Studies of granulosa cell maturation in dominant and subordinate bovine follicles : novel extracellular matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage CYP11A1

During growth of antral ovarian follicles granulosa cells first become associated with a novel type of extracellular matrix, focimatrix, and at larger sizes follicles become either subordinate or dominant. To examine this, bovine subordinate (9.0±s.e.m. 0.4 mm; n=16), partially dominant (12.0±0.6 mm; n=18) and fully dominant (15.0±0.4 mm; n=14) follicles were examined by real time RT-PCR analyses of granulosa cells and by immunohistochemistry of focimatrix. Changes in the expression of FSH receptor, LH receptor, cholesterol side-chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase, aromatase (CYP19A1) and inhibin-α and β-B were observed as expected for follicle sizes examined. After adjusting for size differences, only CYP11A1 was significantly different between the groups, and elevated in dominant follicles. Also after adjusting for differences in size there were no significant differences in expression of focimatrix components collagen type IV α-1 (COL4A1), laminin β-2, nidogen 1 (NID1), and perlecan (HSPG2) or the volume density of NID1 and -2 and HSPG2. The volume density of focimatrix components in laminin 111 was significantly elevated in dominant follicles. Adjusting for analysis of more than one follicle per animal and for multiple correlations, CYP11A1 mRNA levels were highly correlated with the focimatrix genes COL4A1, NID1 and -2 and HSPG2. Thus, focimatrix may potentially regulate CYP11A1 expression, and the regulation of both could be important in follicular dominance.

Detection of bacteriophage and respiratory viruses in droplets

Influenza is a widespread disease occurring in seasonal epidemics, and each year is responsible for up to 500,000 deaths worldwide. Influenza can develop into strains which cause severe symptoms and high mortality rates, and could potentially reach pandemic status if the virus’ properties allow easy transmission. Influenza is transmissible via contact with the virus, either directly (infected people) or indirectly (contaminated objects); via reception of large droplets over short distances (one metre or less); or through inhalation of aerosols containing the virus expelled by infected individuals during respiratory activities, that can remain suspended in the air and travel distances of more than one metre (the aerosol route). Aerosol transmission of viruses involves three stages: production of the droplets containing viruses; transport of the droplets and ability of a virus to remain intact and infectious; and reception of the droplets (via inhalation). Our understanding of the transmission of influenza viruses via the aerosol route is poor, and thus our ability to prevent a widespread outbreak is limited. This study explored the fate of viruses in droplets by investigating the effects of some physical factors on the recovery of both a bacteriophage model and influenza virus. Experiments simulating respiratory droplets were carried out using different types of droplets, generated from a commonly used water-like matrix, and also from an ‘artificial mucous’ matrix which was used to more closely resemble respiratory fluids. To detect viruses in droplets, we used the traditional plaque assay techniques, and also a sensitive, quantitative PCR assay specifically developed for this study. Our results showed that the artificial mucous suspension enhanced the recovery of infectious bacteriophage. We were able to report detection limits of infectious bacteriophage (no bacteriophage was detected by the plaque assay when aerosolised from a suspension of 103 PFU/mL, for three of the four droplet types tested), and that bacteriophage could remain infectious in suspended droplets for up to 20 minutes. We also showed that the nested real-time PCR assay was able to detect the presence of bacteriophage RNA where the plaque assay could not detect any intact particles. Finally, when applying knowledge from the bacteriophage experiments, we reported the quantitative recoveries of influenza viruses in droplets, which were more consistent and stable than we had anticipated. Influenza viruses can be detected up to 20 minutes (after aerosolisation) in suspended aerosols and possibly beyond. It also was detectable from nebulising suspensions with relatively low concentrations of viruses.

An experimental study of a reactive plume in grid turbulence

Experimental results for a reactive non-buoyant plume of nitric oxide (NO) in a turbulent grid flow doped with ozone (O3) are presented. The Damkohler number (Nd) for the experiment is of order unity indicating the turbulence and chemistry have similar timescales and both affect the chemical reaction rate. Continuous measurements of two components of velocity using hot-wire anemometry and the two reactants using chemiluminescent analysers have been made. A spatial resolution for the reactants of four Kolmogorov scales has been possible because of the novel design of the experiment. Measurements at this resolution for a reactive plume are not found in the literature. The experiment has been conducted relatively close to the grid in the region where self-similarity of the plume has not yet developed. Statistics of a conserved scalar, deduced from both reactive and non-reactive scalars by conserved scalar theory, are used to establish the mixing field of the plume, which is found to be consistent with theoretical considerations and with those found by other investigators in non-reative flows. Where appropriate the reactive species means and higher moments, probability density functions, joint statistics and spectra are compared with their respective frozen, equilibrium and reaction-dominated limits deduced from conserved scalar theory. The theoretical limits bracket reactive scalar statistics where this should be so according to conserved scalar theory. Both reactants approach their equilibrium limits with greater distance downstream. In the region of measurement, the plume reactant behaves as the reactant not in excess and the ambient reactant behaves as the reactant in excess. The reactant covariance lies outside its frozen and equilibrium limits for this value of Vd. The reaction rate closure of Toor (1969) is compared with the measured reaction rate. The gradient model is used to obtain turbulent diffusivities from turbulent fluxes. Diffusivity of a non-reactive scalar is found to be close to that measured in non-reactive flows by others.

‘Spotty Dick’ : a research experiment to test design suitability for the use of jewelled spot matrix patterns for UV aware men’s swimwear

The great male Aussie cossie is growing spots. The ‘dick’ tog, as it is colloquially referred to, is linked to Australia’s national identify with overtly masculine bronzed Aussie bodies clothed in this iconic apparel. Yet the reality is our hunger for worshiping the sun and the addiction to a beach lifestyle is tempered by the pragmatic need for neck-to-knee, or more apt head-to-toe, swimwear. Spotty Dick is an irreverent play on male swimwear – it experiments with alternate modes to sheath the body with Lyrca in order to protect it from searing UV’s and at the same time light-heartedly fools around with texture and pattern; to be specific, black Scharovsky crystals, jewelled in spot patterns - jewelled clothing is not characteristically aligned to menswear and even less so to the great Aussie cossie. The crystals form a matrix of spots that attempt to provoke a sense of mischievousness aligned to the Aussie beach larrikin. Ironically, spot patterns are in itself a form of a parody, as prolonged sun exposure ages the skin and sun spots can occur if appropriate sun protection is not used. ‘Spotty Dick’ – a research experiment to test design suitability for the use of jewelled spot matrix patterns for UV aware men’s swimwear. The creative work was paraded at 56 shows, over a 2 week period, and an estimated 50,000 people viewed the work.

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.

The philosophy and practice of water sensitive urban design : is it consistent with a whole system approach

This paper provides a critique of the Water Sensitive Urban Design (WSUD) paradigm by discussing its congruence with an established sustainable design principle called 'whole system design'. It was found that WSUD is congruent with the whole system design approach as a philosophy, but not in practice. Future improvement of WSUD practice may depend on the adoption of a front-loaded, teamwork-based design and planning process that is embedded in the principle of whole system design.