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HRV14 3C acts as a protease and has a role in RNA replication in vivo, interacting with a cloverleaf structure in picornaviral genomic RNA. Picornaviral 3C proteases are able to cleave both N- and C-terminally producing 3CDpro, or, 3Cpro and 3DPol, respectively. In order to investigate the mechanisms whereby these alternative processing pathways are adopted an artificial polyprotein system was constructed, composed of viral sequences from the P3 region of the viral polyprotein flanked by reporter genes. Two antibiotic resistance genes (KanR, TetR) were cloned to act as reporter genes flanking the viral region of interest. Analysis of the cleavage products in a coupled TnT system showed whether N- or C-terminal cleavage had occurred. 3Cpro cleaved preferentially at its N-terminus in [KanR3CproTetR] with a lesser degree of cleavage at its C-terminus. When 3ABC was used as the viral component of the system cleavage at the N-terminus of 3Cpro was also observed. The use of 3CDpro as the viral component also had a regulatory effect on the site (N- or C-terminal) of cleavage by 3Cpro. With 3CDpro as the viral component of the artificial reporter polyprotein cleavage occurred at both the N-and C-termini of 3Cpro as well as at the C-terminus of 3Dpol. This surprising result has led to comparisons with the proteolytic action of viral proteases in the caliciviruses and some plant viruses and the proposal of possible evolutionary links between these viruses. The use of the antibiotic resistance genes as reporters allowed investigation into the use of antibiotic resistance phenotypes in E.coli for monitoring cleavage of the artificial polyprotein. Preliminary results indicated that the system may be useful as a genetic screen to quantify large numbers of mutants.

The adenovirus codes for a protease which is essential for virion infectivity. This protease requires the presence of a peptide cofactor in order to develop optimal activity. This peptide, GVQSLBCRRRCF, originates from the C-terminal of a viral protein, pVI, and some evidence regarding its specificity came from observations showing that neither of the peptides GVQSLKRRRAF or KRRRCF was able to activate the protease, indicating that both the cysteine and the N-terminal were important in the activation process. However, the mechanism by which the peptide activates the protease has never been elucidated. In this project, several factors contributing to the activation mechanism of the human adenovirus type 2 protease were studied, such as the peptide N-terminal length and composition, the environment close to the cysteine and the distance between the N-terminal and the cysteine, in view of assessing the relevance of each of these parameters in the activation process and proposing a mechanism of activation. Based on the above studies, attempts of protease inhibition were also performed based on the activation process rather than on the blocking of the active site, and the relevance of these results was related with the proposed activation mechanism. An attempt to clone an avian adenovirus protease was also performed, in order to try and compare the activation processes between the two proteases.

HIV Proteinase (HIV PR) has proved to be an excellent target for the development of anti-AIDS drugs. Four inhibitors of this enzyme are now approved for clinical use but they, like others, suffer from shortcomings associated with their size and the fact they are peptides. In this thesis the development of small non-peptidic cyclic compounds is described. They were designed to inhibit HIV PR principally by targeting its unique structural features rather than by mimicking its natural substrates. The designed compounds all contained the borinic acid functional group which it was anticipated would interact with the two critical aspartic acid residues of HIV PR. A heteroatom incorporated into these compounds was positioned in such a way that a water molecule which plays a pivotal role in the binding of the enzyme's substrates was displaced. Finally, two benzyl groups were incorporated; these were designed to mimic the side-chains of phenylalanine and tyrosine frequently found in the substrates of HIV PR. The borinic acid functional group has not previously been incorporated into HIV PR inhibitors. Therefore, an analogous series of five 2,6-dibenzylated-4-heterocyclohexanols, where the borinic acid group had been replaced by a hydroxyl group, were prepared and evaluated against the enzyme. These were prepared through bis aldol condensations followed by reduction of the carbonyl group in the bisenones that resulted from dehydration. Although low solubility prevented the analysis of three of these compounds and thereby their effectiveness, two of them were found to be moderately active. Having validated the design of the heterocyclic template, attempts were then made to synthesise borinic acid-containing analogues of these 4-heterocyclohexanols. The attempted syntheses of a directly comparable series of compounds, through application of both bismetallation and bishydroboration strategies, was unsuccessful. Instead, two acyclic diphenyl borinic acids were synthesised. Additionally, several related cyclic borinic acids and acyclic borinic and boronic acids consistent with the design strategy were prepared, from diphenyl sulfone, 2-bromodiphenyl ether and 2-bromodiphenyl sulfide, through the appropriate lithiated species formed by either lithium-hydrogen or lithium-halogen exchange. During the attempted syntheses of a cyclic borinic acid by selective oxidative cleavage of an organoborane derived from diphenyl sulfone, two highly novel borane-amine adducts were synthesised. None of the boron-containing compounds assayed against HIV PR were found to be inhibitors of the enzyme. This is thought to be as a result of the phenyl groups being directly attached to the boron atom, rather than being present in benzyl substituents as had been originally planned.

Production of mature infectious adenovirus type 2 depends on the action of the L3 coded 23kd protease which is known to cleave 7 virus proteins and the host cell proteins cytokeratin K7 and K18. Previous studies have shown the enzyme to be a cysteine proteinase with a novel mechanism of activation requiring the presence of an 11 residue peptide derived from the C-terminus of virus structural protein pVI. It has been proposed that the protease is activated within the assembled virion, although other evidence suggests that the protease is active outwith the virus particle, and is partly responsible for the degradation of the intermediate filament system prior to virion release from the infected cell. The subcellular localisation of the 23kd protease during productive adenovirus infection was investigated using a panel of monoclonal antibodies generated during the course of this study. The monoclonal antibodies were partially characterised and those of interest were epitope mapped using a combination of techniques which included limited and chemical proteolysis, and deletion mutagenesis of the recombinant protein, and screening against overlapping peptides corresponding to specific regions within the 23kd protease. The viral 23kd protease, capsid protein pVIII, and the Ll-52kd probable scaffold protein were shown to colocalise within virus-induced intranuclear clear amorphous inclusions late in infection (24 h.p.i onwards). These inclusions were typically associated with crystalline arrays of assembled virions and are believed to be the same sites which contain relocated PML during the earlier stages of infection. The stmctural appearance of these inclusions varied depending on the fixation method used. The distribution of the viral protease, PML and another cellular protein P80-coilin during the late-phase of nuclear transformation was investigated with possible cleavage of both cellular proteins also partially determined. The viral protease and protein pVIII were also shown to colocalise within cytoplasmic structures but were not found to be associated with cytokeratin K18. The degradation of cytokeratin K18 occurred as early as 20 to 22 h.p.i although the intranuclear distribution of pVI at this stage of infection suggested that the viral protease may be regulated by an as yet unknown mechanism. Isoforms of recombinant wild type 23kd protease (but not Ad2tsl) were detected in vitro which suggested that dimerisation may be an in vivo regulatory mechanism, possibly involving intranuclear trafficking.

A variety of phosphonamidate-containing peptides were synthesised as potential inhibitors of the HIV-1 protease. These transition state analogues were designed using known sequences from HIV-1 protease substrates and incorporated a unique Phe-Pro scissile bond mimic in an attempt to achieve selectivity over the mammalian aspartic proteases. Such compounds were found to be moderate inhibitors of the HIV-1 protease possessing IC50 values in the 1-100 μM range, both in in vitro and in vivo assays. However, the phosphonamidate methyl ester analogues showed a marked ability to enter cells and this feature was highlighted in the 1:1 ratio of in vivo/ in vitro IC50 values (generally for peptidic inhibitors, this ratio is 10-10000 fold higher, indicating poor cell uptake properties). Optimisation of the methyl ester analogues was attempted by alteration of the binding residues flanking either side of the phosphonamidate moiety. However, such alterations had only a small effect on inhibitor potency and the trends observed for the more potent hydroxyl-containing inhibitors were not seen with our compounds. These results suggest hydrogen-bond donating capacity as a key requirement for potent inhibition of the HIV-1 protease, a feature which the methyl ester analogues lack. Due to the associated problems with peptidic inhibitors, the development towards two novel non-peptidic inhibitors of the HIV-1 protease was undertaken. Both cyclic inhibitors were designed to optimise the key interactions at the core of the active site of the enzyme in an attempt for selective, highly potent, low molecular weight inhibitors. Such inhibitors were designed to replace a structural water molecule in the flap region of the enzyme, whilst maintaining the key interactions with the catalytic aspartates. For this purpose cyclic compounds possessing both alcohol functionality and ring heteroatoms were synthesised. The first sulfur-containing analogue produced a moderate inhibitor of the HIV-1 protease and provided a lead compound for further development. The second seven membered ring analogue was designed on the basis of a recent literature compound and the synthesis incorporated a novel bis-ketone, derived from diethyl L-tartrate. This synthesis has yet to be completed and is currently under investigation in our group by Neil Piggot.

It was confirmed that U. urealyticum produces an IgAl protease, which cleaves human IgAl only into intact Fab and Fcalpha fragments. By N-terminal amino acid sequencing of Fcalpha fragments, the site of digestion was identified as a Pro235-Thr236 peptide bond within the a chain hinge-region of IgAl. A number of assay systems were examined for their ability to detect and estimate IgAl protease activity. A reliable and reproducible immunoblotting method was developed, in conjunction with a quantifiable assay utilising [125I] IgAl. By these methods, IgAl protease activity was identified in fourteen serotypes of U. urealyticum, all of which appeared to digest IgAl at the same Pro235-Thr236 peptide bond. The enzyme was active over a broad range of pH (pH 3-10) and was inhibited by the serine-protease inhibitors 3,4-DCI and DFP. The IgAl protease was not located in 'spent' ureaplasma cultivation medium but appeared to be cell-associated. The activity was solubilised by a number of non-ionic detergents which were required in purification buffers to maintain enzyme stability, further suggesting a membrane-bound location. Although the enzyme was not purified to homogeneity, a number of protocols were established which provide a basis for future work. A genomic library of U. urealyticum DNA was produced and a variety of strategies adopted for identification of the iga gene. Radiolabelled DNA probes were generated from a plasmid containing the iga gene for N. gonorrhoeae (pIP503). By Southern blot hybridisation, no significant homology was identified between the heterologous probes and ureaplasma genomic DNA. Based on regions of high nucleotide conservation between the iga genes from N. gonorrhoeae and H. influenzae, degenerate PCR primers were designed. While amplification products did not appear to contain regions of the iga, such an approach may be adapted and extended for use in future studies.

A series of experiments, designed to investigate two mutually incompatible theories of the catalysis carried out by pepsin, the archetypal aspartic protease, were undertaken. Mechanism-activated active-site probes, based on acyl hydrazides, were synthesised, but could not be shown to inactivate pepsin. Experiments designed to trap a covalent intermediate in pepsin catalysis were also carried out, but did not provide any evidence for such an intermediate. A number of methyl hydrogen 1-aminoalkyl phosphonates have been synthesised. They were used, by co-workers in the group, in the synthesis of phosphonamidate-containing penta- and hexapeptide-based inhibitors for the aspartic protease from HIV-1. These compounds were found to be inhibitors in both in vitro and in vivo assays. The best inhibitors had IC50 values in the low micromolar range. Molecular modelling was used to develop a model for the interaction of these compounds with the active site of the protease. This model was used to rationalise some of the results obtained from the inhibitors. Attempts were made to clone, overexpress and purify the protease from E. coli. The protease was purified to homogeneity but no activity could be observed. Various attempts to obtain activity were unsuccessful.

The substrate specificity, classification, expression and control of the adenovirus encoded protease, the activity of which is required for the production of virions, is described. The use of synthetic peptides has shown that the protease cleaves sites of the form (M,L,I)XGG-X or (M,L,I)XGX-G and these consensus sequences have been used to identify potential cleavage sites in all the known substrates of the protease. Putative cleavage sites have also been found in a number of other adenovirus proteins including the major coat proteins, the hexon and the penton, that had not previously been considered as substrates of the protease. The octapeptide, MSGGAFSW, has been used to develop a specific assay for the protease where digestion is monitored by HPLC reverse phase chromatography. Purification of the protease from adenovirus particles and the preparation of antipeptide sera against the L3 23-kDa protein have been used to confirm that the latter is the protease and that it is not proteolytically activated. Inhibitor studies reveal that the enzyme is inhibited by the thiol directed reagents iodoacetate, PCMB and DTDP, and activated by DTT or cysteine suggesting that it is a member of the cysteine class of proteases. Analysis of the sequence of the enzyme, however, shows that it is not a papain-like enzyme and it is suggested that it might be a member of the recently identified subclass of cysteine proteases that are related to trypsin. The 23-kDa protease has been expressed using E.coli and baculovirus expression systems and a purification schedule for the protein was developed using anion exchange and hydrophobic interaction chromatography. The purified enzyme, however, was not able to cleave the peptide substrate MSGGAFSW and the conclusion was drawn that the protein is probably synthesised as an apoenzyme. One of the substrates of the protease, the pre-terminal (pTP) protein was expressed in insect cells using a recombinant baculovirus. Coinfections of cells with recombinant 23-kDa and pTP baculoviruses resulted in efficient processing of the pTP to the intermediate terminal protein (iTP) in situ. The partially purified baculovirus expressed 23-kDa protein was not, however, found to digest the pTP in vitro, supplying further evidence that the adenovirus protease requires a cofactor.

Floral and pollinator microbiomes profoundly impact both ecosystem health and agricultural productivity. However, there are limited integrated studies investigating the shared microbial taxa within these environments, how they shift over time, and how microbes adapt to these habitats. This study aims to bridge this knowledge gap by examining the ecology and evolution of bacterial species that inhabit floral-pollinator environments, and highlighting taxa of interest. We explore the temporal patterns of microbial communities within flowers and pollinators across different ecologically managed environments. 16s rRNA amplicon sequencing of floral, bee crop, and pollen provision samples collected over a growing season from four distinct field sites revealed 279 shared microbial genera, including Acinetobacter, Lactobacillus, Pseudomonas, Bacillus, and Sphingomonas. Temporal analyses unveiled variations in microbial abundance throughout the growing season and distinct temporal patterns between agriculturally managed and unmanaged land. Despite differences across sites, seasons, and land management types, environmental exposure through foraging and land management practices was found to be critical in shaping microbial community structures. The study underscores the integral role of pollinator visitation in shaping floral microbiomes and highlights the importance of understanding these microbial dynamics for the conservation of bee populations and microbial dispersal within agricultural ecosystems. Acinetobacter, a floral nectar specialist, was of particular interest due to its adaptation from soil to floral nectar environments. Phylogenomic analysis of 15 novel Acinetobacter isolates and related strains reveals that nectar-associated Acinetobacter species form a distinct clade, suggesting specialization to these habitats. This ecological shift has driven significant genomic changes, including a reduction in gene number and the acquisition of genes beneficial to colonizing nutrient poor floral nectar environments. Ancestral reconstruction analyses reveal the significant gene losses and gains that optimize nutrient access in nectar, offering insights into the bacterial diversification triggered by extreme changes in selective pressures. These findings represent a significant step towards a more comprehensive understanding of the complexity of microbial communities within pollinator-floral environments.

Industrial applications of ohmic heating are mainly limited to continuous thermal processing of food. Main objectives of this research were to explore its applications in a batch type container, and investigate additional non thermal effects of electric field on bacterial spores, enzymes, carotenoids, flavonoids and quality parameters in food. An ohmic packet made up of multilayered laminate material and capable of sterilizing food was developed. A pouch design optimization exercise was conducted to improve the heating profile and integrity of the hermetic seal at temperature and pressure conditions associated with a sterilization process. A simulation study in 3D was done for sterilization in the ohmic pouch. The mathematical model and sterilization were validated through an inoculated pack study using Geobacillus stearothermophilus spores. Non thermal effects of electricity at frequencies of 10 kHz and 60 Hz during ohmic heating were studied on thermophilic bacterial spores of G. stearothermophilus and Bacillus coagulans. A precise capillary sized ohmic device was invented to eliminate potential source of experimental errors, and to obtain identical time temperature histories for both ohmically and conventionally heated samples. Ohmic heating at both frequencies were found to accelerate inactivation of bacterial spores. It was hypothesized that release of polar dipicolinic acid molecules and spore proteins at higher temperature conditions and their vibrations under the external electric field have resulted in an accelerated inactivation. A linearly increasing temperature analysis of the B. coagulans data suggested that Z values obtained at isothermal conditions may only be valid over a narrow range of temperature.Effects of ohmic heating on pectin methylesterase (PME) and polygalacturonase (PG) enzymes were evaluated in fresh tomato juice at pH of 3.9 and 4.4. PME at pH 3.9 showed a high variability in the inactivation data possibly due to the interaction of multiple isozymes with the electric field. PG data at pH 3.9 showed comparatively less variation and hinted at a higher inactivation rate under ohmic heating. At pH 4.4, PG was found to show accelerated inactivation under ohmic heating in comparison to literature values for its resistant isozyme, PG1. Color values (l, a and b) were found to remain unaffected over the studied pH and temperature ranges. Carotenoids and phenolic compounds of fresh tomato juice remained unaffected by ohmic heating. Inherent amounts of β-carotene, lycopene, phenolic acids and quercetin indicated no or minimal changes at a temperature range from 90 to 110°C at pH of 3.9 and 4.4. Naringenin content showed an interesting behavior with chalconaringenin converting to naringenin in greater proportions. Naringenin equivalents were found to increase at the temperature of 105°C under ohmic heating.

Pectinases are a structurally related yet functionally diverse set of enzymes in Glycosyl Hydrolase Family 28 (GH28) that hydrolyze bonds in galacturonic acid polymer chains. This study examines the long-term evolutionary history of this gene family in completely sequenced fungal genomes in order to elucidate the relationships between GH28 structure and function. Results of this investigation reveal the presence of multiple diverse GH28 gene copies within fungal genomes, as well as a relatively high level of inter-species amino acid sequence divergence, likely due to functional differentiation of GH28 gene family members. Overall, this evolutionary pattern is consistent with the birth-and-death model of gene family evolution and suggests that the diversity of GH28 family can be attributed to multiple rounds of gene duplication followed by functional diversification of gene family members, with some members being lost, resulting in lineage specific expansions of this gene family in various fungal genomes. This lineage specific GH28 expansion is the result of both general genome expansion and evolved niche specificity. Necrotrophic fungi have large genomes and significantly more pectin-degrading enzymes that are used to indiscriminately degrade cell walls and instigate tissue maceration. Conversely, biotrophic fungi have larger genomes than non-pathogens, but their pectin-degrading network is extremely limited. GH28 gene products stimulate the PGIP-mediated hypersensitive immune response, or localized cell death, in host plants. Biotrophic fungi avoid this response by not using GH28 pectinases, which have subsequently been reduced in their genome through purifying selection. Utilizing an evolutionary approach allows us to integrate genetic, genomic, and ecological data to understand principles of molecular evolution and explain the complex molecular arms race resulting from host-parasite interactions.

One of the problems in postharvest handling of sweetpotato roots is the loss of the skin from the surface of the roots, referred to as 'skinning'. During skinning, cuticle, epidermis, and a portion of the outer layers of the periderm separates from underlying tissue, resulting in an increased rate of moisture loss, weight loss, shriveling of the root surface, susceptibility to pathogen attack and poor appearance. Thus, skinning results in a loss of crop value and a reduction in profits for the grower. The following characteristics were measured to determine if they influenced skin adhesion and/or peeling resistance in sweetpotato roots: pectin, lignin, and anthocyanin content, polygalacturonase (PG), pectinmethylesterase (PME) and cellulase activity, periderm dry matter content and root weight. Studies were conducted to determine if there was variation in skin adhesion, cell wall enzyme activity and cell wall components in the periderm of roots (a) during storage, (b) at different physiological ages, (c) when grown under several temperature regimes and (d) when grown in different locations in the Southeastern U.S. The anatomy and histochemical properties of roots grown at different temperatures and locations were characterized. Correlations between the variables were established to determine if skin adhesion and/or peeling resistance might be explained based on cell wall enzyme activity, cell wall composition, and/or anatomy of root periderm. Results indicated that skin adhesion in sweetpotato was highly variable and was affected by cultivar, temperature, humidity, origin and physiological age of the root, and storage. Skin adhesion, cell wall enzyme activity, and pectin content in sweetpotato vary with physiological age of the root. In field experiments, the effect of soil temperature on skin adhesion was not clear, possibly due to the interaction with other environmental conditions. Under controlled conditions, skin adhesion, cell wall enzyme activity, anthocyanin content, periderm and biomass dry matter content, yield, root weight, and root diameter were affected by growth temperature. A growth temperature of 34/31 ºC yielded roots that were more resistant to skin loss, were smaller, and had a thicker periderm, composed of more cell layers and lower dry matter content than roots grown at lower temperatures. A growth temperature of 20/17 ºC increased anthocyanin content of the root periderm. In roots grown in the Southeastern U.S., the degree of skin adhesion and peeling resistance varied depending on root origin. Skin adhesion during storage of sweetpotato roots varied depending on cultivar. Skin adhesion of the roots seemed to increase during the early weeks of storage, but this effect did not appear to be permanent and appeared to be reversible at any time during storage. Curing improved skin adhesion and peeling resistance of the roots. The effect of curing was dependent on the root growth temperature. A similar interaction was detected between curing and growth temperature for some of the studied variables. Skin adhesion did not appear to be directly linked to cell wall enzyme activity. However, enzyme activity levels were affected by cultivar, environmental factors, storage and origin of the roots. Periderm dry matter, lignin, and anthocyanin content did not appear to be significant factors in skin adhesion or peeling resistance. The histochemistry of the periderm of sweetpotato indicated a different anatomical and structural composition of the cell walls depending on growth temperature and where the roots were grown.