Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

Developing a model of care to improve the health and well-being for Indigenous people receiving renal dialysis treatment

The high levels of end-stage renal disease among Indigenous Australians, particularly in remote areas of the country, are a serious public health concern. The magnitude of the problem is reflected in figures from the Australian and New Zealand Transplant and Dialysis Registry that show that Indigenous Australians experience end-stage renal disease at a rate almost 9–10 times higher than other non-Indigenous Australians. A majority of Indigenous Australians have to relocate to receive appropriate renal dialysis treatment. In some Australian states, renal treatment is based on self-care dialysis which allows those Indigenous Australians to be treated back in their community. Evidence clearly shows that reuniting renal patients with community and family improves overall health and well-being for those Indigenous Australians. With the appropriate resources, training, and support, self-care management of renal dialysis treatment is an effective way for Indigenous people with end-stage renal failure to be treated at home. In this context, the study was used to gain insight and further understanding of the impact that end-stage renal disease and renal dialysis treatment has had on the lives of Indigenous community members. The study findings are from 14 individually interviewed people from South East Queensland. Data from the interviews were analysed using a combination of thematic and content analysis. The study methodology was based on qualitative data principles where the Indigenous community members were able to share their experiences and journeys living with end-stage renal disease. Many of the experiences and understanding closely relate to the renal disease pattern and the treatment with other outside influences, such as social, cultural, and environmental influences, all having an equal impact. Each community member’s experience with end-stage renal disease is unique; some manage with family and medical support, while others try to manage independently. From the study, community members who managed their renal dialysis treatment independently were much more aware of their renal health status. The study provides recommendations towards a model of care to improve the health and well-being is based on self-care and self-determination principles.

Adherence to fish oil intervention in patients with chronic kidney disease

Objective: With growing recognition of the role of inflammation in the development of chronic and acute disease, fish oil is increasingly used as a therapeutic agent, but the nature of the intervention may pose barriers to adherence in clinical populations. Our objective was to investigate the feasibility of using a fish oil supplement in hemodialysis patients. ---------- Design: This was a nonrandomized intervention study.---------- Setting: Eligible patients were recruited at the Hemodialysis Unit of Wesley Hospital, Brisbane, Queensland, Australia. Patients The sample included 28 maintenance hemodialysis patients out of 43 eligible patients in the unit. Exclusion criteria included patients regularly taking a fish oil supplement at baseline, receiving hemodialysis for less than 3 months, or being unable to give informed consent.---------- Intervention: Eicosapentaenoic acid (EPA) was administered at 2000 mg/day (4 capsules) for 12 weeks. Adherence was measured at baseline and weekly throughout the study according to changes in plasma EPA, and was further measured subjectively by self-report.---------- Results: Twenty patients (74%) adhered to the prescription based on changes in plasma EPA, whereas an additional two patients self-reported good adherence. There was a positive relationship between fish oil intake and change in plasma EPA. Most patients did not report problems with taking the fish oil. Using the baseline data, it was not possible to characterize adherent patients.---------- Conclusions: Despite potential barriers, including the need to take a large number of prescribed medications already, 74% of hemodialysis patients adhered to the intervention. This study demonstrated the feasibility of using fish oil in a clinical population.

The use of ZIP and CART to model cryptosporidiosis in relation to climatic variables

This research assesses the potential impact of weekly weather variability on the incidence of cryptosporidiosis disease using time series zero-inflated Poisson (ZIP) and classification and regression tree (CART) models. Data on weather variables, notified cryptosporidiosis cases and population size in Brisbane were supplied by the Australian Bureau of Meteorology, Queensland Department of Health, and Australian Bureau of Statistics, respectively. Both time series ZIP and CART models show a clear association between weather variables (maximum temperature, relative humidity, rainfall and wind speed) and cryptosporidiosis disease. The time series CART models indicated that, when weekly maximum temperature exceeded 31°C and relative humidity was less than 63%, the relative risk of cryptosporidiosis rose by 13.64 (expected morbidity: 39.4; 95% confidence interval: 30.9–47.9). These findings may have applications as a decision support tool in planning disease control and risk management programs for cryptosporidiosis disease.

Associations among smoking status, lifestyle and lipoprotein subclasses

BACKGROUND: The relationship between cigarette smoking and cardiovascular disease is well established, yet the underlying mechanisms remain unclear. Although smokers have a more atherogenic lipid profile, this may be mediated by other lifestyle-related factors. Analysis of lipoprotein subclasses by the use of nuclear magnetic resonance spectroscopy (NMR) may improve characterisation of lipoprotein abnormalities. OBJECTIVE: We used NMR spectroscopy to investigate the relationships between smoking status, lifestyle-related risk factors, and lipoproteins in a contemporary cohort. METHODS: A total of 612 participants (360 women) aged 40–69 years at baseline (199021994) enrolled in the Melbourne Collaborative Cohort Study had plasma lipoproteins measured with NMR. Data were analysed separately by sex. RESULTS: After adjusting for lifestyle-related risk factors, including alcohol and dietary intake, physical activity, and weight, mean total low-density lipoprotein (LDL) particle concentration was greater for female smokers than nonsmokers. Both medium- and small-LDL particle concentrations contributed to this difference. Total high-density lipoprotein (HDL) and large-HDL particle concentrations were lower for female smokers than nonsmokers. The proportion with low HDL particle number was greater for female smokers than nonsmokers. For men, there were few smoking-related differences in lipoprotein measures. CONCLUSION: Female smokers have a more atherogenic lipoprotein profile than nonsmokers. This difference is independent of other lifestyle-related risk factors. Lipoprotein profiles did not differ greatly between male smokers and nonsmokers.

Throwing frame and performance of elite male seated shot-putters

Seated shot-putters rely on a customized assistive device called a throwing frame. Currently, the construction of each individual throwing frame is mainly driven by an empirical approach. One way to refine the conception is to improve the basic understanding of performance of seated shot-putters. The relationship between performance and throwing technique has been well described. Remarkably, the relationship between performance and throwing frame characteristics has received limited attention. The primary objective of this study was to present a cataloguing of characteristics of throwing frames used by seated shot-putters. This cataloguing consisted of defining and grouping 26 characteristics into three main categories (i.e., whole body, foot and upper limb specific characteristics) and seven sub-categories. The secondary objective of this study was to provide raw characterisations of the throwing frames for a group of athletes who participated in a world-class event. The characterisation consisted of describing the characteristics of each throwing frame. Potential relationships between characteristics, performance and classification were also identified. The cataloguing was achieved using a 6-step heuristic approach, involving expert opinions and the analysis of 215 attempts produced by 55 male athletes during the 2006 IPC Athletics World Championships. The distribution of samples across characteristics suggested a relevant level of comprehensiveness for the proposed cataloguing. The raw data, the profile of best athletes and the frequency of characteristics provided key benchmark information for construction of a throwing frame as well as coaching, classification and officiating. Analysis of data sets relating to characteristics, performance and classification were inconclusive.

Mesenchymal stem cells in osteoarthritis therapy : current status of theory, technology, and applications

Osteoarthritis (OA) is a chronic, non-inflammatory type of arthritis, which usually affects the movable and weight bearing joints of the body. It is the most common joint disease in human beings and common in elderly people. Till date, there are no safe and effective diseases modifying OA drugs (DMOADs) to treat the millions of patients suffering from this serious and debilitating disease. However, recent studies provide strong evidence for the use of mesenchymal stem cell (MSC) therapy in curing cartilage related disorders. Due to their natural differentiation properties, MSCs can serve as vehicles for the delivery of effective, targeted treatment to damaged cartilage in OA disease. In vitro, MSCs can readily be tailored with transgenes with anti-catabolic or pro-anabolic effects to create cartilage-friendly therapeutic vehicles. On the other hand, tissue engineering constructs with scaffolds and biomaterials holds promising biological cartilage therapy. Many of these strategies have been validated in a wide range of in vitro and in vivo studies assessing treatment feasibility or efficacy. In this review, we provide an outline of the rationale and status of stem-cell-based treatments for OA cartilage, and we discuss prospects for clinical implementation and the factors crucial for maintaining the drive towards this goal.

Optimising collection of footprint evidence from crime scenes and suspects

Analysis of either footprints or footwear impressions which have been recovered from a crime scene is a well known and well accepted part of forensic investigation. When this evidence is obtained by investigating officers, comparative analysis to a suspect’s evidence may be undertaken. This can be done either by the detectives or in some cases, podiatrists with experience in forensic analysis. Frequently asked questions of a podiatrist include; “What additional information should be collected from a suspect (for the purposes of comparison), and how should it be collected?” This paper explores the answers to these and related questions based on 20 years of practical experience in the field of crime scene analysis as it relates to podiatry and forensics. Elements of normal and abnormal foot function are explored and used to explain the high degree of variability in wear patterns produced by the interaction of the foot and footwear. Based on this understanding the potential for identifying unique features of the user and correlating this to footwear evidence becomes apparent. Standard protocols adopted by podiatrists allow for more precise, reliable, and valid results to be obtained from their analysis. Complex data sets are now being obtained by investigating officers and, in collaboration with the podiatrist; higher quality conclusions are being achieved. This presentation details the results of investigations which have used standard protocols to collect and analyse footwear and suspects of recent major crimes.

Geometry vs appearance for discriminating between posed and spontaneous emotions

Spontaneous facial expressions differ from posed ones in appearance, timing and accompanying head movements. Still images cannot provide timing or head movement information directly. However, indirectly the distances between key points on a face extracted from a still image using active shape models can capture some movement and pose changes. This information is superposed on information about non-rigid facial movement that is also part of the expression. Does geometric information improve the discrimination between spontaneous and posed facial expressions arising from discrete emotions? We investigate the performance of a machine vision system for discrimination between posed and spontaneous versions of six basic emotions that uses SIFT appearance based features and FAP geometric features. Experimental results on the NVIE database demonstrate that fusion of geometric information leads only to marginal improvement over appearance features. Using fusion features, surprise is the easiest emotion (83.4% accuracy) to be distinguished, while disgust is the most difficult (76.1%). Our results find different important facial regions between discriminating posed versus spontaneous version of one emotion and classifying the same emotion versus other emotions. The distribution of the selected SIFT features shows that mouth is more important for sadness, while nose is more important for surprise, however, both the nose and mouth are important for disgust, fear, and happiness. Eyebrows, eyes, nose and mouth are important for anger.

Lessons learned from implementation of a demonstration program to reduce the burden of anemia and hookworm in women in Yen Bai Province, Viet Nam

Background Iron deficiency, anemia and hookworm disease are important public health problems for women of reproductive age living in developing countries and affect the health of newborns and infants. Iron supplementation and deworming treatment are effective in addressing these problems in both pregnant and non-pregnant women. Daily iron supplementation and deworming after the first trimester is recommended for pregnant women although these programs usually do not operate efficiently or effectively. Weekly iron-folic acid supplementation and regular deworming for non-pregnant women may be a viable approach for improving iron status and preventing anemia during the reproductive years. Addressing these diseases at a population level before women become pregnant could significantly improve women's health before and during pregnancy, as well as their infants' growth and development. Methods and Results This paper describes the major processes undertaken in a demonstration intervention of preventive weekly iron-folic acid supplementation with regular deworming for all 52,000 women aged 15–45 years in two districts of Yen Bai province, in northern Viet Nam. The intervention strategy included extensive consultation with community leaders and village, commune, district and provincial health staff, and training for village health workers. Distribution of the drugs was integrated with the existing health service infrastructure and the village health workers were the direct point of contact with women. Iron-folic acid tablets and deworming treatment were provided free of charge from May 2006. An independent Vietnamese NGO was commissioned to evaluate compliance and identify potential problems. The program resulted in effective distribution of iron-folic acid tablets and deworming treatment to all villages in the target districts, with full or partial compliance of 85%. Conclusion Training for health staff, the strong commitment of all partners and the use of appropriate educational materials led to broad support for weekly iron-folic acid supplementation and high participation in the regular deworming days. In March 2008 the program was expanded to all districts in the province, a target population of approximately 250,000 WRA, and management was handed over to provincial authorities.

The role of insulin and IGF2 signalling on metabolic pathways in prostate cancer progression

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in Australia, North America, and Europe. If found early and locally confined, CaP can be treated with radical prostatectomy or radiation therapy; however, 25-40% patients will relapse and go on to advanced disease. The most common therapy in these cases is androgen deprivation therapy (ADT), which suppresses androgen production from the testis. Lack of the testicular androgen supply causes cells of the prostate to undergo apoptosis. However, in some cases the regression initially seen with ADT eventually gives way to a growth of a population of cancerous cells that no longer require testicular androgens. This phenotype is essentially fatal and is termed castrate resistant prostate cancer (CRPC). In addition to eventual regression, there are many undesirable side effects which accompany ADT, including development of a metabolic syndrome, which is defined by the U.S. National Library of Medicine as “a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes.” This project will focus on the effect of ADT induced hyperinsulinemia, as mimicked by treating androgen receptor positive CaP cells with insulin in a serum (hormone) deprived environment. While this side effect is not widely explored, in this thesis it is demonstrated for the first time that insulin upregulates pathways important to CaP progression. Our group has previously shown that during CaP progression, the enzymes necessary for de novo steroidogenesis are upregulated in the LNCaP xenograft model, total steroid levels are increased in tumours compared to pre castrate levels, and de novo steroidogenesis from radio-labelled acetate has been demonstrated. Because of the CaP dependence on AR for survival, we and other groups believe that CaP cells carry out de novo steroidogenesis to survive in androgen deprived conditions. Because (a) men on ADT often develop metabolic syndrome, and (b) men with lifestyle-induced obesity and hyperinsulinemia have worse prognosis and faster disease progression, and because (c) insulin causes steroidogenesis in other cell lines, the hypothesis that insulin may contribute to CaP progression through upregulation of steroidogenesis was explored. Insulin upregulates steroidogenesis enzymes at the mRNA level in three AR positive cell lines, as well as upregulating these enzymes at the protein level in two cell lines. It has also been demonstrated that insulin increases mitochondrial (functional) levels of steroid acute regulatory protein (StAR). Furthermore, insulin causes increased levels of total steroids in and induction of de novo steroid synthesis by insulin has been demonstrated at levels induced sufficient to activate AR. The effect of insulin analogs on CaP steroidogenesis in LNCaP and VCaP cells has also been investigated because epidemiological studies suggest that some of the analogs developed may have more cancer stimulatory effects than normal insulin. In this project, despite the signalling differences between glargine, X10, and insulin, these analogs did not appear to induce steroidogenesis any more potently that normal insulin. The effect of insulin of MCF7breast cancer cells was also investigated with results suggesting that breast cancer cells may be capable of de novo steroidogenesis, and that increase in estradiol production may be exacerbated by insulin. Insulin has also been long known to stimulate lipogenesis in the liver and adipocytes, and has been demonstrated to increase lipogenesis in breast cancer cells; therefore, investigation of the effect of insulin on lipogenesis, which is a hallmark of aggressive cancers, was investigated. In CaP progression sterol regulatory element binding protein (SREBP) is dysregulated and upregulates fatty acid synthase (FASN), acetyl CoA-carboxylase, and other lipogenesis genes. SREBP is important for steroidogenesis and in this project has been shown to be upregulated by insulin in CaP cells. Fatty acid synthesis provides building blocks of membrane growth, provides substrates for acid oxidation, the main energy source for CaP cells, provides building blocks for anti-apoptotic and proinflammatory molecules, and provides molecules that stimulate steroidogenesis. In this project it has been shown that insulin upregulates FASN and ACC, which synthesize fatty acids, as well as upregulating hormone sensitive lipase (HSL), diazepam-binding inhibitor (DBI), and long-chain acyl-CoA synthetase 3 (ACSL3), which contribute to lipid activation of steroidogenesis. Insulin also upregulates total lipid levels and de novo lipogenesis, which can be suppressed by inhibition of the insulin receptor (INSR). The fatty acids synthesized after insulin treatment are those that have been associated with CaP; furthermore, microarray data suggests insulin may upregulate fatty acid biosynthesis, metabolism and arachidonic acid metabolism pathways, which have been implicated in CaP growth and survival. Pharmacological agents used to treat patients with hyperinsulinemia/ hyperlipidemia have gained much interest in regards to CaP risk and treatment; however, the scientific rationale behind these clinical applications has not been examined. This thesis explores whether the use of metformin or simvastatin would decrease either lipogenesis or steroidogenesis or both in CaP cells. Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitor, which blocks synthesis of cholesterol, the building block of steroids/ androgens. It has also been postulated to down regulate SREBP in other metabolic disorders. It has been shown in this thesis, in LNCaP cells, that simvastatin inhibited and decreased insulin induced steroidogenesis and lipogenesis, respectively, but increased these pathways in the absence of insulin. Conversely, metformin, which activates AMP-activated protein kinase (AMPK) to shut down lipogenesis, cholesterol synthesis, and protein synthesis, highly suppresses both steroidogenesis and lipogenesis in the presence and absence of insulin. Lastly, because it has been demonstrated to increase steroidogenesis in other cell lines, and because the elucidation of any factors affecting steroidogenesis is important to understanding CaP, the effect of IGF2 on steroidogenesis in CaP cells was investigated. In patient samples, as men progress to CRPC, IGF2 mRNA and the protein levels of the receptors it may signal through are upregulated. It has also been demonstrated that IGF2 upregulates steroidogenic enzymes at both the mRNA and protein levels in LNCaP cells, increases intracellular and secreted steroid/androgen levels in LNCaPs to levels sufficient to stimulate the AR, and upregulated de novo steroidogenesis in LNCaPs and VCaPs. As well, inhibition of INSR and insulin-like growth factor 1 receptor (IGF1R), which IGF2 signals through, suggests that induction of steroidogenesis may be occurring predominantly through IGF1R. In summary, this project has illuminated for the first time that insulin is likely to play a large role in cancer progression, through upregulation of the steroidogenesis and lipogenesis pathways at the mRNA and protein levels, and production levels, and demonstrates a novel role for IGF-II in CaP progression through stimulation of steroidogenesis. It has also been demonstrated that metformin and simvastatin drugs may be useful in suppressing the insulin induction of these pathways. This project affirms the pathways by which ADT- induced metabolic syndrome may exacerbate CaP progression and strongly suggests that the monitoring and modulation of the metabolic state of CaP patients could have a strong impact on their therapeutic outcomes.

Footwear modifies coronal plane forefoot and sagittal plane hallux kinematics during stance phase of walking gait

Footwear is designed to reduce injury, and enhance performance. However, the effect footwear has on foot and ankle kinematics currently remains unknown. Acknowledging the need for improved understanding, multi-segment models of the foot-shoe complex need to be established to both describe and quantify the effect footwear has on the foot and ankle during stance phase of gait. The purpose of this study was to quantify how footwear alters the kinematics of the foot inside the shoe during stance phase of walking gait.

The expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.

A Systematic Review of kinematic models used in foot & ankle biomechanics

Over the past decade our understanding of foot function has increased significantly[1,2]. Our understanding of foot and ankle biomechanics appears to be directly correlated to advances in models used to assess and quantify kinematic parameters in gait. These advances in models in turn lead to greater detail in the data. However, we must consider that the level of complexity is determined by the question or task being analysed. This systematic review aims to provide a critical appraisal of commonly used marker sets and foot models to assess foot and ankle kinematics in a wide variety of clinical and research purposes.

Interleukin-13 promotes susceptibility to chlamydial infection of the respiratory and genital tracts

Chlamydiae are intracellular bacteria that commonly cause infections of the respiratory and genital tracts, which are major clinical problems. Infections are also linked to the aetiology of diseases such as asthma, emphysema and heart disease. The clinical management of infection is problematic and antibiotic resistance is emerging. Increased understanding of immune processes that are involved in both clearance and immunopathology of chlamydial infection is critical for the development of improved treatment strategies. Here, we show that IL-13 was produced in the lungs of mice rapidly after Chlamydia muridarum (Cmu) infection and promoted susceptibility to infection. Wild-type (WT) mice had increased disease severity, bacterial load and associated inflammation compared to IL-13 deficient (−/−) mice as early as 3 days post infection (p.i.). Intratracheal instillation of IL-13 enhanced bacterial load in IL-13−/− mice. There were no differences in early IFN-g and IL-10 expression between WT and IL-13−/− mice and depletion of CD4+ T cells did not affect infection in IL-13−/− mice. Collectively, these data demonstrate a lack of CD4+ T cell involvement and a novel role for IL-13 in innate responses to infection. We also showed that IL-13 deficiency increased macrophage uptake of Cmu in vitro and in vivo. Moreover, the depletion of IL-13 during infection of lung epithelial cells in vitro decreased the percentage of infected cells and reduced bacterial growth. Our results suggest that enhanced IL-13 responses in the airways, such as that found in asthmatics, may promote susceptibility to chlamydial lung infection. Importantly the role of IL-13 in regulating infection was not limited to the lung as we showed that IL-13 also promoted susceptibility to Cmu genital tract infection. Collectively our findings demonstrate that innate IL-13 release promotes infection that results in enhanced inflammation and have broad implications for the treatment of chlamydial infections and IL-13-associated diseases.