Robust pose estimation for a car-like vehicle

In this paper, we outline the sensing system used for the visual pose control of our experimental car-like vehicle, the autonomous tractor. The sensing system consists of a magnetic compass, an omnidirectional camera and a low-resolution odometry system. In this work, information from these sensors is fused using complementary filters. Complementary filters provide a means of fusing information from sensors with different characteristics in order to produce a more reliable estimate of the desired variable. Here, the range and bearing of landmarks observed by the vision system are fused with odometry information and a vehicle model, providing a more reliable estimate of these states. We also present a method of combining a compass sensor with odometry and a vehicle model to improve the heading estimate.

Visual servoing of a car-like vehicle - an application of omnidirectional vision

In this paper, we develop the switching controller presented by Lee et al. for the pose control of a car-like vehicle, to allow the use of an omnidirectional vision sensor. To this end we incorporate an extension to a hypothesis on the navigation behaviour of the desert ant, cataglyphis bicolor, which leads to a correspondence free landmark based vision technique. The method we present allows positioning to a learnt location based on feature bearing angle and range discrepancies between the robot's current view of the environment, and that at a learnt location. We present simulations and experimental results, the latter obtained using our outdoor mobile platform.

Gamma-tocotrienol as an effective agent in targeting prostate cancer stem cell-like population

Emerging evidence supports that prostate cancer originates from a rare sub-population of cells, namely prostate cancer stem cells (CSCs). Conventional therapies for prostate cancer are believed to mainly target the majority of differentiated tumor cells but spare CSCs, which may account for the subsequent disease relapse after treatment. Therefore, successful elimination of CSCs may be an effective strategy to achieve complete remission from this disease. Gamma-tocotrienols (-T3) is one of the vitamin-E constituents which have been shown to have anticancer effects against a wide-range of human cancers. Recently, we have reported that -T3 treatment not only inhibits prostate cancer cell invasion but also sensitizes the cells to docetaxel-induced apoptosis, suggesting that -T3 may be an effective therapeutic agent against advanced stage prostate cancer. Here, we demonstrate for the first time that -T3 can down-regulate the expression of prostate CSC markers (CD133/CD44) in androgen independent (AI) prostate cancer cell lines (PC-3 & DU145), as evident from western blotting analysis. Meanwhile, the spheroid formation ability of the prostate cancer cells was significantly hampered by -T3 treatment. In addition, pre-treatment of PC-3 cells with -T3 was found to suppress tumor initiation ability of the cells. More importantly, while CD133-enriched PC-3 cells were highly resistant to docetaxel treatment, these cells were as sensitive to -T3 treatment as the CD133-depleted population. Our data suggest that -T3 may be an effective agent in targeting prostate CSCs, which may account for its anticancer and chemosensitizing effects reported in previous studies.

Prandial subcutaneous injections of glucagon-like peptide-1 cause weight loss in obese human subjects

Recombinant glucagon-like peptide-1 (7–36)amide (rGLP-1) was recently shown to cause significant weight loss in type 2 diabetics when administered for 6 weeks as a continuous subcutaneous infusion. The mechanisms responsible for the weight loss are not clarified. In the present study, rGLP-1 was given for 5d by prandial subcutaneous injections (PSI) (76nmol 30min before meals, four times daily; a total of 302·4nmol/24h) or by continuous subcutaneous infusion (CSI) (12·7nmol/h; a total of 304·8nmol/24h). This was performed in nineteen healthy obese subjects (mean age 44·2 (sem 2·5) years; BMI 39·0 (sem 1·2)kg/m2) in a prospective randomised, double-blind, placebo-controlled, cross-over study. Compared with the placebo, rGLP-1 administered as PSI and by CSI generated a 15% reduction in mean food intake per meal (P=0·02) after 5d treatment. A weight loss of 0·55 (sem 0·2) kg (P<0·05) was registered after 5d with PSI of rGLP-1. Gastric emptying rate was reduced during both PSI (P<0·001) and CSI (P<0·05) treatment, but more rapidly and to a greater extent with PSI of rGLP-1. To conclude, a 5d treatment of rGLP-1 at high doses by PSI, but not CSI, promptly slowed gastric emptying as a probable mechanism of action of increased satiety, decreased hunger and, hence, reduced food intake with an ensuing weight loss.

The modulation of outdoor running speed : the influence of gradient

This thesis aimed to investigate the way in which distance runners modulate their speed in an effort to understand the key processes and determinants of speed selection when encountering hills in natural outdoor environments. One factor which has limited the expansion of knowledge in this area has been a reliance on the motorized treadmill which constrains runners to constant speeds and gradients and only linear paths. Conversely, limits in the portability or storage capacity of available technology have restricted field research to brief durations and level courses. Therefore another aim of this thesis was to evaluate the capacity of lightweight, portable technology to measure running speed in outdoor undulating terrain. The first study of this thesis assessed the validity of a non-differential GPS to measure speed, displacement and position during human locomotion. Three healthy participants walked and ran over straight and curved courses for 59 and 34 trials respectively. A non-differential GPS receiver provided speed data by Doppler Shift and change in GPS position over time, which were compared with actual speeds determined by chronometry. Displacement data from the GPS were compared with a surveyed 100m section, while static positions were collected for 1 hour and compared with the known geodetic point. GPS speed values on the straight course were found to be closely correlated with actual speeds (Doppler shift: r = 0.9994, p < 0.001, Δ GPS position/time: r = 0.9984, p < 0.001). Actual speed errors were lowest using the Doppler shift method (90.8% of values within ± 0.1 m.sec -1). Speed was slightly underestimated on a curved path, though still highly correlated with actual speed (Doppler shift: r = 0.9985, p < 0.001, Δ GPS distance/time: r = 0.9973, p < 0.001). Distance measured by GPS was 100.46 ± 0.49m, while 86.5% of static points were within 1.5m of the actual geodetic point (mean error: 1.08 ± 0.34m, range 0.69-2.10m). Non-differential GPS demonstrated a highly accurate estimation of speed across a wide range of human locomotion velocities using only the raw signal data with a minimal decrease in accuracy around bends. This high level of resolution was matched by accurate displacement and position data. Coupled with reduced size, cost and ease of use, the use of a non-differential receiver offers a valid alternative to differential GPS in the study of overground locomotion. The second study of this dissertation examined speed regulation during overground running on a hilly course. Following an initial laboratory session to calculate physiological thresholds (VO2 max and ventilatory thresholds), eight experienced long distance runners completed a self- paced time trial over three laps of an outdoor course involving uphill, downhill and level sections. A portable gas analyser, GPS receiver and activity monitor were used to collect physiological, speed and stride frequency data. Participants ran 23% slower on uphills and 13.8% faster on downhills compared with level sections. Speeds on level sections were significantly different for 78.4 ± 7.0 seconds following an uphill and 23.6 ± 2.2 seconds following a downhill. Speed changes were primarily regulated by stride length which was 20.5% shorter uphill and 16.2% longer downhill, while stride frequency was relatively stable. Oxygen consumption averaged 100.4% of runner’s individual ventilatory thresholds on uphills, 78.9% on downhills and 89.3% on level sections. Group level speed was highly predicted using a modified gradient factor (r2 = 0.89). Individuals adopted distinct pacing strategies, both across laps and as a function of gradient. Speed was best predicted using a weighted factor to account for prior and current gradients. Oxygen consumption (VO2) limited runner’s speeds only on uphill sections, and was maintained in line with individual ventilatory thresholds. Running speed showed larger individual variation on downhill sections, while speed on the level was systematically influenced by the preceding gradient. Runners who varied their pace more as a function of gradient showed a more consistent level of oxygen consumption. These results suggest that optimising time on the level sections after hills offers the greatest potential to minimise overall time when running over undulating terrain. The third study of this thesis investigated the effect of implementing an individualised pacing strategy on running performance over an undulating course. Six trained distance runners completed three trials involving four laps (9968m) of an outdoor course involving uphill, downhill and level sections. The initial trial was self-paced in the absence of any temporal feedback. For the second and third field trials, runners were paced for the first three laps (7476m) according to two different regimes (Intervention or Control) by matching desired goal times for subsections within each gradient. The fourth lap (2492m) was completed without pacing. Goals for the Intervention trial were based on findings from study two using a modified gradient factor and elapsed distance to predict the time for each section. To maintain the same overall time across all paced conditions, times were proportionately adjusted according to split times from the self-paced trial. The alternative pacing strategy (Control) used the original split times from this initial trial. Five of the six runners increased their range of uphill to downhill speeds on the Intervention trial by more than 30%, but this was unsuccessful in achieving a more consistent level of oxygen consumption with only one runner showing a change of more than 10%. Group level adherence to the Intervention strategy was lowest on downhill sections. Three runners successfully adhered to the Intervention pacing strategy which was gauged by a low Root Mean Square error across subsections and gradients. Of these three, the two who had the largest change in uphill-downhill speeds ran their fastest overall time. This suggests that for some runners the strategy of varying speeds systematically to account for gradients and transitions may benefit race performances on courses involving hills. In summary, a non – differential receiver was found to offer highly accurate measures of speed, distance and position across the range of human locomotion speeds. Self-selected speed was found to be best predicted using a weighted factor to account for prior and current gradients. Oxygen consumption limited runner’s speeds only on uphills, speed on the level was systematically influenced by preceding gradients, while there was a much larger individual variation on downhill sections. Individuals were found to adopt distinct but unrelated pacing strategies as a function of durations and gradients, while runners who varied pace more as a function of gradient showed a more consistent level of oxygen consumption. Finally, the implementation of an individualised pacing strategy to account for gradients and transitions greatly increased runners’ range of uphill-downhill speeds and was able to improve performance in some runners. The efficiency of various gradient-speed trade- offs and the factors limiting faster downhill speeds will however require further investigation to further improve the effectiveness of the suggested strategy.

Performance analysis of adaptive lattice filters for FM signals and alpha-stable processes

The performance of an adaptive filter may be studied through the behaviour of the optimal and adaptive coefficients in a given environment. This thesis investigates the performance of finite impulse response adaptive lattice filters for two classes of input signals: (a) frequency modulated signals with polynomial phases of order p in complex Gaussian white noise (as nonstationary signals), and (b) the impulsive autoregressive processes with alpha-stable distributions (as non-Gaussian signals). Initially, an overview is given for linear prediction and adaptive filtering. The convergence and tracking properties of the stochastic gradient algorithms are discussed for stationary and nonstationary input signals. It is explained that the stochastic gradient lattice algorithm has many advantages over the least-mean square algorithm. Some of these advantages are having a modular structure, easy-guaranteed stability, less sensitivity to the eigenvalue spread of the input autocorrelation matrix, and easy quantization of filter coefficients (normally called reflection coefficients). We then characterize the performance of the stochastic gradient lattice algorithm for the frequency modulated signals through the optimal and adaptive lattice reflection coefficients. This is a difficult task due to the nonlinear dependence of the adaptive reflection coefficients on the preceding stages and the input signal. To ease the derivations, we assume that reflection coefficients of each stage are independent of the inputs to that stage. Then the optimal lattice filter is derived for the frequency modulated signals. This is performed by computing the optimal values of residual errors, reflection coefficients, and recovery errors. Next, we show the tracking behaviour of adaptive reflection coefficients for frequency modulated signals. This is carried out by computing the tracking model of these coefficients for the stochastic gradient lattice algorithm in average. The second-order convergence of the adaptive coefficients is investigated by modeling the theoretical asymptotic variance of the gradient noise at each stage. The accuracy of the analytical results is verified by computer simulations. Using the previous analytical results, we show a new property, the polynomial order reducing property of adaptive lattice filters. This property may be used to reduce the order of the polynomial phase of input frequency modulated signals. Considering two examples, we show how this property may be used in processing frequency modulated signals. In the first example, a detection procedure in carried out on a frequency modulated signal with a second-order polynomial phase in complex Gaussian white noise. We showed that using this technique a better probability of detection is obtained for the reduced-order phase signals compared to that of the traditional energy detector. Also, it is empirically shown that the distribution of the gradient noise in the first adaptive reflection coefficients approximates the Gaussian law. In the second example, the instantaneous frequency of the same observed signal is estimated. We show that by using this technique a lower mean square error is achieved for the estimated frequencies at high signal-to-noise ratios in comparison to that of the adaptive line enhancer. The performance of adaptive lattice filters is then investigated for the second type of input signals, i.e., impulsive autoregressive processes with alpha-stable distributions . The concept of alpha-stable distributions is first introduced. We discuss that the stochastic gradient algorithm which performs desirable results for finite variance input signals (like frequency modulated signals in noise) does not perform a fast convergence for infinite variance stable processes (due to using the minimum mean-square error criterion). To deal with such problems, the concept of minimum dispersion criterion, fractional lower order moments, and recently-developed algorithms for stable processes are introduced. We then study the possibility of using the lattice structure for impulsive stable processes. Accordingly, two new algorithms including the least-mean P-norm lattice algorithm and its normalized version are proposed for lattice filters based on the fractional lower order moments. Simulation results show that using the proposed algorithms, faster convergence speeds are achieved for parameters estimation of autoregressive stable processes with low to moderate degrees of impulsiveness in comparison to many other algorithms. Also, we discuss the effect of impulsiveness of stable processes on generating some misalignment between the estimated parameters and the true values. Due to the infinite variance of stable processes, the performance of the proposed algorithms is only investigated using extensive computer simulations.

The mechanism of maturation of insulin-like growth factor-1

This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l- 3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however, detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed unlikely to participate in the formation of des(1-3)IGF-I. In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of an in vivo role for des(1-3)IGF-I, as reported by others. The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the presence of the dipeptide Gly-Leu. As part of attempts to detect tissue production of des(1-3)IGF-I, a monoclonal antibody (MAb ), directed towards the GPE- end ofiGF-I was produced by immunisation with a 10-mer covalently attached to a carrier protein. By the use of indirect ELISA and inhibitor studies, the MAb was shown to selectively recognise peptides with anNterminal GPE- sequence, and applied to the indirect detection of des(1-3)IGF-I. The concentration of GPE in brain, measured by mass spectrometry ( MS), was low, and the concentration of total IGF-I (measured by ELISA with a commercial polyclonal antibody [P Ab]) was 40 times higher at 50 nmol/kg. This also, was not consistent with the action of a tripeptidyl peptidase in brain that converted all IGF-I to des(1-3)IGF-I plus GPE. Contrasting ELISA results, using the MAb prepared in this study, suggest an even higher concentration of intact IGF-I of 150 nmollkg. This would argue against the presence of any des( 1-3 )IGF-I in brain, but in turn, this indicates either the presence of other substances containing a GPE amino-terminus or other cross reacting epitope. Although the results of the specificity studies reported in Chapter 5 would make this latter possibility seem unlikely, it cannot be completely excluded. No GP was detected in brain by MS. No GPE was detected in colostrum by capillary electrophoresis (CE) but the interference from extraneous substances reduced the detectability of GPE by CE and this approach would require further, prior, purification and concentration steps. A molecule, with a migration time equal to that of the peptide GP, was detected in colostrum by CE, but the concentration (~ 10 11mo/L) was much higher than the IGF-I concentration measured by radio-immunoassay using a PAb (80 nmol/L) or using a Mab (300-400 nmolL). A DPP IV enzyme was detected in colostrum and this could account for the GP, derived from substrates other than IGF-1. Based on the differential results of the two antibody assays, there was no indication of the presence of des(1-3)IGF-I in brain or colostrum. In the absence of any enzyme activity directed towards the amino terminus of IGF-I and the absence any potential products, IGF-I, therefore, does not appear to "mature" via des(1-3)IGF-I in the brain, nor in the neutral colostrum. In spite of these results which indicate the absence of an enzymic attack on IGF-I and the absence of the expected products in tissues, the possibility that the conversion of IGF-I may occur in neutral conditions in limited amounts, cannot be ruled out. It remains possible that in the extracellular environment of the membrane, a complex interaction of IGF-I, binding protein, aminopeptidase(s) and receptor, produces des(1- 3)IGF-I as a transient product which is bound to the receptor and internalised.

The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.