Excessive muscle tone or stiffness secondary to stroke frequently involves the ankle plantarflexors and has been associated with decreased mobility and reduced function. Although becoming more common in clinical practice, the effectiveness of botulinum toxin A (BTX-A) injected in the ankle plantarflexors on gait biomechanics is not well established. The primary objective of this study was to describe the kinematic and kinetic changes that occur during walking following BTX-A treatment of the hypertonic ankle plantarflexors. As well, the study explored whether there were clinical characteristics uniquely associated with subjects that exhibited biomechanical improvement. The study was a single group, open label trial with repeated measures, including multiple baseline and three post-intervention time points. Seven chronic hemiparetic stroke subjects with ankle hypertonicity were included in the study. Full lower limb bilateral gait analysis provided joint kinematic and kinetic information throughout stance. As well, clinical measures of ankle range of motion and spasticity were assessed pre and post treatment. Data were analyzed using paired samples t-tests and repeated measures ANOVA with Least Significant Difference adjustment for post-hoc analysis as necessary (significance level p≤0.05). Of the kinematic variables, significant improvements in peak dorsiflexion and plantarflexion and the ankle angle at initial contact were found 10 weeks post-injection relative to baseline. No significant kinetic changes were detected, however 2 subjects showed improved positive work at the ankle post-injection and 5 subjects demonstrated increased positive work at the hip post-treatment. Although subjects were classified as “responders” or “non-responders” based on clinical improvement observed 2 weeks post-injection, there was no observable association between those who responded clinically and those who demonstrated improved gait. The major findings suggest that BTX-A injection results in tone reduction and in some cases improves the biomechanical efficiency of gait. In cases where kinetic variables remained unchanged following treatment, perhaps the increased tone was not the limiting factor of reduced function. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2007-08-30 09:41:03.24

No description available.

Ricin toxin is a very deadly plant protein that is synthesized by the plant Ricinus communis. The molecular structure of ricin toxin places it in a group of similar proteins classified as a Type II RIP due to its heterodimeric construction; it is composed of a toxic A-chain possessing enzymatic action, and a receptor binding B-chain. Monoclonal antibodies were obtained with binding activities against either the A-chain or B-chain, and a surrogate non-toxic ricin analogue, TST10114, was determined to be suitable for characterization of the anti-ricin monoclonal antibodies. One potent anti-ricin A-chain neutralizing monoclonal antibody was chosen for chimerization, RAC18, which exhibited strong binding affinity and neutralizing properties. The constant regions of a human immunoglobulin G2 (IgG2) were used as the backbone for the recombinant chimeric antibody. The resulting chimeric RAC18-huG2 was transiently expressed in human-derived HEK 293F cells, purified, and assessed for binding characteristics and functional attributes.

Type II toxin-antitoxin (TA) systems are composed of a stable toxin and its cognate unstable antitoxin that impedes the toxin through direct interaction. The human oral pathogen Streptococcus mutans uses a quorum-sensing peptide (CSP) as a stress-inducible pheromone to synchronize gene expression in response to specific stressors. The objectives of this study were to investigate the role of S. mutans MazEF TA in cell survival and characterize the functionality of CSP-inducible chromosomal type II TAs. Our results suggest that MazEF represents a stress-response element. Interestingly, S. mutans negatively regulates its MazEF system under high-cell-density environment that is characteristic of oral biofilms. S. mutans also encodes a novel chromosomal type II TA involved in biofilm formation and development of dormant persister cells. The results from this study suggest a complex interplay between quorum-sensing (signal), type II TA activation (response), and persister formation (phenotype) as a reaction to environmental perturbations.

Ricin toxin is a very deadly plant protein that is synthesized by the plant Ricinus communis. The molecular structure of ricin toxin places it in a group of similar proteins classified as a Type II RIP due to its heterodimeric construction; it is composed of a toxic A-chain possessing enzymatic action, and a receptor binding B-chain. Monoclonal antibodies were obtained with binding activities against either the A-chain or B-chain, and a surrogate non-toxic ricin analogue, TST10114, was determined to be suitable for characterization of the anti-ricin monoclonal antibodies. One potent anti-ricin A-chain neutralizing monoclonal antibody was chosen for chimerization, RAC18, which exhibited strong binding affinity and neutralizing properties. The constant regions of a human immunoglobulin G2 (IgG2) were used as the backbone for the recombinant chimeric antibody. The resulting chimeric RAC18-huG2 was transiently expressed in human-derived HEK 293F cells, purified, and assessed for binding characteristics and functional attributes.

Botulism is a foodborne intoxication caused by ingestion of Clostridium botulinum neurotoxin (BoNT). Preliminary studies focussed on the production of polyclonal antisera against BoNT/E by immunizing a rabbit with botulinal toxoid type E. The antiserum was subsequently used to detect BoNT/E using the slot blot immunoassay where samples were applied to a slot blot filtration manifold and drawn by vacuum through a membrane. The membrane was then immunologically processed before chemiluminescent detection. However, the antisera lacked specificity and cross-reacted with closely related clostridia strains. / The specificity of the antisera was increased by adsorbing cross-reactive antibodies from whole antisera with affinity columns made with total proteins from culture supernatants of closely related clostridia. Alternatively, specific antibodies were isolated with an affinity column prepared with C. botulinum type E toxoid. / Different methods of concentrating BoNT/E in each sample prior to testing them were evaluated to increase the sensitivity of the assay. / The slot blot immunoassay was then evaluated for detection of BoNT/E in mixed cultures and in food samples. (Abstract shortened by UMI.)

Thesis (Ph. D.)--University of Nevada, Reno, 2007. / "May, 2007." Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 210-222).

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-29Bitstream added on 2014-06-13T20:44:10Z : No. of bitstreams: 1 pigatto_cp_dr_jabo.pdf: 841661 bytes, checksum: 288fcbc74e176eac0befc1827d1bc15e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Escherichia coli produtoras de toxina Shiga (STEC) são reconhecidas como agentes causadores de infecções em humanos em todo o mundo. O principal reservatório é o bovino. Neste trabalho, cepas de STEC previamente isoladas de fezes bovinas foram caracterizadas usando PCR multiplex para determinar os genes de virulência (stx1, stx2, ehxA, eaeA e saa), soroaglutinação passiva reversa em látex (RPLA-VTEC screen) para avaliar a expressão da toxina Shiga, PCR-RFLP e sequenciamento para obter os subtipos e a variabilidade dos genes stx2, respectivamente. Foram determinados também os sorotipos, o perfil de sensibilidade e a viabilidade das cepas de STEC em queijo minas frescal. A freqüência de STEC nas amostras de fezes bovinas foi de 37%. Foram encontrados trinta e quatro sorotipos de STEC sendo os mais freqüentes o ONT:H7 (10%), O22:H8, O22:H16 e ONT:H21 (7% cada). Onze sorotipos encontrados não tinham sido associados com STEC até o momento. A maioria das STEC (96%) foi susceptível a todos os antimicrobianos testados. A produção de toxina Shiga determinada pelo ensaio RPLA foi de 89%. Os marcadores de virulência foram encontrados em 11 diferentes combinações, a mais freqüente foi stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% cada). Foram detectados 8 subtipos de stx2: stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc e stx2O48. Os genes que apresentaram maior freqüência foram: stx2 e stx2c. As seqüências parciais obtidas sugerem a presença de elevada variabilidade nos genes do tipo stx2 nas STEC analisadas. A viabilidade de STEC não-O157 em queijo minas revelou que diferentes cepas de STEC podem ser detectadas nos queijos após 10 dias de armazenamento sob refrigeração. Os dados encontrados neste trabalho sugerem isolados com alto potencial de patogenicidade oferecendo risco de desencadear graves infecções à população. / Shiga toxin-producing Escherichia coli (STEC) is recognize worldwide as an organism capable to cause human diseases. Cattle are the main source of STEC. In this research, STEC strains previously isolated were analyzed using multiplex-PCR for virulence genes, the RPLA assay to detect the Shiga toxin production and serotyping. PCR-RFLP and nucleotide sequence were analyzed to detect stx2 genes subtypes and their variability. Moreover tests for antimicrobial susceptibility and the vialbility of STEC in Minas Frescal cheese were done. The frequency of cattle shedding STEC was 37%. Thirty-four serotypes of STEC were found, the most frequent being ONT:H7 (10%), O22:H8, O22:H16 and ONT:H21 (7% each). Eleven serotypes had not been associate with STEC until the moment. Most of the strains (96%) were susceptible to all antimicrobial agents tested. Production of Shiga toxin by the RPLA assay was detected in most (89%) of the STEC strains. The frequency of virulence markers were found in 11 diferent combinations: stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% each). Eigth stx2 subtypes were detect (stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc; stx2O48) and the most frequent were: stx2; stx2c. The partial sequences of stx2 genes suggested a high variability of stx2 types in the STEC analyzed. The STEC viability in cheese could be detected after 10 days of storage under refrigeration. The results found in this work suggest strains with high potential of pathogenicity offering risk to lead serious infections to the population.

Doctor of Philosophy / Genetics Interdepartmental Program / Revathi Govind / Clostridium difficile is a gram-positive anaerobic, motile, spore-forming opportunistic bacterium. It is a nosocomial pathogen, and the symptoms of C. difficile infection (CDI) range from mild diarrhea to life-threatening pseudomembranous colitis and toxic megacolon. Antibiotic use is the primary risk factor for the development of CDI as it disrupts the healthy protective gut flora which enables C. difficile to colonize and establish in the colon. C. difficile damages the host tissue by secreting toxins and disseminates in the environment by forming spores. The two-major toxin-encoding genes, tcdA, and tcdB are located within a 19.6 kb pathogenicity locus (PaLoc), which also includes the gene encoding an RNA polymerase sigma factor TcdR, that is essential for toxin gene expression. We created a site-directed mutation in tcdR in the epidemic-type C. difficile R20291 strain and found that disruption of tcdR affected sporulation in addition to toxin production. Spores of the tcdR mutant were more heat- sensitive and required nearly three-fold higher taurocholate to germinate when compared to the wild-type (WT). Transmission Electron Microscopic analysis of the tcdR mutant spores also revealed a weakly assembled exosporium. Consistent with our phenotypic assays, our comparative transcriptome analysis also showed significant downregulation of sporulation genes in the tcdR mutant when compared to the WT strain. Our findings on tcdR suggest that the regulatory networks of toxin production and sporulation in C. difficile R20291 strain are interlinked with each other. Transcriptome analysis revealed the sin operon to be significantly downregulated in the tcdR mutant which made us hypothesize the link between sin operon regulation and sporulation. The sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition in B. subtilis. SinR in B. subtilis mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, and SinI/SinR interaction determines whether or not the SinR can inhibit target gene expression. The C. difficile genome carries two sinR homologs in the operon, and we named it as sinR and sinR’, coding for SinR (112 aa) and SinR’ (105 aa), respectively. To identify the regulation of sin on sporulation, we created a site-directed mutation in the sin locus in two different C. difficile strains R20291 and JIR8094. Comparative transcriptome analysis of the sinRR’ mutants revealed their pleiotropic roles in controlling several essential pathways including sporulation, toxin production, and motility (STM) in C. difficile. We performed several genetic and biochemical experiments, to prove that SinR regulates transcription of crucial regulators in STM pathways, which includes sigD, spo0A, and codY. Unlike B. subtilis, SinR’ acts as an antagonist of SinR and SinR’/SinR determines SinR activity. Our in vivo experiment using hamster model also demonstrated the importance of sin locus for successful C. difficile colonization. Our findings above reveal that sin locus acts as a central link that regulates essential pathways including sporulation, toxin production, and motility, which are critical for C. difficile pathogenesis. The final section of this dissertation analyzes a variant codY gene in the epidemic C. difficile R20291 strain. In this strain the CodY, a global nutrient sensor-regulator carry a missense mutation where the 146th tyrosine residue is replaced with asparagine (CodY[superscript Y146N]). Our preliminary study with the mutated CodY[superscript Y146N] suggested its differential role in its regulatory activity. Further analysis of CodY[superscript Y146N] might give some possible clues behind the hypervirulent nature of epidemic R20291 strain. Taken together, studies performed on both tcdR and sinR mutants reveal a significant amount of crosstalk occurring between the powerful regulators of STM pathways under the directionality of TcdR and SinR in determining their ultimate cell fate. Our findings on CodY[superscript Y146N] suggest how the bacteria could switch to a hypervirulence mode by manipulating one of its vital regulators like CodY.