Monitoring and analysis of internet traffic targeting unused address spaces

Today’s evolving networks are experiencing a large number of different attacks ranging from system break-ins, infection from automatic attack tools such as worms, viruses, trojan horses and denial of service (DoS). One important aspect of such attacks is that they are often indiscriminate and target Internet addresses without regard to whether they are bona fide allocated or not. Due to the absence of any advertised host services the traffic observed on unused IP addresses is by definition unsolicited and likely to be either opportunistic or malicious. The analysis of large repositories of such traffic can be used to extract useful information about both ongoing and new attack patterns and unearth unusual attack behaviors. However, such an analysis is difficult due to the size and nature of the collected traffic on unused address spaces. In this dissertation, we present a network traffic analysis technique which uses traffic collected from unused address spaces and relies on the statistical properties of the collected traffic, in order to accurately and quickly detect new and ongoing network anomalies. Detection of network anomalies is based on the concept that an anomalous activity usually transforms the network parameters in such a way that their statistical properties no longer remain constant, resulting in abrupt changes. In this dissertation, we use sequential analysis techniques to identify changes in the behavior of network traffic targeting unused address spaces to unveil both ongoing and new attack patterns. Specifically, we have developed a dynamic sliding window based non-parametric cumulative sum change detection techniques for identification of changes in network traffic. Furthermore we have introduced dynamic thresholds to detect changes in network traffic behavior and also detect when a particular change has ended. Experimental results are presented that demonstrate the operational effectiveness and efficiency of the proposed approach, using both synthetically generated datasets and real network traces collected from a dedicated block of unused IP addresses.

Heavy vehicle suspensions : testing and analysis

Transport regulators consider that, with respect to pavement damage, heavy vehicles (HVs) are the riskiest vehicles on the road network. That HV suspension design contributes to road and bridge damage has been recognised for some decades. This thesis deals with some aspects of HV suspension characteristics, particularly (but not exclusively) air suspensions. This is in the areas of developing low-cost in-service heavy vehicle (HV) suspension testing, the effects of larger-than-industry-standard longitudinal air lines and the characteristics of on-board mass (OBM) systems for HVs. All these areas, whilst seemingly disparate, seek to inform the management of HVs, reduce of their impact on the network asset and/or provide a measurement mechanism for worn HV suspensions. A number of project management groups at the State and National level in Australia have been, and will be, presented with the results of the project that resulted in this thesis. This should serve to inform their activities applicable to this research. A number of HVs were tested for various characteristics. These tests were used to form a number of conclusions about HV suspension behaviours. Wheel forces from road test data were analysed. A “novel roughness” measure was developed and applied to the road test data to determine dynamic load sharing, amongst other research outcomes. Further, it was proposed that this approach could inform future development of pavement models incorporating roughness and peak wheel forces. Left/right variations in wheel forces and wheel force variations for different speeds were also presented. This led on to some conclusions regarding suspension and wheel force frequencies, their transmission to the pavement and repetitive wheel loads in the spatial domain. An improved method of determining dynamic load sharing was developed and presented. It used the correlation coefficient between two elements of a HV to determine dynamic load sharing. This was validated against a mature dynamic loadsharing metric, the dynamic load sharing coefficient (de Pont, 1997). This was the first time that the technique of measuring correlation between elements on a HV has been used for a test case vs. a control case for two different sized air lines. That dynamic load sharing was improved at the air springs was shown for the test case of the large longitudinal air lines. The statistically significant improvement in dynamic load sharing at the air springs from larger longitudinal air lines varied from approximately 30 percent to 80 percent. Dynamic load sharing at the wheels was improved only for low air line flow events for the test case of larger longitudinal air lines. Statistically significant improvements to some suspension metrics across the range of test speeds and “novel roughness” values were evident from the use of larger longitudinal air lines, but these were not uniform. Of note were improvements to suspension metrics involving peak dynamic forces ranging from below the error margin to approximately 24 percent. Abstract models of HV suspensions were developed from the results of some of the tests. Those models were used to propose further development of, and future directions of research into, further gains in HV dynamic load sharing. This was from alterations to currently available damping characteristics combined with implementation of large longitudinal air lines. In-service testing of HV suspensions was found to be possible within a documented range from below the error margin to an error of approximately 16 percent. These results were in comparison with either the manufacturer’s certified data or test results replicating the Australian standard for “road-friendly” HV suspensions, Vehicle Standards Bulletin 11. OBM accuracy testing and development of tamper evidence from OBM data were detailed for over 2000 individual data points across twelve test and control OBM systems from eight suppliers installed on eleven HVs. The results indicated that 95 percent of contemporary OBM systems available in Australia are accurate to +/- 500 kg. The total variation in OBM linearity, after three outliers in the data were removed, was 0.5 percent. A tamper indicator and other OBM metrics that could be used by jurisdictions to determine tamper events were developed and documented. That OBM systems could be used as one vector for in-service testing of HV suspensions was one of a number of synergies between the seemingly disparate streams of this project.

Swept frequency biompedance analysis for the determination of body water compartments

Bioelectrical impedance analysis, (BIA), is a method of body composition analysis first investigated in 1962 which has recently received much attention by a number of research groups. The reasons for this recent interest are its advantages, (viz: inexpensive, non-invasive and portable) and also the increasing interest in the diagnostic value of body composition analysis. The concept utilised by BIA to predict body water volumes is the proportional relationship for a simple cylindrical conductor, (volume oc length2/resistance), which allows the volume to be predicted from the measured resistance and length. Most of the research to date has measured the body's resistance to the passage of a 50· kHz AC current to predict total body water, (TBW). Several research groups have investigated the application of AC currents at lower frequencies, (eg 5 kHz), to predict extracellular water, (ECW). However all research to date using BIA to predict body water volumes has used the impedance measured at a discrete frequency or frequencies. This thesis investigates the variation of impedance and phase of biological systems over a range of frequencies and describes the development of a swept frequency bioimpedance meter which measures impedance and phase at 496 frequencies ranging from 4 kHz to 1 MHz. The impedance of any biological system varies with the frequency of the applied current. The graph of reactance vs resistance yields a circular arc with the resistance decreasing with increasing frequency and reactance increasing from zero to a maximum then decreasing to zero. Computer programs were written to analyse the measured impedance spectrum and determine the impedance, Zc, at the characteristic frequency, (the frequency at which the reactance is a maximum). The fitted locus of the measured data was extrapolated to determine the resistance, Ro, at zero frequency; a value that cannot be measured directly using surface electrodes. The explanation of the theoretical basis for selecting these impedance values (Zc and Ro), to predict TBW and ECW is presented. Studies were conducted on a group of normal healthy animals, (n=42), in which TBW and ECW were determined by the gold standard of isotope dilution. The prediction quotients L2/Zc and L2/Ro, (L=length), yielded standard errors of 4.2% and 3.2% respectively, and were found to be significantly better than previously reported, empirically determined prediction quotients derived from measurements at a single frequency. The prediction equations established in this group of normal healthy animals were applied to a group of animals with abnormally low fluid levels, (n=20), and also to a group with an abnormal balance of extra-cellular to intracellular fluids, (n=20). In both cases the equations using L2/Zc and L2/Ro accurately and precisely predicted TBW and ECW. This demonstrated that the technique developed using multiple frequency bioelectrical impedance analysis, (MFBIA), can accurately predict both TBW and ECW in both normal and abnormal animals, (with standard errors of the estimate of 6% and 3% for TBW and ECW respectively). Isotope dilution techniques were used to determine TBW and ECW in a group of 60 healthy human subjects, (male. and female, aged between 18 and 45). Whole body impedance measurements were recorded on each subject using the MFBIA technique and the correlations between body water volumes, (TBW and ECW), and heighe/impedance, (for all measured frequencies), were compared. The prediction quotients H2/Zc and H2/Ro, (H=height), again yielded the highest correlation with TBW and ECW respectively with corresponding standard errors of 5.2% and 10%. The values of the correlation coefficients obtained in this study were very similar to those recently reported by others. It was also observed that in healthy human subjects the impedance measured at virtually any frequency yielded correlations not significantly different from those obtained from the MFBIA quotients. This phenomenon has been reported by other research groups and emphasises the need to validate the technique by investigating its application in one or more groups with abnormalities in fluid levels. The clinical application of MFBIA was trialled and its capability of detecting lymphoedema, (an excess of extracellular fluid), was investigated. The MFBIA technique was demonstrated to be significantly more sensitive, (P<.05), in detecting lymphoedema than the current technique of circumferential measurements. MFBIA was also shown to provide valuable information describing the changes in the quantity of muscle mass of the patient during the course of the treatment. The determination of body composition, (viz TBW and ECW), by MFBIA has been shown to be a significant improvement on previous bioelectrical impedance techniques. The merit of the MFBIA technique is evidenced in its accurate, precise and valid application in animal groups with a wide variation in body fluid volumes and balances. The multiple frequency bioelectrical impedance analysis technique developed in this study provides accurate and precise estimates of body composition, (viz TBW and ECW), regardless of the individual's state of health.

Basin analysis and economic geology of the Northern Mount Isa Basin

The Mount Isa Basin is a new concept used to describe the area of Palaeo- to Mesoproterozoic rocks south of the Murphy Inlier and inappropriately described presently as the Mount Isa Inlier. The new basin concept presented in this thesis allows for the characterisation of basin-wide structural deformation, correlation of mineralisation with particular lithostratigraphic and seismic stratigraphic packages, and the recognition of areas with petroleum exploration potential. The northern depositional margin of the Mount Isa Basin is the metamorphic, intrusive and volcanic complex here referred to as the Murphy Inlier (not the "Murphy Tectonic Ridge"). The eastern, southern and western boundaries of the basin are obscured by younger basins (Carpentaria, Eromanga and Georgina Basins). The Murphy Inlier rocks comprise the seismic basement to the Mount Isa Basin sequence. Evidence for the continuity of the Mount Isa Basin with the McArthur Basin to the northwest and the Willyama Block (Basin) at Broken Hill to the south is presented. These areas combined with several other areas of similar age are believed to have comprised the Carpentarian Superbasin (new term). The application of seismic exploration within Authority to Prospect (ATP) 423P at the northern margin of the basin was critical to the recognition and definition of the Mount Isa Basin. The Mount Isa Basin is structurally analogous to the Palaeozoic Arkoma Basin of Illinois and Arkansas in southern USA but, as with all basins it contains unique characteristics, a function of its individual development history. The Mount Isa Basin evolved in a manner similar to many well described, Phanerozoic plate tectonic driven basins. A full Wilson Cycle is recognised and a plate tectonic model proposed. The northern Mount Isa Basin is defined as the Proterozoic basin area northwest of the Mount Gordon Fault. Deposition in the northern Mount Isa Basin began with a rift sequence of volcaniclastic sediments followed by a passive margin drift phase comprising mostly carbonate rocks. Following the rift and drift phases, major north-south compression produced east-west thrusting in the south of the basin inverting the older sequences. This compression produced an asymmetric epi- or intra-cratonic clastic dominated peripheral foreland basin provenanced in the south and thinning markedly to a stable platform area (the Murphy Inlier) in the north. The fmal major deformation comprised east-west compression producing north-south aligned faults that are particularly prominent at Mount Isa. Potential field studies of the northern Mount Isa Basin, principally using magnetic data (and to a lesser extent gravity data, satellite images and aerial photographs) exhibit remarkable correlation with the reflection seismic data. The potential field data contributed significantly to the unravelling of the northern Mount Isa Basin architecture and deformation. Structurally, the Mount Isa Basin consists of three distinct regions. From the north to the south they are the Bowthorn Block, the Riversleigh Fold Zone and the Cloncurry Orogen (new names). The Bowthom Block, which is located between the Elizabeth Creek Thrust Zone and the Murphy Inlier, consists of an asymmetric wedge of volcanic, carbonate and clastic rocks. It ranges from over 10 000 m stratigraphic thickness in the south to less than 2000 min the north. The Bowthorn Block is relatively undeformed: however, it contains a series of reverse faults trending east-west that are interpreted from seismic data to be down-to-the-north normal faults that have been reactivated as thrusts. The Riversleigh Fold Zone is a folded and faulted region south of the Bowthorn Block, comprising much of the area formerly referred to as the Lawn Hill Platform. The Cloncurry Orogen consists of the area and sequences equivalent to the former Mount Isa Orogen. The name Cloncurry Orogen clearly distinguishes this area from the wider concept of the Mount Isa Basin. The South Nicholson Group and its probable correlatives, the Pilpah Sandstone and Quamby Conglomerate, comprise a later phase of now largely eroded deposits within the Mount Isa Basin. The name South Nicholson Basin is now outmoded as this terminology only applied to the South Nicholson Group unlike the original broader definition in Brown et al. (1968). Cored slimhole stratigraphic and mineral wells drilled by Amoco, Esso, Elf Aquitaine and Carpentaria Exploration prior to 1986, penetrated much of the stratigraphy and intersected both minor oil and gas shows plus excellent potential source rocks. The raw data were reinterpreted and augmented with seismic stratigraphy and source rock data from resampled mineral and petroleum stratigraphic exploration wells for this study. Since 1986, Comalco Aluminium Limited, as operator of a joint venture with Monument Resources Australia Limited and Bridge Oil Limited, recorded approximately 1000 km of reflection seismic data within the basin and drilled one conventional stratigraphic petroleum well, Beamesbrook-1. This work was the first reflection seismic and first conventional petroleum test of the northern Mount Isa Basin. When incorporated into the newly developed foreland basin and maturity models, a grass roots petroleum exploration play was recognised and this led to the present thesis. The Mount Isa Basin was seen to contain excellent source rocks coupled with potential reservoirs and all of the other essential aspects of a conventional petroleum exploration play. This play, although high risk, was commensurate with the enormous and totally untested petroleum potential of the basin. The basin was assessed for hydrocarbons in 1992 with three conventional exploration wells, Desert Creek-1, Argyle Creek-1 and Egilabria-1. These wells also tested and confrrmed the proposed basin model. No commercially viable oil or gas was encountered although evidence of its former existence was found. In addition to the petroleum exploration, indeed as a consequence of it, the association of the extensive base metal and other mineralisation in the Mount Isa Basin with hydrocarbons could not be overlooked. A comprehensive analysis of the available data suggests a link between the migration and possible generation or destruction of hydrocarbons and metal bearing fluids. Consequently, base metal exploration based on hydrocarbon exploration concepts is probably. the most effective technique in such basins. The metal-hydrocarbon-sedimentary basin-plate tectonic association (analogous to Phanerozoic models) is a compelling outcome of this work on the Palaeo- to Mesoproterozoic Mount lsa Basin. Petroleum within the Bowthom Block was apparently destroyed by hot brines that produced many ore deposits elsewhere in the basin.

Genetic Analysis of Tolerance to Rice Tungro Bacilliform Virus in Rice (Oryza sativa L.) Through Agroinoculation

Balimau Putih [an Indonesian cultivar tolerant to rice tungro bacilliform virus (RTBV)] was crossed with IR64 (RTBV, susceptible variety) to produce the three filial generations F1, F2 and F3. Agroinoculation was used to introduce RTBV into the test plants. RTBV tolerance was based on the RTBV level in plants by analysis of coat protein using enzyme-linked immunosorbent assay. The level of RTBV in cv. Balimau Putih was significantly lower than that of IR64 and the susceptible control, Taichung Native 1. Mean RTBV levels of the F1, F2 and F3 populations were comparable with one another and with the average of the parents. Results indicate that there was no dominance and an additive gene action may control the expression of tolerance to RTBV. Tolerance based on the level of RTBV coat protein was highly heritable (0.67) as estimated using the mean values of F3 lines, suggesting that selection for tolerance to RTBV can be performed in the early selfing generations using the technique employed in this study. The RTBV level had a negative correlation with plant height, but positive relationship with disease index value

Polybrominated diphenyl ether (PBDE) concentrations decrease with age : analysis of pooled human blood serum in the Australian population

Introduction Polybrominated diphenyl ethers (PBDEs) are considered to be a cost effective and efficient way to reduce the possibility of product ignition and inhibit the spread of fire, thereby limiting harm caused by fires. PBDEs are incorporated into a wide variety of manufactured products and are now considered an ubiquitous contaminant found worldwide in biological and environmental samples1 . In comparison to “traditional” persistent organic pollutants (POPs), the exposure modes of PBDEs in humans are less well defined, although dietary sources, inhalation (air/particulate matter) and dust ingestion have been reported 2-4. Limited investigations of population specific factors such as age or gender and PBDE concentrations report: no conclusive correlation by age in adults; higher concentrations in children ; similar concentrations in maternal and cord blood; and no gender differences. After preliminary findings of higher PBDE concentrations in children than in adults in Australia11 we sought to investigate at what age the PBDE concentrations peaked in an effort to focus exposure studies. This investigation involved the collection of blood samples from young age groups and the development of a simple model to predict PBDE concentrations by age in Australia.

Reliability analysis and economic equipment replacement appraisal for substation and sub-transmission systems with explicit inclusion of non-repairable failures

The modern society has come to expect the electrical energy on demand, while many of the facilities in power systems are aging beyond repair and maintenance. The risk of failure is increasing with the aging equipments and can pose serious consequences for continuity of electricity supply. As the equipments used in high voltage power networks are very expensive, economically it may not be feasible to purchase and store spares in a warehouse for extended periods of time. On the other hand, there is normally a significant time before receiving equipment once it is ordered. This situation has created a considerable interest in the evaluation and application of probability methods for aging plant and provisions of spares in bulk supply networks, and can be of particular importance for substations. Quantitative adequacy assessment of substation and sub-transmission power systems is generally done using a contingency enumeration approach which includes the evaluation of contingencies, classification of the contingencies based on selected failure criteria. The problem is very complex because of the need to include detailed modelling and operation of substation and sub-transmission equipment using network flow evaluation and to consider multiple levels of component failures. In this thesis a new model associated with aging equipment is developed to combine the standard tools of random failures, as well as specific model for aging failures. This technique is applied in this thesis to include and examine the impact of aging equipments on system reliability of bulk supply loads and consumers in distribution network for defined range of planning years. The power system risk indices depend on many factors such as the actual physical network configuration and operation, aging conditions of the equipment, and the relevant constraints. The impact and importance of equipment reliability on power system risk indices in a network with aging facilities contains valuable information for utilities to better understand network performance and the weak links in the system. In this thesis, algorithms are developed to measure the contribution of individual equipment to the power system risk indices, as part of the novel risk analysis tool. A new cost worth approach was developed in this thesis that can make an early decision in planning for replacement activities concerning non-repairable aging components, in order to maintain a system reliability performance which economically is acceptable. The concepts, techniques and procedures developed in this thesis are illustrated numerically using published test systems. It is believed that the methods and approaches presented, substantially improve the accuracy of risk predictions by explicit consideration of the effect of equipment entering a period of increased risk of a non-repairable failure.

Multiscale analysis of protein functions and stochastic modelling of gene transcriptional regulatory networks

Genomic and proteomic analyses have attracted a great deal of interests in biological research in recent years. Many methods have been applied to discover useful information contained in the enormous databases of genomic sequences and amino acid sequences. The results of these investigations inspire further research in biological fields in return. These biological sequences, which may be considered as multiscale sequences, have some specific features which need further efforts to characterise using more refined methods. This project aims to study some of these biological challenges with multiscale analysis methods and stochastic modelling approach. The first part of the thesis aims to cluster some unknown proteins, and classify their families as well as their structural classes. A development in proteomic analysis is concerned with the determination of protein functions. The first step in this development is to classify proteins and predict their families. This motives us to study some unknown proteins from specific families, and to cluster them into families and structural classes. We select a large number of proteins from the same families or superfamilies, and link them to simulate some unknown large proteins from these families. We use multifractal analysis and the wavelet method to capture the characteristics of these linked proteins. The simulation results show that the method is valid for the classification of large proteins. The second part of the thesis aims to explore the relationship of proteins based on a layered comparison with their components. Many methods are based on homology of proteins because the resemblance at the protein sequence level normally indicates the similarity of functions and structures. However, some proteins may have similar functions with low sequential identity. We consider protein sequences at detail level to investigate the problem of comparison of proteins. The comparison is based on the empirical mode decomposition (EMD), and protein sequences are detected with the intrinsic mode functions. A measure of similarity is introduced with a new cross-correlation formula. The similarity results show that the EMD is useful for detection of functional relationships of proteins. The third part of the thesis aims to investigate the transcriptional regulatory network of yeast cell cycle via stochastic differential equations. As the investigation of genome-wide gene expressions has become a focus in genomic analysis, researchers have tried to understand the mechanisms of the yeast genome for many years. How cells control gene expressions still needs further investigation. We use a stochastic differential equation to model the expression profile of a target gene. We modify the model with a Gaussian membership function. For each target gene, a transcriptional rate is obtained, and the estimated transcriptional rate is also calculated with the information from five possible transcriptional regulators. Some regulators of these target genes are verified with the related references. With these results, we construct a transcriptional regulatory network for the genes from the yeast Saccharomyces cerevisiae. The construction of transcriptional regulatory network is useful for detecting more mechanisms of the yeast cell cycle.

Correlation of the expression of human kallikrein-related peptidases 4 and 7 with the prognosis in oral squamous cell carcinoma

Background Very few articles have been written about the expression of kallikreins (KLK4 and KLK7) in oral cancers. Therefore, the purpose of this study was to examine and report on their prognostic potential. Methods Eighty archival blocks of primary oral cancers were sectioned and stained for KLK4 and KLK7 by immunohistochemistry. The percentage and the intensity of malignant keratinocyte staining were correlated with patient survival using Cox regression analysis. Results Both kallikreins were expressed strongly in the majority of tumor cells in 68 of 80 cases: these were mostly moderately or poorly differentiated neoplasms. Staining was particularly intense at the infiltrating front. Patients with intense staining had significantly shorter overall survival (p < .05). Conclusion This is the first observation on the patient survival influenced by kallikrein expression in oral carcinoma. The findings are consistent with those for carcinomas at other sites, in particular the prostate and ovary. KLK4 and/or KLK7 immunohistochemistry seems to have diagnostic and prognostic potential in this disease.

Particulate texture image analysis with applications

Texture analysis and textural cues have been applied for image classification, segmentation and pattern recognition. Dominant texture descriptors include directionality, coarseness, line-likeness etc. In this dissertation a class of textures known as particulate textures are defined, which are predominantly coarse or blob-like. The set of features that characterise particulate textures are different from those that characterise classical textures. These features are micro-texture, macro-texture, size, shape and compaction. Classical texture analysis techniques do not adequately capture particulate texture features. This gap is identified and new methods for analysing particulate textures are proposed. The levels of complexity in particulate textures are also presented ranging from the simplest images where blob-like particles are easily isolated from their back- ground to the more complex images where the particles and the background are not easily separable or the particles are occluded. Simple particulate images can be analysed for particle shapes and sizes. Complex particulate texture images, on the other hand, often permit only the estimation of particle dimensions. Real life applications of particulate textures are reviewed, including applications to sedimentology, granulometry and road surface texture analysis. A new framework for computation of particulate shape is proposed. A granulometric approach for particle size estimation based on edge detection is developed which can be adapted to the gray level of the images by varying its parameters. This study binds visual texture analysis and road surface macrotexture in a theoretical framework, thus making it possible to apply monocular imaging techniques to road surface texture analysis. Results from the application of the developed algorithm to road surface macro-texture, are compared with results based on Fourier spectra, the auto- correlation function and wavelet decomposition, indicating the superior performance of the proposed technique. The influence of image acquisition conditions such as illumination and camera angle on the results was systematically analysed. Experimental data was collected from over 5km of road in Brisbane and the estimated coarseness along the road was compared with laser profilometer measurements. Coefficient of determination R2 exceeding 0.9 was obtained when correlating the proposed imaging technique with the state of the art Sensor Measured Texture Depth (SMTD) obtained using laser profilometers.

Serum bone formation marker correlation with improved osseointegration in osteoporotic rats treated with simvastatin

Objective: Simvastatin has been shown to enhance osseointegration of pure titanium implants in osteoporotic rats. This study aimed to evaluate the relationship between the serum level of bone formation markers and the osseointegration of pure titanium implants in osteoporotic rats treated with simvastatin. Materials and methods: Fifty-four female Sprague Dawley rats, aged 3 months old, were randomly divided into three groups: Sham-operated group (SHAM; n=18), ovariectomized group (OVX; n=18), and ovariectomized with Simvastatin treatment group (OVX+SIM; n=18). Fifty-six days after ovariectomy, screw-shaped titanium implants were inserted into the tibiae. Simvastatin was administered orally at 5mg/kg each day after the placement of the implant in the OVX+SIM group. The animals were sacrificed at either 28 or 84 days after implantation and the undecalcified tissue sections were processed for histological analysis. Total alkaline phosphatase (ALP), bone specific alkaline phosphatase (BALP) and bone Gla protein (BGP) were measured in all animal sera collected at the time of euthanasia and correlated with the histological assessment of osseointegration. Results: The level of ALP in the OVX group was higher than the SHAM group at day 28, with no differences between the three groups at day 84. The level of BALP in the OVX+SIM group was significantly higher than both OVX and SHAM groups at days 28 and 84. Compared with day 28, the BALP level of all three groups showed a significant decrease at day 84. There were no significant differences in BGP levels between the three groups at day 28, but at day 84 the OVX+SIM group showed significantly higher levels than both the OVX and SHAM groups. There was a significant increase in BGP levels between days 28 and 84 in the OVX+SIM group. The serum bone marker levels correlated with the histological assessment showing reduced osseointegration in the OVX compared to the SHAM group which is subsequently reversed in the OVX+SIM group.

A review of road traffic noise indicators and their correlation with the LA10(18hour)

This paper outlines a study to determine the correlation between the LA10(18hour) and other road traffic noise indicators. It is based on a database comprising of 404 measurement locations including 947 individual days of valid noise measurements across numerous circumstances taken between November 2001 and November 2007. This paper firstly discusses the need and constraints on the indicators and their nature of matching a suitable indicator to the various road traffic noise dynamical characteristics. The paper then presents a statistical analysis of the road traffic noise monitoring data, correlating various indicators with the LA10(18hour) statistical indicator and provides a comprehensive table of linear correlations. There is an extended analysis on relationships across the night time period. The paper concludes with a discussion on the findings.

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.