cells? The hypothesis is that growth factor signaling is dependent on Rab5 activity in mediating the aggressiveness of cancer cells. The goal is to demonstrate that IGF-1 signaling is dependent on Rab5 function in breast cancer progression. Here, the results thus far, have shown that while activation of Rab5 may mediate increased cell proliferation, migration and invasion in breast cancer cells, the Rab5 GEF, RIN1 interacts with the IGFR thereby facilitating migration and invasion activities in breast cells. Furthermore, endocytosis of the IGFR in breast cancer cells seems to be caveolin dependent as the data has shown. This taken together, the data shows that IGF-1 signaling in breast cancer cells relies on IGF-1R phosphorylation, caveolae internalization and sequestration to the early endosome RIN1 function and Rab5 activation.

This dissertation utilized electrospray ion mobility mass spectrometry (ESI-IMS-MS) to develop methods necessary for the separation of chiral compounds of forensic interest. The compounds separated included ephedrines and pseudoephedrines, that occur as impurities in confiscated amphetamine type substances (ATS) in an effort to determine the origin of these substances. The ESI-IMS-MS technique proved to be faster and more cost effective than traditional chromatographic methods currently used to conduct chiral separations such as gas and liquid chromatography. Both mass spectrometric and computational analysis revealed the separation mechanism of these chiral interactions allowing for further development to separate other chiral compounds by IMS. Successful separation of chiral compounds was achieved utilizing a variety of modifiers injected into the IMS drift tube. It was found that the modifiers themselves did not need to be chiral in nature and that achiral modifiers were sufficient in performing the required separations. The ESI-IMS-MS technique was also used to detect thermally labile compounds which are commonly found in explosive substances. The methods developed provided mass spectrometric identification of the type of ionic species being detected from explosive analytes as well as the appropriate solvent that enhances detection of these analytes in either the negative or positive ion mode. An application of the developed technique was applied to the analysis of a variety of low explosive smokeless powder samples. It was found that the developed ESI-IMS-MS technique not only detected the components of the smokeless powders, but also provided data that allowed the classification of the analyzed smokeless powders by manufacturer or make.

This research examined the effects of acclimation to lowered salinity, elevated temperature, and hypoxia on aerobic metabolism of the Gulf killifish, Fundulus grandis, a common estuarine resident of the Gulf of Mexico. Standard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS), and critical oxygen tension (Pcrit) were each influenced by one or more acclimation treatments. Assessing the consistency of these traits measured in the same individuals over time, all were found to be significantly repeatable with no indication that the repeatability of any traits was affected by acclimation conditions. Significant correlations were found between SMR and Pcrit (positively correlated), between SMR and AAS (negatively correlated), between MMR and AAS (positive), and between AAS and Pcrit (negative). This study, therefore, documents the effects of acclimation on these traits, their repeatability, and correlations among them. It further suggests that repeatability of these traits is not context dependent.

INTER-SEGMENT COORDINATION VARIABILITY POST ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION SEPTEMBER 2015 DEVIN K. KELLY, B.S., UNIVERSITY OF MASSACHUSETTS AMHERST M.S., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Dr. Joseph Hamill There is an increased risk for ipsilateral graft rupture and contralateral ACL rupture following ACL reconstruction surgery (ACLR) despite return to sport clearance. The reason for this increased risk is not well understood. Previous literature has shown that decreased coordination variability is indicative of an injured system regardless of the absence of pain. PURPOSE: To quantify inter-segment coordination variability during three portions of the stance phase of gait in athletes at three progressive time points post-surgery compared to the contralateral limb (NI) and healthy controls. METHODS: Three-dimensional kinematic and kinetic data were collected for 10 ACLR and 10 healthy athletes matched for age, gender, and activity level. The ACLR group was measured at 4 weeks, 12 weeks, and when cleared to run post-surgery. Kinematic data were used in a modified vector coding technique to determine inter-segment coordination variability of lower extremity couples of interest. Statistical significance was determined using two factor multivariate ANOVAs (limb x visit) for early (1-33%), mid (34-66%), and late (67-100%) stance with alpha level set at .05. Tukey post-hoc tests were performed where appropriate. RESULTS: ACLR athletes have decreased inter-segment coordination variability of the involved lower extremity during the late stance phase of gait compared to both the contralateral limb and healthy controls at 4 weeks post-surgery. By 12 weeks post-surgery there were improvements in joint function as exemplified by inter-segment coordination variability of the ACLR involved limb becoming similar to the healthy control limb. CONCLUSION: Inter-segment coordination variability during late stance in the present study is not an indication for the increased risk for ipsilateral graft rupture and contralateral ACL rupture in ACLR athletes.