Jin, Dawei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / ABSTRACT (IN CHINESE) --- p.ii / ABSTRACT (IN ENGLISH) --- p.iv / ACKNOWLEDGEMENTS --- p.vii / CONTENTS --- p.ix / LIST OF TABLES --- p.vxiii / LIST OF FIGURES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- Biology of C. albicans --- p.2 / Chapter 1.1.1 --- Taxonomy --- p.2 / Chapter 1.1.2 --- Basic cell biology --- p.2 / Chapter 1.1.2.1 --- Cell cycle and phenotypic switch --- p.2 / Chapter 1.1.2.2 --- Cell wall --- p.3 / Chapter 1.1.3 --- "Morphological, culture and biochemical characteristics" --- p.4 / Chapter 1.1.4 --- Genomics --- p.5 / Chapter 1.1.5 --- Pathogenecity --- p.6 / Chapter 1.2 --- Catheter-related bloodstream infections (CRBSI) caused by C. albicans --- p.7 / Chapter 1.2.1 --- Intravenous catheter type --- p.7 / Chapter 1.2.2 --- Epidemiology of CRBSI caused by C. albicans --- p.8 / Chapter 1.2.3 --- Pathogenesis of intravascular catheter-related infections --- p.9 / Chapter 1.2.4 --- Diagnosis of catheter-related infections --- p.10 / Chapter 1.2.5 --- Prevention and control --- p.11 / Chapter 1.3 --- Mechanism of C. albicans adhesion to catheters --- p.12 / Chapter 1.3.1 --- The definition of microbial adhesion --- p.12 / Chapter 1.3.2 --- Relationship between microbial adhesion and biofilm formation --- p.12 / Chapter 1.4 --- Agglutinin-like sequence (ALS) gene family of C. albicans --- p.14 / Chapter 1.4.1 --- Members of ALS gene family --- p.14 / Chapter 1.4.2 --- Chromosomal location of ALS genes --- p.14 / Chapter 1.4.3 --- ALS gene organization --- p.14 / Chapter 1.4.3.1 --- Three-domain structure of ALS genes --- p.15 / Chapter 1.4.3.2 --- Characterization of ALS genes. --- p.15 / Chapter 1.4.4 --- ALS gene allelic variation --- p.17 / Chapter 1.5 --- Experimental models for catheter adhesion study of C. albicans --- p.17 / Chapter 1.5.1 --- "Static adhesion model for C, albicans" --- p.18 / Chapter 1.5.1.1 --- Advantage of static adhesion model --- p.19 / Chapter 1.5.1.2 --- Limitation of static adhesion model --- p.19 / Chapter 1.5.2 --- Dynamic adhesion model for C. albicans --- p.19 / Chapter 1.5.2.1 --- Advantage of dynamic adhesion model --- p.20 / Chapter 1.5.2.2 --- Limitation of dynamic adhesion model --- p.20 / Chapter 1.5.3 --- Quantification methods of adherent cells --- p.21 / Chapter 1.5.4 --- ALS gene expression study in the in vitro model --- p.22 / Chapter 1.6 --- Aim of study --- p.22 / Chapter CHAPTER II --- MATERIALS & METHODS --- p.24 / Chapter 2.1 --- Strains used in this study --- p.25 / Chapter 2.2 --- Design of an in vitro dynamic adhesion model for C. albicans --- p.26 / Chapter 2.2.1 --- Flask --- p.26 / Chapter 2.2.2 --- Peristaltic pump --- p.26 / Chapter 2.2.3 --- Glass tube and vascular catheters. --- p.27 / Chapter 2.2.4 --- Sterility check of in vitro dynamic adhesion model --- p.27 / Chapter 2.3 --- Construction of C. albicans growth curve --- p.27 / Chapter 2.4 --- Measurement of C. albicans adhesion to catheters --- p.29 / Chapter 2.5 --- Detection of C. albicans ALS genes --- p.30 / Chapter 2.5.1 --- DNA extraction of C. albicans --- p.30 / Chapter 2.5.2 --- ALS primers design --- p.31 / Chapter 2.5.3 --- PCR reaction --- p.32 / Chapter 2.5.4 --- Gel electrophoresis --- p.32 / Chapter 2.5.5 --- Purification of PCR products --- p.33 / Chapter 2.6 --- Construction of E. coli plasmid containing gene --- p.34 / Chapter 2.6.1 --- Ligation using the pGEM®-T Easy Vector --- p.34 / Chapter 2.6.2 --- Preparation of E. coli DH5a electro-competent cells --- p.35 / Chapter 2.6.3 --- Clean up of DNA ligation reaction for electro-transformation --- p.36 / Chapter 2.6.4 --- Electro-transformation of E. coli DH5a electro-competent cells --- p.37 / Chapter 2.6.5 --- Blue / white screening for positive transformation of E. coli DH5a. --- p.37 / Chapter 2.6.6 --- Extraction of plasmid containing ALS1 gene --- p.39 / Chapter 2.6.7 --- Plasmid validation by PCR and gel electrophoresis --- p.39 / Chapter 2.6.8 --- Serial dilution of plasmid solutions for ALS1 standard curve construction --- p.40 / Chapter 2.7 --- C. albicans ALS1 gene expression in dynamic adhesion model --- p.41 / Chapter 2.7.1 --- Design of real-time PCR primers specific for C. albicans ALS1 --- p.41 / Chapter 2.7.2 --- Validation of primers specificity --- p.42 / Chapter 2.7.3 --- RNA extraction of C. albicans cells adhered on catheters --- p.43 / Chapter 2.7.4 --- Complementary DNA (cDNA) synthesis --- p.45 / Chapter 2.7.5 --- Quantitative real-time RT-PCR --- p.46 / Chapter 2.8 --- Statistical analyses --- p.48 / Chapter CHAPTER III --- RESULTS --- p.49 / Chapter 3.1. --- Validation of the in vitro dynamic adhesion model for C. albicans --- p.50 / Chapter 3.2. --- C. albicans growth curve construction --- p.50 / Chapter 3.3. --- Measurement of C. albicans adhesion on catheters --- p.50 / Chapter 3.4. --- Detection of C. albicans SC5314 ALS genes --- p.52 / Chapter 3.5. --- Validation of E. coli plasmid containing ALS1 gene --- p.54 / Chapter 3.6. --- C. albicans ALS 1 gene expression in dynamic adhesion model --- p.54 / Chapter 3.6.1. --- Specificity validation of ALS1 real-time primers --- p.55 / Chapter 3.6.2. --- Quantitative real-time RT-PCR --- p.55 / Chapter CHAPTER IV --- DISCUSSION --- p.57 / Chapter 4.1 --- Experimental design of the in vitro dynamic adhesion model --- p.58 / Chapter 4.1.1 --- Advantages of this in vitro dynamic adhesion model --- p.58 / Chapter 4.1.2 --- Limitation of this in vitro dynamic adhesion model --- p.58 / Chapter 4.1.3 --- Catheter arrangement inside the glass tube --- p.60 / Chapter 4.1.4 --- Reproducibility of experiments in the model --- p.62 / Chapter 4.1.5 --- Identification of potential contamination in the model --- p.63 / Chapter 4.1.6 --- Advantages of removing method for C. albicans adherent cells --- p.64 / Chapter 4.1.7 --- Limitation of removing method for C. albicans adherent cells --- p.64 / Chapter 4.1.8 --- Limitation of statistical analysis --- p.66 / Chapter 4.1.9 --- Primers design --- p.67 / Chapter 4.1.9.1 --- Primers of C. albicans ALS gene detection --- p.67 / Chapter 4.1.9.2 --- Validation of ALS 1 real-time primers specificity --- p.69 / Chapter 4.2 --- C. albicans adhesion to catheters --- p.70 / Chapter 4.2.1 --- Theoretical explanation of C. albicans adhesion to different catheters --- p.71 / Chapter 4.3 --- C. albicans ALS gene expression --- p.74 / Chapter 4.3.1 --- Functions of Als proteins --- p.75 / Chapter 4.3.1.1 --- Adhesive functions --- p.75 / Chapter 4.3.1.2 --- Other functions in C. albicans pathogenesis --- p.75 / Chapter 4.3.2 --- Analysis of ALS1 gene expression pattern in the in vitro model --- p.76 / Chapter 4.4 --- Clinical application of our study --- p.78 / Chapter 4.5 --- Future study --- p.80 / Chapter 4.6 --- Conclusion --- p.81 / REFERENCES --- p.83

"March 2004" / Includes bibliographical references (leaves 213-272) / xxiii, 272 leaves ; ill. (some col.), plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2004

Amyotrophic lateral sclerosis (ALS) is a devastating disease characterized by the progressive degeneration of motor neurons. Dominantly-inherited mutations to the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) cause 3-6% of all ALS cases. The complete mechanism behind the toxicity of mutant SOD1 remains unclear, although significant evidence points to aberrant or incomplete metal-binding having a role in a toxic gain-of-function. However, the relevance of the metal-binding of SOD1 to mutant-SOD1-linked ALS remains controversial. Direct assessments of protein metal-binding from transgenic, SOD1-overexpressing rodent models of the disease are difficult to acquire due to the non-covalent nature of the interaction. The relatively small amount of disease-afflicted spinal cord tissue in which the motor neurons reside compounds the difficulty of measuring the protein metal binding of SOD1 from transgenic mice. This dissertation addresses the metals bound to SOD1 throughout the disease course in transgenic mice using a novel mass spectrometry assay. The methodology developed here offers the first detailed examination of partially unfolded intermediates of SOD1 present in the spinal cord of pre-symptomatic, symptomatic, and end-stage transgenic mice overexpressing the ALS-associated SOD1 mutation G93A (glycine mutated to alanine at position 93). These results were compared to age-matched transgenic mice expressing wild-type SOD1 that do not develop ALS symptoms. To extract SOD1 from relevant spinal cord tissue, a 300 ��m necropsy punch was used to remove a small piece of tissue from the ventral or dorsal gray matter of a 1 mm-thick slice of spinal cord. Physiological salts that interfere with electrospray mass spectrometry were removed by binding the proteins to a C4 Ziptip��, a pipette tip containing hydrophobic, reversed-phase packing material. Washing the Ziptip-bound proteins with water eliminated interfering salts. Bound proteins could then be eluted into a mass spectrometer with low concentrations of acetonitrile plus formic acid. Electrospray ionization conditions were determined that could keep both copper and zinc bound to SOD1. Using a high-resolution Fourier transform-ion cyclotron resonance mass spectrometer, we used the assay to collect isotopically-resolved protein mass data. Theoretical protein isotope distributions were calculated from the empirical formulas of SOD1 and matched to the experimental data with a least squares fitting algorithm to determine the multiple intermediates of SOD1 present. Spinal cord tissue, wild-type in particular, was notable for containing significantly more one-metal SOD1 than any other tissue, despite having 3-fold less SOD1 than liver. We quantitatively compared the levels of soluble, partially unfolded intermediates of SOD1 from wild-type and G93A SOD1 spinal cords. Wild-type mouse spinal cord contained significantly more of all of the partially unfolded intermediates copper-deficient SOD1, disulfide reduced SOD1, and apo SOD1. The amount of zinc-containing SOD1 was exceptionally high in wild-type mice, comprising 60% of the total SOD1 in wild-type spinal cord. The larger amounts of these SOD1 intermediates in wild-type transgenic mice indicate that they are not directly responsible for toxicity in vivo. However, copper-containing, zinc-deficient SOD1 was the one species found in higher concentrations in G93A SOD1 spinal cord. The concentration was on average 0.6-0.8 ��M in G93A spinal cord, compared to 0.1-0.3 ��M zinc-deficient SOD1 found in the wild-type mouse spinal cord. A concentration above 0.5 ��M zinc-deficient SOD1 was sufficient to induce motor neuron death in vitro. These results suggest that copper-containing, zinc-deficient SOD1 could be the toxic species responsible for motor neuron death in ALS. / Graduation date: 2013

La seqüenciació del genoma humà ha marcat una fita important en la història de la biologia. Com a conseqüència, la genètica i la genòmica han experimentat un progrés enorme. Això ha permès un millor coneixement tant de les causes genètiques de malalties humanes, com del per què de les diferències comunes entre individus. Com a sistemes complexos que som tots els éssers vius, hem de considerar el paper que tenen les interaccions entre les diferents parts del genoma a l'hora d'especificar el resultat final, és a dir, el fenotip. Igualment, podem dir que el genoma conté un conjunt d'instruccions, però que la forma en què aquestes es porten a terme depèn, també de contingències ambientals i històriques. Per tant, la naturalesa de les instruccions genètiques no és completament determinista en tots els casos, si bé hi ha una sèrie de processos en què sí que es compleix aquesta perfecta relació entre herència i expressió final. Aquesta mateixa situació es presenta amb certes alteracions genètiques i amb el desenvolupament de patologies, la qual cosa facilita enormement el diagnòstic precoç i obre les possibilitats per a la teràpia genètica. Però la gran majoria de fenotips, incloent-hi moltes condicions d'interès per a la medicina, tenen una base complexa, és a dir, no existeix "el gen" que determina el caràcter de forma unívoca, sinó que aquest és el resultat de l'acció simultània de molts gens, no tots amb la mateixa participació, juntament amb l'efecte de l'ambient. Aquesta tesi doctoral va arrencar en aquest punt, tenint com a objectiu l'aprofundiment en les bases genètiques d'un tipus de sordesa lligada a mutacions al vi Preface DNA mitocondrial i de la qual se'n tenien evidències de la implicació tant de factors ambientals com diversos factors genètics. D'altra banda, els tests basats en l'ADN són un dels primers usos comercials i d'aplicació mèdica d'aquests nous descobriments de la genètica. Aquests tests poden ser utilitzats per al diagnòstic de malalties, confirmació diagnòstica, informació del pronòstic, així com del curs de la malaltia, confirmar la presència de malaltia en pacients assimptomàtics i amb diferents graus de certesa, predir el risc de futures malalties en persones sanes i en la seva descendència. Aquest és l'objectiu final, i sovint encara utòpic, de la recerca en biomedicina: una millor comprensió del procés biològic, que derivi en un millor tractament i prevenció de la malaltia. Aquesta tesi també ha volgut contribuir humilment en aquest aspecte. Durant aquests anys s'han recollit centenars de mostres de famílies sordes, amb la finalitat de donar un "diagnòstic" de la causa genètica. Poques vegades ho hem conseguit, però en qualsevol cas, si això alguna vegada ha ajudat a algú d'alguna manera, ja em dono per satisfeta.

This thesis examines the effects that Pleistocene glaciation had on the population structure and contemporary genetic patterns of the hairy woodpecker (Picoides villosus). A combination of molecular markers, revealed reduced levels of gene flow among groups of hairy woodpeckers. Microsatellite analyses suggest barriers to gene flow have influenced contemporary population structure, with higher structure found in western North America where barriers to gene flow are more prevalent. MtDNA analyses revealed three distinct genetic lineages, two in North America and a third in Central America. Results indicate these lineages separated prior to the Wisconsin glaciation (~100 kya) and that contemporary population structure is the result of post-glacial expansion from multiple refugia following deglaciation. Current taxonomy recognizes 17 subspecies (Jackson et al., 2002), but molecular analyses in this study do not support current subspecies designations. / xii, 117 leaves ; 29 cm

No description available.

Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Scarabaeid pests in South Africa and especially KwaZulu-Natal are characterised by a very long larval life cycle and short pupal and adult periods. However, it has nearly always been the adults of the species that have been identified, with very little attention paid to the larval identification of the species. This is unfortunate as it is nearly always the larval stage that is found to be associated with crop damage. Accurate identification of the species of these larvae is important for the management of scarabaeid pest species, as it unlocks the necessary information on the biology and ecology of many species, which allows the adaptation of control methods for different species. Inadequate keys for the taxonomy of larvae of these groups, as well as the lack of morphological taxonomists working on these groups have been identified as constraints. When a species is difficult to identify using traditional taxonomic methods, DNA diagnostic tools can be useful. Chapter 2 investigated the feasibility of identifying scarabaeid larvae using mitochondrial DNA data. Variation in the base pair sequence of the mitochondrial cytochrome c oxidase sub unit I (cox 1) gene was used. DNA sequences of cox 1 from scarabaeid larvae collected from sugarcane fields were compared with sequences from scarabaeid adults of known species in order to identify the species attacking sugarcane. Neighbour-joining and maximum parsimony analyses of 658 bp cox 1 sequences identified groups of larvae that linked to adult specimens. The major groupings delimited specimens belonging to the subfamilies Dynastinae, Melolonthinae and Rutelinae. Within-group sequence divergence ranged from 0 - 3.4 % and divergence between sister groups ranged from 2.6 - 25.1 %. The recorded divergence range within and between tribes was 0 - 21.3 % and 17.3 - 28.5% respectively. Similarly, the divergence range observed within and between genera was 0 - 19.2 % and 17.1 - 25.4% respectively. The maximum sequence divergence observed within subfamilies was 23.7 % and divergence between subfamilies ranged from 16.8 - 26.7 %. Examination of pairwise sequence divergence levels as well as node support allowed 68% of the unidentified larval specimens to be associated with identified adult specimens. Phylogenetic analysis matched identified adult mtDNA with unidentified larval mtDNA. This allowed the identification of those larvae through morphological characteristics unique to certain species. To create a field key to the subfamilies of Dynastinae, Melolonthinae and Rutelinae the most useful character distinguishing larvae of different species was the raster but additional morphological characteristics were included. These relationships between larval and adult scarabaeid specimens from sugarcane were examined using various phylogenetic tools. The data set included a total of 19 morphological characters as well as 166 partial cox 1 gene sequences. Maximum parsimony analyses were performed on morphological, molecular and combined data. The same morphological and molecular data sets were run both separately and as a combined analysis with MrBayes. In both types of analyses the morphological data performed poorly and crude groupings resulted, dividing taxa to tribe level only. Molecular data showed greater resolution than the morphological data and taxa were separated into groups equivalent to species and morphospecies designated in Chapter 2. A partition homogeneity test indicated that both data types could be combined. It is recommended that both morphological and molecular data be utilised in identification of scarabaeid sugarcane pests and that a character-based approach be implemented. Further molecular data from other genes should be included to test the accuracy of these results. The keys produced during this study will allow workers to focus on a single species biology, and subsequently allow an analysis of between species interactions, and within species control. These advances are a start to the improvement of knowledge of the species composition of scarabaeid larvae in sugarcane fields, thus making management and biological control of these pests a greater possibility. Further recommendations for future work are discussed in Chapter 5. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Photosystem II (PSII) is the membrane-bound oxidoreductase peptide complex responsible for the oxidation of water to molecular oxygen and reduction of plastoquinone to plastoquinol. Primary electron transfer is initiated upon absorption of a photon by the primary donor chl resulting in electron transfer and production of a P680+QA charge separated state. P680+ is reduced by YZ (Y161 of the D1 polypeptide subunit), one of two redox-active tyrosine residues found in PSII. This produces a neutral tyrosyl radical (YZ ) which is subsequently reduced by electrons derived from water at the oxygen-evolving complex (OEC). The OEC is composed of four manganese, one calcium ion, and one chloride ion. Four photons are required to convert water to O2, each photon advancing the OEC through successive oxidation states or S states. The exact chemical mechanism of water oxidation in PSII is not known. However, proton-coupled electron transfer (PCET) is thought to be one of the fundamental steps in driving the extraction of electrons and protons from water. Here, the mechanism of water oxidation is investigated with focus on PCET events using vibrational spectroscopy. Vibrational spectroscopy is sensitive to changes in protein structure, charge, and hydrogen bonding, and is ideal for the study of fast events coupled with light-induced electron transfer. The results presented here demonstrate the utility of time-resolved infrared spectroscopy in the detection of intermediates of photosynthetic water oxidation. We suggest that proton transfer may precede manganese oxidation during water oxidation based on time-resolved infrared and difference FT-IR spectroscopic results. The mechanism of PCET associated with YZ reduction is investigated. Using reaction-induced difference FT-IR spectroscopy, the identity of the chloride binding site is speculated through the use of bromide exchange at the OEC. Also, proton transfer reactions at the OEC are investigated using azide as a vibrational probe. The advances in the understanding of photosynthetic water oxidation gained in this work will aid in the elucidation of the chemical mechanism of this important reaction. Understanding the details of photosynthetic water oxidation will assist in the development of technology aimed at harnessing the energy of the sun for the benefit of humankind.

Shigella is a genus of Gram-negative bacteria responsible for bacillary dysentery in humans. Shigella flexneri type 2a in particular is responsible for the majority of incidents in developing countries. The S. flexneri protease IcsP, is a member of the Omptin family of outer membrane (OM) proteases which cleaves IcsA, a polarly localised OM protein required for Shigella virulence. Mutations in icsP have been shown to effect the observed distribution of IcsA, however the significance of IcsP in Shigella virulence is incompletely understood. In this study, aspects of IcsP biology were investigated. S. flexneri 2457T and M90T icsP mutants were constructed to investigate the role of IcsP in Shigella intercellular spread, and it was found that icsP in both S. flexneri backgrounds did not appear to be essential for cell-tocell spread in human cervical cancer HeLa cells, but enhanced cell-to-cell spread in monkey kidney CV-1 cells (as determined by plaque assays). Complementation with icsP returned the mutant phenotype to wild-type. The results suggest IcsP does play a role in Shigella intercellular spread. The 2457T icsP mutant was subsequently complemented with an altered icsP gene encoding a haemagglutinin epitope tagged IcsP (IcsPHA) to determine the distribution of IcsP on the cell surface. In both S. flexneri and E. coli K-12 possessing smooth and rough lipopolysaccharide (LPS), the distribution of IcsPHA was found to be punctate across the cell surface. Deconvolution analysis revealed that IcsP distribution was punctate and banded in both LPS backgrounds. A smooth LPS E. coli K-12 yfdI mutant strain expressing IcsPHA was also constructed, and experiments involving treatment of this strain with bacteriophage Sf6 tail spike protein suggested that LPS O antigen chains masked IcsP in smooth LPS strains. During these studies, double-labelling of IcsPHA and LPS in a S. flexneri 5a M90T strain revealed a helical distribution of LPS in this strain. Overall, the results suggest IcsP has a punctate, banded distribution across the cell surface. The effect of virK and rmlD mutations on IcsP was then investigated by constructing a virK, rmlD and virK/rmlD double mutant in S. flexneri 2457T. Western immunoblotting showed no change in IcsP expression levels in either the virK, rmlD or virK/rmlD mutants compared to wild-type. Surprisingly, the virK mutant showed no change in IcsA expression levels by Western immunoblotting and plaque assays (using HeLa and CV-1 cells) suggested that virK was not essential for Shigella intercellular spread (contradicting the published data on this gene). No effect was also observed on IcsP expression level or on IcsP’s ability to cleave IcsA into culture supernatants. Finally alternative substrates for the protease activity of IcsP were investigated against known Omptin substrates (plasminogen, α2-antiplasmin, complement, protamine and colicins). However, IcsP appeared to have no effect on these substrates as determined by proteolytic cleavage assays and antimicrobial assay. Interestingly, Plg cleavage by rough LPS S. flexneri, and α2AP cleavage by both smooth and rough LPS S. flexneri, was observed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339487 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008