M.Tech. / Low Level Laser Therapy (LLLT), also known as photo-biostimulation or simply phototherapy, has widely been used in the treatment of wounds, with its history dating back to the early 1960s (Ohshiro and Calderhead, 1991). Despite some literature reporting negative and non-existent cellular responses to LLLT, a growing body of literature reports the positive and beneficial effects of LLLT. LLLT has proved to be efficient in speeding and improving the quality of wound healing. Stressed cells respond more favourably to LLLT by recovering to their most natural state and functional capability (Bernett, 1998; Karu, 1998). When healing appears to be impaired, these tissues respond positively to the appropriate doses of light, especially light that is within 600 to 1,000 nm wavelengths (Enwemeka et al., 2004). Cellular responses to LLLT include changes in mitochondrial intracellular calcium ion (Ca2+) levels, Mitochondrial Membrane Potential (MMP), Adenine Triphosphate (ATP) concentration, and cyclic 5’, 3’ Adenosine Monophosphate (cAMP) (Karu, 1998). The mitochondrion is the power house of a cell and the major location of cellular ATP synthesis (Bayens and Dominiczak, 1999). ATP is an energy rich molecule that drives processes responsible for cell growth or proliferation (Klug et al., 2003). LLLT alters intracellular pH which is related to activation of ATPase leading to an increase in ATP production in the mitochondria of the cell (Alexandratou et al., 2002; Karu, 1998). However the mechanisms by which the beneficial effects are attained by cells in stress or injury state are not clear.

Epithelia act as protective barriers and it is therefore essential that wounded epithelia are rapidly repaired to maintain barrier function. Cells surrounding epithelial wounds become motile following wounding, which involves generating dynamic actin structures that drive closure of the wound. These actin structures include filopodia which are important in the final stage of epithelial closure in which the opposing epithelial edges are joined together. The molecular mechanisms that trigger wound edge cells to become motile are not well understood. Using Drosophila wound healing and the morphogenetic process dorsal closure as models, we find that phosphatidylinositol 3,4,5-triphosphate (PIP3) regulates epithelial closure by promoting the formation of filopodia at epithelial edges. PIP3 accumulates at epithelial edges and genetically depleting PIP3 results in reduced filopodia and defects in epithelial closure. We demonstrate that the GTPase Rac and guanine nucleotide exchange factor Myoblast City function downstream of PIP3 to promote filopodia formation. We also demonstrated that the scaffolding protein Par3/Bazooka and the lipid phosphatase PTEN are responsible for restricting the localisation of PIP3 and consequently the downstream signals to the epithelial leading edge, so acting to determine the location of filopodia formation. This project reveals a novel mechanism by which actin protrusions, required for epithelial closure, are formed in response to epithelial damage. Additionally, we have identified an additional role for PIP3 in regulating the extrusion of cells from epithelial sheets in the Drosophila embryo. This finding implicates PIP3 in the regulation of tissue homoeostasis, and could contribute to our understanding of tumour initiation as unregulated tissue growth can result in the formation of tumours.

The complex process of wound repair becomes disrupted in the elderly with a profound effect on patient morbidity and huge financial implications for the NHS. While age itself is a risk factor for delayed healing recent work implicates estrogen decline, rather than intrinsic ageing per se, as the critical regulator of delayed healing in elderly subjects. In women estrogen levels fall dramatically post-menopause and with increasing life expectancy most women in the developed now world spend at least a third of their lives in a state of estrogen deprivation. Estrogen replacement can reverse this delay, but unfortunately long term estrogen treatment (HRT) increases breast cancer risk such that steroidal estrogen is now listed as a carcinogen. The aim of this study has been to functionally dissect the role of estrogen signalling during repair at the molecular, cellular and physiological levels. New data presented within this thesis reveal estrogen to be a global regulator of healing with pleiotropic effects on multiple wound cell types. By combining pharmacological manipulation and genetic ablation my data reveals novel diametrically opposed roles for the two estrogen receptor isoforms, ERalpha and ERbeta, during healing. I have further exploited this to demonstrate the in vivo therapeutic potential of compounds with receptor selective agonistic/antagonistic activity. Additionally, I have further investigated the mechanism of action of estrogen and these selective compounds implicating the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) in beneficial effects on healing. This research leads the way toward translation into human studies with the ultimate aim of developing targeted therapeutics for the treatment of delayed acute and chronic wounds, particularly in the elderly.

The molecular mechanisms underlying tissue regeneration and wound healing are still poorly understood despite their importance. In this paper we develop a bioinformatics approach, combining biology and network theory to drive experiments for better understanding the genetic underpinnings of wound healing mechanisms and for selecting potential drug targets. We start by selecting literature-relevant genes in murine wound healing, and inferring from them a Protein-Protein Interaction (PPI) network. Then, we analyze the network to rank wound healing-related genes according to their topological properties. Lastly, we perform a procedure for in-silico simulation of a treatment action in a biological pathway. The findings obtained by applying the developed pipeline, including gene expression analysis, confirms how a network-based bioinformatics method is able to prioritize candidate genes for in vitro analysis, thus speeding up the understanding of molecular mechanisms and supporting the discovery of potential drug targets.

At the outset of my PhD, my aim was to investigate the mechanisms responsible for the directed migration of primitive myeloid cells (PMCs) to wounds in Xenopus embryos. PMCs are the first blood cells to differentiate and become functional in Xenopus embryos, and have a notable migratory ability to be recruited by embryonic wounds before a functional vasculature is established. To find the mechanism underlying PMCs migration toward embryonic wounds, I first performed a screen to identify candidate cytoskeleton related genes, which might be responsible for facilitating the inflammatory response to injury in embryos. In situ hybridization and RT-PCR showed that coronin1a and l-plastin were specifically expressed in PMCs. I carried out loss-of-function experiments for coronin 1a and l-plastin in Xenopus embryos. Unfortunately neither knockdown affected the ability for PMCs to migrate during embryonic development or during the wound healing process. Loss-of-function experiments on coronin 1a and l-plastin also did not affect epidermal wound closure speed. Thus, although coronin 1a and l- plastin are expressed specifically in PMCs, they do not appear to be necessary for the migration of PMCs during development and during wound healing in Xenopuos embryos. Since my initial aim failed to provide insight into the mechanisms that mediate 9the inflammatory response to embryonic wounds, I decided to investigate the function of a previously identified monomeric actin protein during embryonic wound healing and appendage regeneration: namely Thymosin beta4 (Tβ4). In situ hybridization experiments showed that Tβ4 is expressed exclusively in the epidermis of developing frog embryos. Tβ4 knockdown embryos resulted in a significantly delay in the speed of wound closure during the early phase of wound healing. This delay correlated with a decrease in the actin contractile ring at the wound margin. Furthermore I found that the cell shapes of epidermal cells in the Tβ4 knockdown embryos were different from epidermal cells in control embryos. I hypothesize that this reduction caused the actin filaments changes in the epidermal cells, and were responsible for the failure of the cells to form an actin contractile ring, thus delaying the initial speed of wound closure. I tried to confirm that most of these defects specific to Tβ4, by performing rescue experiments with Tβ4 mRNA injections. Furthermore, I discovered that Tβ4 knockdown embryos displayed defects in tail development, including the absence of blood vessel branching within the fin of the tail. Finally, I found that the tails in Tβ4 knocked-down tadpoles failed to regenerate, while tails in control embryos regenerated completely following amputation. Both in situ hybridization and real-time PCR showed that Tβ4 was up regulated in the regenerated part of the tail in Xenopus tadpoles. Together with the tail amputation results, Tβ4 might be important for tail development and regeneration. These findings suggest that Tβ4 might play an important roles in the modulation of the actin cytoskeleton, which are essential for the proper behavior of epidermal cells during wound healing and appendage regeneration.

For years, it has been appreciated that embryos have remarkable abilities to heal wounds efficiently and perfectly, without scar formation. However, the molecular mechanisms underlying embryonic wound healing, especially how they coordinate and function in an efficient way, remains poorly understood. The primary aim of my PhD thesis was to use Xenopus as a model system to investigate the molecular and cellular mechanisms which are responsible for the regulation and coordination of embryonic wound healing. More specifically, my thesis includes the study of three signalling pathways during embryonic wound healing; namely the Erk MAPK pathway, PI3K pathway and inositol phosphate pathways. Erk and PI3K signalling are sequentially activated post injury, during separate phases of wound closure. The initial activation of Erk signalling governs the initial stage of wound closure, by mediating myosin-2 phosphorylation and actomyosin contraction through Rho activity. PI3K signalling increases in the late stage of wound closure, promotes leading edge migration and zippering via Rac and Cdc42 activity (Manuscript #1). From the findings of this study, I proposed a novel model, which suggests a cooperation of these two signalling pathways in orchestrating distinct cytoskeletal events during in tissue morphogenesis. In the second part of my thesis, I studied the role of inositol phosphate signalling during wound healing. In particular, I studied the role of the enzyme Itpkb and its product InsP4, in promoting rapid wound healing (Manuscript #2). Itpkb colocalizes with F-actin cable and promotes its formation at the wound edge in both single cell and multicellular wounds, enhancing the activity of three Rho GTPases Rac, Cdc42 and Rho at the same time. In addition, itpkb is required for calcium propagation from the wound edge to distant cells, suggesting a role in transmitting the wound signal across the tissue, resulting in the coordination of healing in multicellular wounds. Together, these PhD work provided more insights into the in vivo regulation of intracellular and intercellular signals in coordinating cell behavior in tissue movement during embryonic wound healing.

Many researchers have failed to find a relationship between experience and judgment accuracy. In this study the purpose was to understand the relationship between experience and expertise in clinical judgment. Common sense suggests that experienced subjects make better quality judgments, compared to novices. Clinical judgments, however, are ill-structured and characterized by uncertainty; they take place in a dynamic context, with delayed or nonexistent feedback and are difficult to learn. Cognitive operations that translate "cues" (such as risk factors, signs, and symptoms) into judgments are not fully understood. Cognitive constructs (conceptual structure, sensitivity to patterns in data, and judgment process) and individual differences in age, education, and experience were explored to identify their relationship to judgment expertise. Indicators of judgment quality were: accuracy, consistency, latency, confidence, calibration, and knowledge accessibility. In phase 1 of this study, cues were identified that best predicted healing time for 258 surgical patients with abdominal incisions. In Phase 2, the subjects were 36 nurses with a range of experience caring for surgical patients. Generating both quantitative and qualitative data, subjects made judgments about incisional healing on the basis of information from actual patients. Multidimensional scaling was used to reveal conceptual structure, and lens modeling was applied to assess sensitivity to broad patterns. An information board task with think-aloud protocols demonstrated judgment process. The selection of tasks was based on their analysis- or intuition-inducing features, using K. R. Hammond's (1990) cognitive continuum theory. Experience accounted for a only a small proportion of variance in performance, whereas confidence in judgment was more strongly related to experience. Taken together, these findings replicated previous research. Protocol data showed that metacognition, knowledge accessibility, and reflectivity increased with experience. Conceptual structure predicted judgment accuracy under intuitive conditions. Support was found for Dreyfus and Dreyfus' (1986) hypothesized transition in cognition, from deliberate processing of discrete cues, to intuitive processing of patterns of cues encoded in memories for specific cases. This study has theoretical significance by adding to knowledge about clinical judgment, and by increasing understanding of cognitive changes associated with expertise. This study has practical significance in providing direction for the development of teaching methods aimed to increase learning from experience in probabilistic contexts. / Education, Faculty of / Educational and Counselling Psychology, and Special Education (ECPS), Department of / Graduate

High molecular weight mouse nerve growth factor(H M W-NGF), in addition to its effects on certain neural elements, is also chemotactic for human polymorphonuclear leukocytes. One of the subunits of H M W-NGF is a protease of the serine family and its active site contains a serine residue and a closely-neighboring histidine residue that are both essential for proteolysis. Elimination of enzyme activity by irreversibly blocking the single serine has no effect on leukotaxis, but blocking the histidine abolishes leukotaxis. These results suggest the possibility that part of the proteolytic active site of this enzyme may have evolved to perform more than one, completely different, biologic function - proteolysis as well as nonproteolytically mediated chemotaxis.

BACKGROUND: Diabetes is a worldwide epidemic, and the number of those affected is only growing. Diabetes is characterized by hyperglycemia due to the body’s inability to produce or properly use insulin. Hyperglycemia contributes to diabetic complications in several ways, one of which is promoting glycation. Glycation is the non-enzymatic glucosylation of proteins, and because glycation is adventitious, the process most commonly occurs on proteins with long half-lives, such as collagen. Glycation greatly changes collagen’s mechanical and biochemical properties. Glycation leads to the production of advanced glycation end products (AGEs) that have been shown to contribute to the complications seen in diabetes in one of two ways: establishment of crosslinks between molecules in the basement membrane of the extracellular matrix, altering cellular function, or interactions between AGEs and AGE receptors on the cell surface. Diabetes greatly impairs the body’s ability to heal wounds, and it is thought that the AGEs produced by glycation greatly contribute this phenomenon. However, it is not fully understood, what direct role AGEs and glycated collagen plays in the wound healing process. Three-dimensional microtissue models have been developed for the purpose of studying wound healing, and the creation of a three-dimensional microtissue with glycated collagen allows for investigation into the specific role that glycated collagen plays on both the mechanical and biochemical properties of the wound closure and the healing process. METHODS: In order to study the effect of glycated collagen on wound healing, a protocol to make glycated collagen must first be developed. To make glycated collagen, soluble rat-tail type I collagen will be incubated with 250mM ribose at 4°C for a minimum of five days to allow the collagen to become glycated. The glycated collagen will be used to make a collagen gel, and then papain buffer will digest the gel. The extent of glycation will be determined through quantifying the digested glycated collagen gel’s autofluorescence, absorbance, and changes that can be perceived visually. Once it is confirmed that the collagen has been glycated, it will be incorporated into a microtissue model based on a previously published protocol. The microtissue will then be wounded with a micromanipulator and 16-gauge needle, and visualized via time-lapse microscopy. The rate at which the wound closes will be compared in microtissues made with glycated collagen to those made with non-glycated collagen. RESULTS: Glycation of collagen was unable to be confirmed consistently by measuring the autofluorescence of the collagen gel digests. However, the absorbance of the collagen gel digest was used to determine that the collagen was 43.16% glycated and visual changes in the collagen gels made with glycated collagen was also observed. Microtissues were able to successfully form with the glycated collagen, and were able to be used to compare wound healing in normal microtissues against those made with glycated collagen.

Bats (Order Chiroptera) are the only mammals capable of power flight. The flight membranes of bats are not only essential for locomotion, but also play vital roles in homeostasis. Although understanding wound healing in the flight membranes of bats is important because injuries in the wild are common, with the recent emergence of white-nose syndrome, understanding wound healing in bat flight membranes has become even more important. In order to conduct my studies on wound healing in the flight membranes of bats, it was necessary to manually restrain bats. In this thesis I present a novel bat restrainer that I designed and that reduces stress experienced by restrained bats during experimentation and data collection. Wound healing is an energy dependent process, as such it is expected that wound healing times will vary during periods of energy constraint (i.e. hibernation) and/or at times of peak demand (i.e. lactation). However, previous studies on wound healing have only looked at healing at times when there are no energy constraints. In thesis I aimed to better understand the effects of seasonality and reproduction on wound healing. Using an 8 mm circular punch, I inflicted biopsy wounds to the chiropatagium of healthy captive big brown bats, Eptesicus fuscus. I compared wound healing times between winter and summer seasons, and between reproductive (i.e. lactating) and non-reproductive females. As expected, wound healing times were longer during the winter months when bats are conserving energy. On the other hand, reproductive status did not have an effect on wound healing times. Although most bats heal, I observed impaired wound healing. This finding is important because it is the first time that impaired wound healing is reported in healthy bats. / Thesis / Master of Science (MSc)