A comparison of the 27-item and 12-item intolerance of uncertainty scales

The 27-item Intolerance of Uncertainty Scale (IUS) has become one of the most frequently used measure of Intolerance of Uncertainty. More recently, an abridged, 12-item version of the IUS has been developed. The current research used clinical (n = 50) and non-clinical (n = 56) samples to examine and compare the psychometric properties of both versions of the IUS. The two scales showed good internal consistency at both the total and subscale level and had satisfactory test-retest reliability. Both versions were correlated with worry and trait anxiety and had satisfactory concurrent validity. Significant differences between the scores of the clinical and non-clinical sample supported discriminant validity. Predictive validity was also supported for the two scales. Total scores, in the case of the clinical sample, and a subscale, in the case of the non-clinical sample, significantly predicted pathological worry and trait anxiety. Overall, the clinicians and researchers can use either version of the IUS with confidence, due to their sound psychometric properties.

Subchondral bone osteoblasts can induce chondrocyte mineralization during osteoarthritis and this process is relevant to cartilage degradation

Pathological mineralization of articular cartilage is a characteristic feature of osteoarthritis (OA); however, the underlying mechanisms, and their relevance to cartilage degeneration, are not clear. The involvement of subchondral bone changes in OA have been reported previously with the characterization of abnormal subchondral bone mineral density (BMD), osteiod volume, altered bone mechanical parameters and an increase in bone turnover markers. A number of osteoarthritic animal models have demonstrated that subchondral bone changes often precede cartilage degeneration. In this study site specific localization of mineralization markers were detected in the OA cartilage. Chondrocytes and osteoblasts derived from OA cartilage and subchondral bone showed a significant increase in the mRNA expressions of mineralization markers. Interestingly, osteoblasts from OA subchondral bone could significantly decrease cartilage matrix expression; whereas, increase mineralization of chondrocytes (Figure 1). Osteogenic factors, such as CBFA1, ALP, and type X collagen (Col-X), were detected in chondrocytes under mineralization conditions (Figure 2). Furthermore, chondrocyte mineralization was followed by increased mRNA and protein levels of MMP-2, MMP-9 and MMP-13, all of which are detrimental to cartilage integrity in vivo. The data reported here suggests that the upregulation of subchondral bone-mineralization, typical of OA progression, causes cartilage mineralization, and that the mineralization of chondrocytes induce increased MMP levels with a subsequent degradation of the articular cartilage.

Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo a typical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA related disorders.

Modeling prostate cancer: a perspective on transgenic mouse models

Despite considerable success in treatment of early stage localized prostate cancer (PC), acute inadequacy of late stage PC treatment and its inherent heterogeneity poses a formidable challenge. Clearly, an improved understanding of PC genesis and progression along with the development of new targeted therapies are warranted. Animal models, especially, transgenic immunocompetent mouse models, have proven to be the best ally in this respect. A series of models have been developed by modulation of expression of genes implicated in cancer-genesis and progression; mainly, modulation of expression of oncogenes, steroid hormone receptors, growth factors and their receptors, cell cycle and apoptosis regulators, and tumor suppressor genes have been used. Such models have contributed significantly to our understanding of the molecular and pathological aspects of PC initiation and progression. In particular, the transgenic mouse models based on multiple genetic alterations can more accurately address the inherent complexity of PC, not only in revealing the mechanisms of tumorigenesis and progression but also for clinically relevant evaluation of new therapies. Further, with advances in conditional knockout technologies, otherwise embryonically lethal gene changes can be incorporated leading to the development of new generation transgenics, thus adding significantly to our existing knowledge base. Different models and their relevance to PC research are discussed.

Replication of the Frank-Starling response in a Mock Circulation loop

Mock circulation loops (MCLs) are used to evaluate cardiovascular devices prior to in-vivo trials; however they lack the vital autoregulatory responses that occur in humans. This study aimed to develop and implement a left and right ventricular Frank-Starling response in a MCL. A proportional controller based on ventricular end diastolic volume was used to control the driving pressure of the MCL’s pneumatically operated ventricles. Ventricular pressure-volume loops and end systolic pressure-volume relationships were produced for a variety of healthy and pathological conditions and compared with human data to validate the simulated Frank-Starling response. The non-linear Frank-Starling response produced in this study successfully altered left and right ventricular contractility with changing preload and was validated with previously reported data. This improvement to an already detailed MCL has resulted in a test rig capable of further refining cardiovascular devices and reducing the number of in-vivo trials.

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

Clinical strategies for the alleviation of contractures from a predictive mathematical model of dermal repair

Hypertrophic scars arise when there is an overproduction of collagen during wound healing. These are often associated with poor regulation of the rate of programmed cell death(apoptosis) of the cells synthesizing the collagen or by an exuberant inflammatory response that prolongs collagen production and increases wound contraction. Severe contractures that occur, for example, after a deep burn can cause loss of function especially if the wound is over a joint such as the elbow or knee. Recently, we have developed a morphoelastic mathematical model for dermal repair that incorporates the chemical, cellular and mechanical aspects of dermal wound healing. Using this model, we examine pathological scarring in dermal repair by first assuming a smaller than usual apoptotic rate for myofibroblasts, and then considering a prolonged inflammatory response, in an attempt to determine a possible optimal intervention strategy to promote normal repair, or terminate the fibrotic scarring response. Our model predicts that in both cases it is best to apply the intervention strategy early in the wound healing response. Further, the earlier an intervention is made, the less aggressive the intervention required. Finally, if intervention is conducted at a late time during healing, a significant intervention is required; however, there is a threshold concentration of the drug or therapy applied, above which minimal further improvement to wound repair is obtained.

Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

Development of novel vaccine strategies to prevent genital tract chlamydial infections

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection in the developed world and the leading cause of preventable blindness worldwide. As reported by the World Health Organization in 2001, there are approximately 92 million new infections detected annually, costing health systems billions of dollars to treat not only the acute infection, but also to treat infection-associated sequelae. The majority of genital infections are asymptomatic, with 50-70% going undetected. Genital tract infections can be easily treated with antibiotics when detected. Lack of treatment can lead to the development of pelvic inflammatory disease, ectopic pregnancies and tubal factor infertility in women and epididymitis and prostatitis in men. With infection rates on the continual rise and the large number of infections going undetected, there is a need to develop an efficacious vaccine which prevents not only infection, but also the development of infection-associated pathology. Before a vaccine can be developed and administered, the pathogenesis of chlamydial infections needs to be fully understood. This includes the kinetics of ascending infection and the effects of inoculating dose on ascension and development of pathology. The first aim in this study was to examine these factors in a murine model. Female BALB/c mice were infected intravaginally with varying doses of C. muridarum, the mouse variant of human C. trachomatis, and the ascension of infection along the reproductive tract and the time-course of infection-associated pathology development, including inflammatory cell infiltration, pyosalpinx and hydrosalpinx, were determined. It was found that while the inoculating dose did affect the rate and degree of infection, it did not affect any of the pathological parameters examined. This highlighted that the sexual transmission dose may have minimal effect on the development of reproductive sequelae. The results of the first section enabled further studies presented here to use an optimal inoculating dose that would ascend the reproductive tract and cause pathology development, so that vaccine efficacy could be determined. There has been a large amount of research into the development of an efficacious vaccine against genital tract chlamydial infections, with little success. However, there have been no studies examining the effects of the timing of vaccination, including the effects of vaccination during an active genital infection, or after clearance of a previous infection. These are important factors that need to be examined, as it is not yet known whether immunization will enhance not only the individual's immune response, but also pathology development. It is also unknown whether any enhancement of the immune responses will cause the Chlamydia to enter a dormant, persistent state, and possibly further enhance any pathology development. The second section of this study aimed to determine if vaccination during an active genital tract infection, or after clearance of a primary infection, enhanced the murine immune responses and whether any enhanced or reduced pathology occurred. Naïve, actively infected, or previously infected animals were immunized intranasally or transcutaneously with the adjuvants cholera toxin and CpG-ODN in combination with either the major outer membrane protein (MOMP) of C. muridarum, or MOMP and ribonucleotide reductase small chain protein (NrdB) of C. muridarum. It was found that the systemic immune responses in actively or previously infected mice were altered in comparison to animals immunized naïve with the same combinations, however mucosal antibodies were not enhanced. It was also found that there was no difference in pathology development between any of the groups. This suggests that immunization of individuals who may have an asymptomatic infection, or may have been previously exposed to a genital infection, may not benefit from vaccination in terms of enhanced immune responses against re-exposure. The final section of this study aimed to determine if the vaccination regimes mentioned above caused in vivo persistence of C. muridarum in the upper reproductive tracts of mice. As there has been no characterization of C. muridarum persistence in vitro, either ultrastructurally or via transcriptome analysis, this was the first aim of this section. Once it had been shown that C. muridarum could be induced into a persistent state, the gene transcriptional profiles of the selected persistent marker genes were used to determine if persistent infections were indeed present in the upper reproductive tracts of the mice. We found that intranasal immunization during an active infection induced persistent infections in the oviducts, but not the uterine horns, and that intranasal immunization after clearance of infection, caused persistent infections in both the uterine horns and the oviducts of the mice. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. It is possible that the induction of persistent infections in the reproductive tract might enhance the development of pathology and thereby enhance the risk of infertility, factors that need to be prevented by vaccination, not enhanced. Overall, this study has shown that the inoculating dose does not affect pathology development in the female reproductive tract of infected mice, but does alter the degree and rate of ascending infection. It has also been shown that intranasal immunization during an active genital infection, or after clearance of one, induces persistent infections in the uterine horns and oviducts of mice. This suggests that potential vaccine candidates will need to have these factors closely examined before progressing to clinical trials. This is significant, because if the same situation occurs in humans, a vaccine administered to an asymptomatic, or previously exposed individual may not afford any extra protection and may in fact enhance the risk of development of infection-associated sequelae. This suggests that a vaccine may serve the community better if administered before the commencement of sexual activity.

Modelling the interaction of keratinocytes and fibroblasts during normal and abnormal wound healing processes

The crosstalk between fibroblasts and keratinocytes is a vital component of the wound healing process, and involves the activity of a number of growth factors and cytokines. In this work, we develop a mathematical model of this crosstalk in order to elucidate the effects of these interactions on the regeneration of collagen in a wound that heals by second intention. We consider the role of four components that strongly affect this process: transforming growth factor-beta, platelet-derived growth factor, interleukin-1 and keratinocyte growth factor. The impact of this network of interactions on the degradation of an initial fibrin clot, as well as its subsequent replacement by a matrix that is mainly comprised of collagen, is described through an eight-component system of nonlinear partial differential equations. Numerical results, obtained in a two-dimensional domain, highlight key aspects of this multifarious process such as reepithelialisation. The model is shown to reproduce many of the important features of normal wound healing. In addition, we use the model to simulate the treatment of two pathological cases: chronic hypoxia, which can lead to chronic wounds; and prolonged inflammation, which has been shown to lead to hypertrophic scarring. We find that our model predictions are qualitatively in agreement with previously reported observations, and provide an alternative pathway for gaining insight into this complex biological process.

Do anti-inflammatory agents promote linear ablation lesion discontinuities? : an electrophysiological examination

Background: Catheter ablation for atrial fibrillation (AF) is more efficacious than antiarrhythmic therapy. Post ablation recurrences reduce ablation effectiveness and are contributed by lesion discontinuity in the fibrotic linear ablation lesions. The anti-fibrotic role of statins in reducing AF is being assessed in current trials. By reducing the chronic pathological fibrosis that occurs in AF they may reduce AF. However if statins also have an effect on the acute therapeutic fibrosis of an ablation, this could exacerbate lesion discontinuity and AF recurrence. We tested the hypothesis that statins attenuate ablation lesion continuity in a recognised pig atrial linear ablation model. Aims: To assess whether Atorvastatin diminishes the bi-directional conduction block produced by a linear atrial ablation lesion. Methods: Sixteen pigs were randomised to statin (n=8) or placebo (n=8) with drug pre-treatment for 3 days and a further 4 weeks. At initial electrophysiological study (EPS1) 3D right atrium (RA) mapping and a vertical ablation linear lesion in the posterior RA with bidirectional conduction block were completed (Gepstein Circ 1999). Follow-up electrophysiological assessment (EPS2) at 28 days assessed bidirectional conduction block maintenance. Results: Data of 15/16 (statin=7) pigs were analysed. Mean lesion length was 3.7 ± 0.8cm with a mean of 17.9 ± 5.7 lesion applications. Bi-directional conduction block was confirmed in 15/15 pigs (100%) at EPS1 and EPS2. Conclusions: Atorvastatin did not affect ablation lesion continuity in this pig atrial linear ablation model. If patients are on long-term statins for AF reduction, periablation cessation is probably not necessary.

Investigating the potential value of individual parameters of histological grading systems in a sheep model of cartilage damage : the modified Mankin method

A total histological grade does not necessarily distinguish between different manifestations of cartilage damage or degeneration. An accurate and reliable histological assessment method is required to separate normal and pathological tissue within a joint during treatment of degenerative joint conditions and to sub-classify the latter in meaningful ways. The Modified Mankin method may be adaptable for this purpose. We investigated how much detail may be lost by assigning one composite score/grade to represent different degenerative components of the osteoarthritic condition. We used four ovine injury models (sham surgery, anterior cruciate ligament/medial collateral ligament instability, simulated anatomic anterior cruciate ligament reconstruction and meniscal removal) to induce different degrees and potentially 'types' (mechanisms) of osteoarthritis. Articular cartilage was systematically harvested, prepared for histological examination and graded in a blinded fashion using a Modified Mankin grading method. Results showed that the possible permutations of cartilage damage were significant and far more varied than the current intended use that histological grading systems allow. Of 1352 cartilage specimens graded, 234 different manifestations of potential histological damage were observed across 23 potential individual grades of the Modified Mankin grading method. The results presented here show that current composite histological grading may contain additional information that could potentially discern different stages or mechanisms of cartilage damage and degeneration in a sheep model. This approach may be applicable to other grading systems.

Differential expression of chemokine and matrix re-modelling genes is associated with contrasting schistosome-induced hepatopathology in murine models

The pathological outcomes of schistosomiasis are largely dependent on the molecular and cellular mechanisms of the host immune response. In this study, we investigated the contribution of variations in host gene expression to the contrasting hepatic pathology observed between two inbred mouse strains following Schistosoma japonicum infection. Whole genome microarray analysis was employed in conjunction with histological and immunohistochemical analysis to define and compare the hepatic gene expression profiles and cellular composition associated with the hepatopathology observed in S. japonicum-infected BALB/c and CBA mice. We show that the transcriptional profiles differ significantly between the two mouse strains with high statistical confidence. We identified specific genes correlating with the more severe pathology associated with CBA mice, as well as genes which may confer the milder degree of pathology associated with BALB/c mice. In BALB/c mice, neutrophil genes exhibited striking increases in expression, which coincided with the significantly greater accumulation of neutrophils at granulomatous regions seen in histological sections of hepatic tissue. In contrast, up-regulated expression of the eosinophil chemokine CCL24 in CBA mice paralleled the cellular influx of eosinophils to the hepatic granulomas. Additionally, there was greater down-regulation of genes involved in metabolic processes in CBA mice, reflecting the more pronounced hepatic damage in these mice. Profibrotic genes showed similar levels of expression in both mouse strains, as did genes associated with Th1 and Th2 responses. However, imbalances in expression of matrix metalloproteinases (e.g. MMP12, MMP13) and tissue inhibitors of metalloproteinases (TIMP1) may contribute to the contrasting pathology observed in the two strains. Overall, these results provide a more complete picture of the molecular and cellular mechanisms which govern the pathological outcome of hepatic schistosomiasis. This improved understanding of the immunopathogenesis in the murine model schistosomiasis provides the basis for a better appreciation of the complexities associated with chronic human schistosomiasis.

Near infrared spectroscopy for non-destructive evaluation of articular cartilage

There is a need for an accurate real-time quantitative system that would enhance decision-making in the treatment of osteoarthritis. To achieve this objective, significant research is required that will enable articular cartilage properties to be measured and categorized for health and functionality without the need for laboratory tests involving biopsies for pathological evaluation. Such a system would provide the capability of access to the internal condition of the cartilage matrix and thus extend the vision-based arthroscopy that is currently used beyond the subjective evaluation of surgeons. The system required must be able to non-destructively probe the entire thickness of the cartilage and its immediate subchondral bone layer. In this thesis, near infrared spectroscopy is investigated for the purpose mentioned above. The aim is to relate it to the structure and load bearing properties of the cartilage matrix to the near infrared absorption spectrum and establish functional relationships that will provide objective, quantitative and repeatable categorization of cartilage condition outside the area of visible degradation in a joint. Based on results from traditional mechanical testing, their innovative interpretation and relationship with spectroscopic data, new parameters were developed. These were then evaluated for their consistency in discriminating between healthy viable and degraded cartilage. The mechanical and physico-chemical properties were related to specific regions of the near infrared absorption spectrum that were identified as part of the research conducted for this thesis. The relationships between the tissue's near infrared spectral response and the new parameters were modeled using multivariate statistical techniques based on partial least squares regression (PLSR). With significantly high levels of statistical correlation, the modeled relationships were demonstrated to possess considerable potential in predicting the properties of unknown tissue samples in a quick and non-destructive manner. In order to adapt near infrared spectroscopy for clinical applications, a balance between probe diameter and the number of active transmit-receive optic fibres must be optimized. This was achieved in the course of this research, resulting in an optimal probe configuration that could be adapted for joint tissue evaluation. Furthermore, as a proof-of-concept, a protocol for obtaining the new parameters from the near infrared absorption spectra of cartilage was developed and implemented in a graphical user interface (GUI)-based software, and used to assess cartilage-on-bone samples in vitro. This conceptual implementation has been demonstrated, in part by the individual parametric relationship with the near infrared absorption spectrum, the capacity of the proposed system to facilitate real-time, non-destructive evaluation of cartilage matrix integrity. In summary, the potential of the optical near infrared spectroscopy for evaluating articular cartilage and bone laminate has been demonstrated in this thesis. The approach could have a spin-off for other soft tissues and organs of the body. It builds on the earlier work of the group at QUT, enhancing the near infrared component of the ongoing research on developing a tool for cartilage evaluation that goes beyond visual and subjective methods.

The roles of the preproghrelin-derived peptides - ghrelin, desacyl ghrelin and obestatin - in prostate cancer

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.