Aim: A study to investigate the potential feasibility (including estimated effect-size), acceptability and health benefits of DEFO and physiotherapy in treatment of spasticity following intramuscular injection of BT. Participants: Adults living in the community with focal spasticity of the upper or lower limb (Modified Ashworth Scale 2-3) recruited at a regional Spasticity Clinic. Intervention: provision of an individually fitted DEFO (worn daily up to 8 hours) usual care and physiotherapy (as required) for 6 weeks. Methods: Mixed methods embedded design feasibility study: Quantitative: Feasibility single-blind RCT: Intervention Group: DEFO intervention protocol, usual care and physiotherapy, Control Group: usual care and physiotherapy. Qualitative: Topic guided interviews of the intervention group and clinicians. Measures: Goal Attainment Scale (GAS) primary measure and secondary measures for function and care benefit; Arm Activity measure (ArmA), Leeds Arm Impact Score (LASIS), VAS for pain, European Quality of Life-5 Dimensions (EQ-5D), gait velocity (10MTT). Variance and fidelity was captured with: DEFO wearing record, Activity Log, clinical records and Physiotherapy modalities. Analysis: ANCOVA adjusted means and statistical comparison for significance of measures (at baseline, after six weeks and twelve weeks) between groups and to inform power calculations. Thematic Analysis of clinician and participant transcribed interviews. Quantitative and qualitative findings were integrated and triangulated to inform a larger study. Results: Participants (n=25) recruited over twelve months, (n=22) completed study. Statistical analysis showed improvements in both groups with greater health benefit in the intervention group with mean difference in the GAS of 12.17 (95% CI: 3.16 to 21.18; p = 0.014) but no statistical significance in the secondary measures. Effect-size was estimated from the GAS findings for 200 per group for a larger study. Physiotherapy modalities for spasticity were linked to 'passive' and 'active' function. Feasibility and acceptability was established with Thematic Analysis providing valuable insight into patient and clinician perspectives on disability. Conclusions: Findings indicated potential added health benefits including carer benefit. Feasibility, acceptability and clinical application of DEFO as a potential new intervention were established. This has implications for future spasticity management with patient benefit for passive and active function. Further research is indicated with a fully powered study (based on the GAS sample results) to evaluate DEFO efficacy in people with spasticity following BT.

Genes are able to replicate horizontally and vertically- a given gene may be more successful on horizontally mobile elements than others. This includes genes that exhibit a post-segregational killing (PSK) phenotype. PSK is generated by expression of a toxin and antitoxin from a mobile element, such that if a bacterium loses the element the toxin becomes active in the cell and the cell dies. All PSKs described to date involve a toxin and an antitoxin function, though within a given group of toxin and antitoxin gene pairs only some are likely to exhibit this phenotype. Here, I investigate what differentiates genes that induce PSK from biochemically similar genes that do not. One group of genes of which some are known to induce PSK is toxinantitoxin (TA) systems, composed of a stable toxin and an unstable antitoxin. I analyzed computational data on the distribution of type I TA systems (RNA antitoxin), which appear to be less mobile than type II TA systems (protein toxin). Data on validated TAs suggests a correlation between distribution, mobility and the PSK phenotype. Differences in phylogeny could be due to differences in tendency to exhibit PSK in different environments. This connection between distribution and PSK was explored by experimentally testing a computationally described operon, plasmid_Toxin-ptaRNA1, that exhibited structural and distributional similarities to a mobile type I TA system. Despite this, expression of the predicted toxin ORFs did not reduce growth (as measured by saturation density) in E. coli, and the operon did not induce PSK. The conditions of PSK were further tested with the toxin (barnase) and antitoxin (barstar), which are not known to have the phenotype. A number of heterologous expression systems were developed with these genes in E. coli to test their ability to exhibit PSK in a manner akin to both type II TA systems, with a cytoplasmic toxin, and bacteriocins,which have a secreted toxin. I used equations of logarithmic decay to model the necessary expression of the proteins in the cell and their rate of decay after plasmid loss to enable PSK. My results suggest there is likely to be an evolutionary trend toward TA systems with high expression levels of very unstable antitoxins. Secreted barnase was also tested experimentally for its ability to induce PSK similar to bacteriocins, which exhibit a PSK-like phenotype in monoculture by driving maintenance of the immunity encoding plasmid. Barnase did not induce PSK, possibly due to its inability to cause antibiosis in our test system. Structural similarities and biochemical similarities are not sufficient to determine whether a given system will act as a PSK because numerous contextual factors have an effect on whether the genes are addictive. A given set of genes may have the phenotype in one species but not another, under one set of environmental conditions but not another, or on one replicon but not another. This is consistent with the competition hypothesis, which states that genes will be selected for on mobile elements due to their ability to increase horizontal reproductive success, depending on the environmental conditions.

2 pp. / Arsenic is the twentieth most abundant element in the earth's crust and frequently occurs in rock formations of the Southwestern United States. Arsenic remains in the environment over long periods and when it occurs in high concentrations, it can be toxic to many life forms, but it also has been shown to be an essential nutrient for many animal species and may be to humans, too. This publication provides information about the impact arsenic in drinking water has over human and plant health and the ways to remove it.

The cell envelope of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogenic bacterium implicated in human oral and systemic disease, plays a critical role in maintenance of cellular homeostasis, resistance to external stress, and host'pathogen interactions. Our laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function. The MorC sequence was determined to be conserved in Gammaproteobacteria. Based on this bioinformatic analysis, the functional conservation of this protein was investigated utilizing an A. actinomycetemcomitans morC mutant as a model system to express homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. MorC from all organisms restored at least one of the A. actinomycetemcomitans mutant phenotypes, implying that the protein is functionally conserved across Gammaproteobacteria. Further, deletion mutagenesis indicated that the last 10 amino acids of the carboxyl terminus were necessary to maintain the integrity of the membrane. The observed pleiotropic effects suggested alterations in the membrane protein composition of the morC mutant. Stable isotope dimethyl labeling in conjunction with mass spectrometry was employed to quantitatively determine the differences in the abundance of membrane proteins of the isogenic mutant and wild-type strains. A total of 665 envelope associated proteins were identified and functionally annotated using bioinformatic tools. All proteins, except MorC, were detected in the mutant strain. However, 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain. These proteins were ascribed functions associated with protein quality control, oxidative stress response, and protein secretion systems. One protein found to be reduced was a component of the fimbrial secretion system of A. actinomycetemcomitans. The significance of this finding was unclear due to the afimbriated nature of the laboratory strain used in the study. Therefore, the defect in fimbriation was identified and complemented in trans. The transformed strain displayed all of the hallmarks of a naturally fimbriated strain including: a distinct star-like colony morphology; robust biofilm formation; and presence of fimbriae as detected by electron microscopy. The isogenic morC mutant strain transformed with an identical plasmid did not display any fimbriated phenotypes. The role of MorC in fimbriae production of a naturally fimbriated strain was investigated by inactivation of morC in a clinical isolate. The mutant strain displayed phenotypes typically associated with inactivation of morC. However, fimbriae were still observed on the surface, although in lesser amounts on some individual bacteria, and this strain formed a biofilm with volume similar to the parent. Interestingly, significant changes in microcolony architecture of the biofilm were observed by confocal microscopy. MorC plays a critical role in maintaining secretion of major virulence determinants of A. actinomycetemcomitans. Specific changes in the protein composition of the cell envelope indicate a direct or compensatory role of these proteins in maintaining membrane physiology. The functional conservation of MorC also implies an important role for this protein in other Gram-negative bacteria. This work suggests a role of MorC as an accessory or a scaffold protein involved in secretion.

Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) is an emerging food- and water-borne pathogen, causing approximately 73,000 annual infections in the United States and an estimated 1.5 million infections globally. E. coli O157:H7, the most frequently associated EHEC strain, is primarily transmitted through consumption of contaminated ground beef and produce and leads to hemorrhagic colitis in humans. In 5% to 15% of infected patients, circulating Shiga toxins (Stx1, Stx2) cause hemolytic uremic syndrome (HUS), characterized by the presence of thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy, contributing to acute kidney injury (AKI). Current treatment is supportive and antibiotic therapy is contraindicative as it increases toxin production. Therapeutics for EHEC-induced HUS need to be identified to minimize kidney injury and uncontrolled coagulopathy. Well-characterized animal models of HUS and EHEC infection are available and provide avenues for potential therapeutic discovery. Baboons (Papio) challenged with endotoxin-free Shiga toxins develop full spectrum HUS, and mice infected with Stx2-producing Citrobacter rodentium (Cr Stx2+), a genetically modified enteric mouse pathogen, develop severe Stx2-mediated kidney injury. Initial studies have shown that soluble thrombomodulin (sTM), an anti-coagulant, is a promising therapeutic in preventing severe kidney injury in pediatric patients. In these studies, we determined whether complement was activated in baboons challenged with Shiga toxins, and evaluated whether intraperitoneal injection of sTM would reduce disease severity from mice infected with Cr Stx2+. D-dimer and cell injury markers (HMGB1, histones) confirmed the presence of coagulopathy and cell injury in Stx challenged baboons. Studies revealed that complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these pre-clinical models. Soluble thrombomodulin treatment in Cr Stx2+ infected mice significantly decreased colonization but did not alter mortality. However, gene expression of kidney injury markers (NGAL, KIM-1) decreased significantly compared to no treatment indicating sTM-associated cytoprotectivity. The C. rodentium mouse model does not develop the coagulopathy seen in HUS patients and sTM treatment may be more effective in the baboon toxemia model. Soluble thrombomodulin is a promising therapeutic for EHEC-induced HUS and should be further evaluated in Stx challenged baboons.

O desenvolvimento de biossensores é um tema de pesquisa bastante promissor, uma vez que permite monitorar diversas classes de substâncias, que muitas vezes apresentam grande interesse nas mais diversas áreas da ciência. Biossensores são pequenos dispositivos que utilizam componentes biológicos como elementos de reconhecimento, ligados a um sistema dedetecção, transdução e amplificação do sinal gerado na reação com o analito-alvo. Podem ser utilizados diversos elementos, sendo os principais, atualmente, aqueles baseados em aptâmeros e nanomateriais, por sua alta especificidade e sensibilidade. Seu potencial de utilização varia desde a detecção e tratamento de doenças ou a medição de componentes nos fluidos biológicos, até o monitoramento ambiental e prevenção de contaminação e bioterrorismo. Neste projeto foi desenvolvido um biossensor eletroquímico cujo objetivo é identificar toxinas inoculadas em pacientes que sofreram acidentes com animais peçonhentos. Foram utilizados os anticorpos/imuniglobulinas comerciais e a peçonha bruta de jararaca (Bothrops) para fazer o estudo/desenvolvimento do biossensor. Neste trabalho foram apresentados os resultados da imobilização dos anticorpos (imunoglobulinas) sobre o eletrodo de trabalho, bem como sua resposta eletroquímica utilizando voltametria cíclica. As soluções utilizadas foram de NaCl 0,9% e tampão fosfato (0,1 mol.L-1) com pH=7,4 por ser bem próximo ao pH fisiológico, pois, posteriormente quer se investigar em plasma sanguíneo. Os voltamogramas cíclicos e a microscopia eletrônica de varredura mostraram a diferença do eletrodo com e sem a imobilização das imunoglobulinas, evidenciando que o biossensor é eficaz para o sistema analisado, sendo promissor aos estudos. Com o biossensor construído, foi investigada a resposta eletroquímica relativa à interação antígeno-anticorpo (veneno-soro) para analisar a interação específica entre eles, sendo o resultado positivamente o esperado. / The development of biosensors is a research topic very promising, since it allows you to monitor various classes of substances, which often exhibit great interest in several areas of science. Biosensors are small devices that use biological recognition elements and components, bound to a selfAdetecting system, transduction and amplification of the signal generated in the reaction with the target analyte it. Various elements, the main currently those based on aptamers and nanomaterials for their high specificity and sensitivity can be used. Their potential use varies from the detection and treatment of diseases or measurement of components in biological fluids to the environmental monitoring and contamination prevention and bioterrorism. In this project we developed an electrochemical biosensor whose objetico is inoculated identify toxins in patients who have suffered accidents with poisonous animals. Antibodies / commercial imuniglobulinas and the crude venom of pit viper (Bothrops) were used to study / development of the biosensor. In this work the results of immobilization of antibodies (immunoglobulins) on the working electrode and its electrochemical response using cyclic voltammetry were presented. The solutions used were 0.9% NaCl and phosphate buffer (0.1 mol L-1) of pH = 7.4 to be close to physiological pH and therefore further investigated whether in blood plasma. Cyclic voltammetry and scanning electron microscopy showed the difference of the electrode with and without immobilization of immunoglobulins, indicating that the biosensor is effective for the system analyzed, and promising to studies. With the biosensor constructed, we investigated the electrochemical response on the antigen-antibody (venom antiserum) interaction to analyze the specific interaction between them, the result being positively expected.

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulent factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two subunits namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut, these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. This thesis is a step towards understanding structural and functional aspects of the binary toxin produced by C. difficile. The first half of this thesis (chapter I and II) provides a brief introduction to the method of structure determination of proteins molecules, i. e. X-ray crystallography and a detailed overview of C. difficile and the three known toxins from C. difficile namely – Toxin A, Toxin B and the binary toxin. Chapter II further focuses on C. difficile binary toxin and other related toxins. These toxins, known as the ADP-ribosylating toxins (ADPRTs) form a big family of potent toxins which includes Cholera, Pertussis and Diphtheria toxins and are capable of transferring the ADP-ribose part of NAD/NADPH to a varity of substrates in the target cell which ultimately results in cell death. The second half of the thesis comprises of experimental procedures that were carried out during the course of this study and their results. Cloning and expression methods for recombinant CDTa and CDTb in bacterial system followed by their purification are described with the abnormal behaviour exhibited by CDTb (chapter III). We show for the first time that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells (Chapter IV). The purification processes described yielded milligram quantities of binary toxin fragments of high purity that led to the successful crystallisation of the proteins (chapter IV) for further functional and structural studies. High resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5 and 9.0) and in complex with the ADP ribose donors -NAD and NADPH (at pH 9.0) have been determined (chapter V). The crystal structures of the native protein show ‘pronounced conformational flexibility’ confined to the active site region of the protein and ‘enhanced’ disorder at low pH while the complex structures highlight significant differences in ‘ligand specificity’ compared with the enzymatic subunit of a close homologue, Clostridium perfringens Iota toxin (Ia). These structural data provide the first detailed information on protein-donor substrate complex stabilisation in CDTa which may have implications in understanding CDT recognition. Crystallisation of CDTb yielded preliminary crystals. The optimisation of these crystallisation conditions is underway. The thesis concludes with some thoughts and discussion on future directions of this research.

Cosmetic treatment with botulinum toxin type A injections is the top non-surgical cosmetic procedure in the U.S. Many patients are beginning treatment at a younger age to prevent the development of facial wrinkles associated with aging. However, there is limited data to support the use of prophylactic botulinum toxin injections. Patients beginning treatment at a younger age have fewer wrinkles requiring fewer units to treat, which reduces the overall cost of treatment. Patients also maintain higher levels of self- esteem by preventing or delaying the onset of facial wrinkles that can negatively impact their appearance. This study proposes that patients receiving botulinum toxin injections at a younger age (< 35) will have higher satisfaction with treatment outcomes. By demonstrating an association between starting age of injections and patient satisfaction, this study aims to provide merit for clinical trials studying the effectiveness of prophylactic botulinum toxin injections for cosmetic indications.

Shiga toxin (Stx) is a bacteriophage (phage)-encoded virulence factor of the Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 implicated in the pathogenesis of renal tissue damage and bloody diarrhoea in human. Cattle are the main asymptomatic reservoir for EHEC O157:H7 with the lymphoid-follicle rich areas of the terminal rectum identified as the primary colonisation site. However, the significance of Stx during bovine intestinal colonisation by EHEC O157:H7 remains unclear with mixed findings described in published studies. The objective of this study was to investigate if Stx and the Stx-encoding phage significantly contribute to EHEC O157:H7 colonisation particularly at the bovine terminal rectum. The expression of Stx receptor, Globotrioasylceramide (Gb3) at the bovine terminal rectum was analysed by fluorescence microscopy, revealing a similar pattern of Gb3 detection in the bovine colon with scattered positive detections limited to sub-epithelial, mesenchymal-associated cells. Purified Stx2 treatment of Gb3+ and Gb3- epithelial cell lines for 6 to 18 hours produced no effect on the cell cycle and proliferation. CD3+/CD8+, CD3+/γδ+ and CD21+ cells were significantly different between calves infected with EHEC O157:H7 Strain 9000 (Stx2a+/Stx2c+) and the uninfected calves, but not in calves with Strain 10671 (Stx2c+). Stx did not interfere with IFN-gamma (IFN-γ)-activation of the JAK/STAT1 pathway in epithelial cells. Bovine EHEC O157:H7 strains isolated from Scottish cattle farms in the IPRAVE study (Phage type 21/28 and 32) were used for a series of bacterial phenotypic characterisation assays. Total Stx production, Verocytotoxicity, growth in a competitive environment, epithelial cell adherence and Galleria mellonella virulence assay were performed to compare the IPRAVE EHEC O157:H7 strains (PT21/28 and PT32) and the isogenic Stx-phage mutants. Stx levels produced by the bovine-originated EHEC O157:H7 strains were significantly lower than that of the human isolated strains. The absence of Gb3 on the bovine terminal rectal epithelium, the non-significant changes in the cell cycle along with the uninterrupted IFN-γ activation of the JAK/STAT1 pathway in intestinal epithelial cells and the minute quantities of Stx generated by EHEC O157:H7 bovine strains suggest that the toxin is not involved in colonisation directly, at least at the intestinal epithelial level. Although future work is required to explain the mechanisms underlying the observed EHEC O157:H7 phenotypic changes particularly in the Stx-phage mutant strains, the work done has proven that the Stx-encoding phage indeed has the ability to exert changes in the bacterial cell leading to changes in bacterial phenotypes, which in turn, might affect the colonisation of the bovine intestine.

As toxinas de veneno de serpentes têm como principal função alterar a homeostase das presas para fins de alimentação ou defesa. O estudo aprofundado da composição do veneno de serpentes é importante para a produção de soros antiofídicos mais eficientes, para a descoberta de novos fármacos e na compreensão de processos biológicos, ecológicos e evolutivos. As pesquisas com toxinas têm mostrado uma versatilidade natural, refinada pela evolução, na diversificação de funções em famílias de proteínas recrutadas de suas funções endógenas, por meio de sucessivas duplicações e acumulo de mutações levando a uma evolução acelerada. A miríade de toxinas disponíveis e sua diversidade de funções ainda não foram completamente descritas. A combinação das análises em larga escala do transcriptoma de novo da glândula de veneno e do proteoma do veneno permite elaborar um perfil mais completo do toxinoma do veneno, permitindo inclusive um aumento na sensibilidade da detecção de toxinas pouco representadas e inesperadas nos venenos. O objetivo geral deste estudo foi analisar o toxinoma do veneno de uma das mais perigosas espécies australianas, a Pseudonaja textilis (Elapidae). Foi possível identificar no veneno as toxinas: fatores de coagulação de veneno do complexo protrombinase, subunidades de fosfolipases A2 (PLA2) da neurotoxina textilotoxin e PLA2 de atividade procoagulante, neurotoxinas tipo three-finger toxin (3FTx), inibidores de protease do tipo-kunitz textilinin, e pela primeira vez, uma nova variante de 3FTx, lectinas tipo C, CRiSP além de indícios de toxinas de lagarto Heloderma e outras proteínas candidatas a toxinas como calreticulin e dipeptidase 2. Metaloproteinases, pouco estudadas em Elapidae, foram clonadas e detectadas no veneno por ensaios de fracionamento e imunoreatividade. A análise do transcriptoma identificou novas isoformas e variantes de toxinas, principalmente das 3FTx e dos inibidores de serinoproteases, assim como transcritos de toxinas que não foram detectadas no veneno e que merecem mais investigações. O quadro de sintomas com acidentes em humanos é bem explicado pelas toxinas identificadas, porém, em seu habitat natural, as toxinas pouco conhecidas e até então não descritas devem ter funções importantes e específicas na predação. Identificar esta diversidade de variantes é importante para entender o modo de ação das toxinas. / Snake venom toxins alter prey homeostasis for feeding or defense. In depth studies of venom composition are important for better antivenom production, for new drugs lead and discovery and for better understanding of biological, ecological and evolutionary processes. Research on toxins have shown the natures way of innovating, refined by evolution, diversifying functions of protein families recruited from their endogenous function to the venom gland by successive gene duplication and mutation accumulation, leading to an accelerated evolution. A myriad of available toxins and diversity of functions is still available for discovery. Combining high throughput techniques such as venom gland de novo transcriptomics and venom proteomics, one can assess and observe a more complete profile of the snake toxinome, additionally allowing an upscale in low represented and unexpected toxin detection. The aim of this project was to investigate the venom toxinome of one of the most dangerous Australian species, Pseudonaja textilis (Elapidae). The toxins identified in it venom was: protrombinase complex coagulation factors, neurotoxic textilotoxin phospholipase A2 (PLA2) subunits and procoagulant PLA2, neurotoxic three-finger toxins (3FTx), Kunitz-type protease inhibitor textilinin, and for the first time, a new long 3FTx, C-type lectins, CRiSPs, as well as evidences of lizard toxins from Heloderma genus and other toxin candidates calreticulin and dipeptidase 2. Metalloproteinases, little investigated in Elapidae, was cloned and detected in the venom after fractionation and immunoassay. The transcriptome revealed new toxin variants and isoforms, specially 3FTx and serine protease inhibitors, as well as transcripts from toxins not detected in the venom that deserves further investigation. Human accident symptoms are well explained by the identified toxins, however, in its natural environment, little known and undescribed toxins must have specific and important role in predation. Identifying this diversity is important to better understand toxins ways of action.