While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2008-09-26 12:45:02.924

No description available.

Capsule Endoscopy (CE) is a unique technique for facilitating non-invasive and practical visualization of the entire small intestine. It has attracted a critical mass of studies for improvements. Among numerous studies being performed in capsule endoscopy, tremendous efforts are being made in the development of software algorithms to identify clinically important frames in CE videos. This thesis presents a computer-assisted method which performs automated detection of CE video-frames that contain bleeding. Specifically, a methodology is proposed to classify the frames of CE videos into bleeding and non-bleeding frames. It is a Support Vector Machine (SVM) based supervised method which classifies the frames on the basis of color features derived from image-regions. Image-regions are characterized on the basis of statistical features. With 15 available candidate features, an exhaustive feature-selection is followed to obtain the best feature subset. The best feature-subset is the combination of features that has the highest bleeding discrimination ability as determined by the three performance-metrics: accuracy, sensitivity and specificity. Also, a ground truth label annotation method is proposed in order to partially automate delineation of bleeding regions for training of the classifier. The method produced promising results with sensitivity and specificity values up to 94%. All the experiments were performed separately for RGB and HSV color spaces. Experimental results show the combination of the mean planes in red and green planes to be the best feature-subset in RGB (Red-Green-Blue) color space and the combination of the mean values of all three planes of the color space to be the best feature-subset in HSV (Hue-Saturation-Value).

Capsule Endoscopy (CE) is a unique technique for facilitating non-invasive and practical visualization of the entire small intestine. It has attracted a critical mass of studies for improvements. Among numerous studies being performed in capsule endoscopy, tremendous efforts are being made in the development of software algorithms to identify clinically important frames in CE videos. This thesis presents a computer-assisted method which performs automated detection of CE video-frames that contain bleeding. Specifically, a methodology is proposed to classify the frames of CE videos into bleeding and non-bleeding frames. It is a Support Vector Machine (SVM) based supervised method which classifies the frames on the basis of color features derived from image-regions. Image-regions are characterized on the basis of statistical features. With 15 available candidate features, an exhaustive feature-selection is followed to obtain the best feature subset. The best feature-subset is the combination of features that has the highest bleeding discrimination ability as determined by the three performance-metrics: accuracy, sensitivity and specificity. Also, a ground truth label annotation method is proposed in order to partially automate delineation of bleeding regions for training of the classifier. The method produced promising results with sensitivity and specificity values up to 94%. All the experiments were performed separately for RGB and HSV color spaces. Experimental results show the combination of the mean planes in red and green planes to be the best feature-subset in RGB (Red-Green-Blue) color space and the combination of the mean values of all three planes of the color space to be the best feature-subset in HSV (Hue-Saturation-Value).

Lange Zeit galt der Dünndarm als „Blackbox“ des Gastrointestinaltraktes. Seit Einführung der Videokapselendoskopie im Jahr 2001 eröffnete sich eine Methode, den Dünndarm zu visualisieren. An einem Kollektiv von 203 Patienten habe ich Indikationen, Ergebnisse und klinischen Nutzen von Dünndarmkapselendoskopien in einem Zeitraum von 4 Jahren untersucht. Der Dünndarm ist in der Gastroduodeno- und Koloskopie nicht komplett zugänglich, so dass bei entsprechender Indikation die nicht-invasive Videokapselendoskopie vorgenommen werden kann. Sie ist in der Lage 2-4 Bilder pro Sekunde in einem Zeitraum von 8-9 Stunden aufzunehmen, die als Film von ca. 50.000 Bildern zusammengestellt und interpretiert werden kann. Die Daten zur diagnostischen Ausbeute dieser Untersuchung variieren und sind abhängig von der entsprechenden Indikation. Zur Überprüfung des klinischen Nutzens habe ich daher in meiner Arbeit speziell die Passagezeiten und die erhobenen Befunde, wie Erosionen, Ulzerationen, Angiodysplasien, Petechien, Venektasien, Lymphangiektasien, Erytheme, Ödeme, Zottenreliefveränderungen, extrinsische Engen und Erhabenheiten im Hinblick für ihre diagnostische Bedeutung ausgewertet. Berücksichtigt wurden die Auswertbarkeit, Komplikationsrate sowie Vor- und Nachuntersuchungen. Das Aufklärungsgespräch erfolgte mindestens einen Tag vor der Videokapselendoskopie. Die Abführmaßnahmen entsprachen einer Koloskopievorbereitung. Das Studienkollektiv (203 Patienten) bestand aus 58% männlichen und 42% weiblichen Patienten. Der Altersdurchschnitt betrug 58 Jahre, die Altersspanne reichte von 8-90 Jahren. Über 93% nahmen die Videokapsel selbstständig ein, eine Applikation erfolgte bei 7% der Patienten in den Bulbus duodeni. Folgende Indikationen führten bei unserer Patientenklientel zu der Videokapselendoskopie: unklare gastrointestinale Blutung (45,3%), unklare abdominelle Schmerzen (24,1%), unklare Anämie (11,3%), Verdacht auf/ oder Komplikation bei Morbus Crohn (6,5%), unklare Diarrhoe (6,4%), Polyp- und Tumorsuche (5,4%), rezidivierendes unklares Erbrechen und Eiweißverlustsyndrom (jeweils 0,5%). Eine komplette Dünndarmpassage konnte innerhalb der Aufzeichnungszeit von 8-9 Stunden bei 84% der Patienten erreicht werden. Der Mittelwert der Magenpassagezeit lag bei 21 Minuten und der Dünndarmpassagezeit bei 6 Stunden. Die Komplikation Kapselretention trat bei 2% auf. Pathologische Befunde im Dünndarm wurden bei 85% detektiert. Die höchste diagnostische Ausbeute ergab sich bei der Abklärung der unklaren gastrointestinalen Blutung (80%) und bei der unklaren Anämie (78%), als häufigste Ursache wurden Schleimhautläsionen (43%) gefunden. Unklare abdominelle Schmerzen wiesen eine niedrigere diagnostische Ausbeute (41%) auf. Therapeutische Maßnahmen resultierten bei 73% der untersuchten Patienten aus den Kapselergebnissen. Eine medikamentöse Therapie wurde bei 66% eingeleitet oder verändert, Endoskopien wurden bei 4% und eine operative Therapie bei 4,4% durchgeführt. Damit ist die Dünndarmkapselendoskopie bei klarer Fragestellung und guter Darmvorbereitung eine sichere und sinnvolle Untersuchungsmethode, insbesondere zur Klärung unklarer gastrointestinaler Blutungen. Spezifische Dünndarmerkrankungen, wie der M. Crohn oder Tumore können relativ sicher ausgeschlossen werden.

Vibrio vulnificus are marine bacteria causing fatal septicemia through wound infections or consumption of contaminated seafood. V. vulnificus is an excellent model for the study of surface polysaccharides, as it is capable of synthesizing capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide (EPS). V. vulnificus strains exhibit a multitude of carbotypes that evolve through unknown mechanisms. CPS is a confirmed virulence factor, but the genetics of its biosynthesis are unknown. The main objective of these experiments was to gain insight into the biosynthesis, regulation and evolution of ATCC 27562 outer surface polysaccharides. A miniTn10 transposon (Tn) system was used for mutagenesis and single insertions were confirmed through Southern analysis. A novel 25 kb CPS biosynthesis locus was identified through sequencing of regions surrounding Tn insertions; a region encoding putative LPS core biosynthetic functions was identified adjacent to the CPS cluster. The CPS locus contained features of O-antigen biosynthetic loci and was unusual in carrying characteristics of both group I and IV capsular biosynthetic loci. Mutations in this region resulted in elimination of CPS and LPS, and both were shown to be dependent on the activity of the polymerase Wzy. Evidence is presented here supporting horizontal transfer (HT) as a contributor to V. vulnificus CPS evolution. CPS regions of V. vulnificus 27562, YJ016 and CMCP6 contain strain specific genes surrounded by conserved regions, suggestive of HT. Moreover, a CPS locus virtually identical to that of 27562 was discovered in Shewanella putrefaciens strain 200. 27562 CPS is distinctive as it contains N-acetylmuramic acid. Genes encoding murA and murB activities were identified within the cluster and shown to be functionally redundant, supporting HT acquisition of this region. A screen of V. vulnificus gDNA library using CPS biosynthesis and transport mutants identified a cyclic diguanylate cyclase, dcpA. dcpA-mediated increase in cyclic diguanylate lead to EPS production, rugosity phenotypes and enhanced biofilm formation. Interestingly, virulence and motility were not affected suggesting complexity of cyclic diguanylate regulation in V. vulnificus, supported by the large number of cyclic diguanylate related proteins in Vulnificus strains.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 6 uppsatser.

O apelo pela conservação ambiental e o significativo aumento no número de organismos cultivados de alto valor genético demandam tecnologias que permitam conservar sua genética, mesmo após a morte do animal. A criopreservação de gametas possibilita a preservação da genética de espécies ameaçadas e de interesse comercial, prolongando sua vida reprodutiva evitando assim a perda de material genético por doenças, catástrofes, transferência de animais ou perda do habitat natural. A criopreservação tem sido aplicada à conservação de ovários e tecido ovariano, no entanto, há muitas controvérsias acerca de qual seria o melhor protocolo a ser utilizado. Tendo isso em vista, o presente estudo teve como objetivo avaliar a morfologia do tecido ovariano de zebrafish criopreservado em cápsula de metal com o uso de diferentes soluções crioprotetoras. As soluções crioprotetoras utilizadas foram: 1,5 M metanol + 4,5 M propileno glicol (SC1); 1,5 M metanol + 5,5 M Me2SO (SC2); 1,5 M metanol + 4,5 M propileno glicol + 0,5 M sacarose (SC3); 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose (SC4). Após o descongelamento a integridade de cinco estágios de desenvolvimento folicular foi avaliada em cada grupo. A morfologia celular foi observada através de análise histológica. A análise dos dados mostrou que os folículos em estágio I e II foram os melhores criopreservados em todos os grupos experimentais. Sendo que, os grupos SC4 e SC2 foram os que apresentaram os melhores resultados, respectivamente com 88,26% e 84,2% de folículos sem alterações morfológicas. Já os estágios foliculares mais avançados de desenvolvimento (estágios IV e V) apresentaram-se com alterações em todos os grupos. Portanto, apesar do sucesso na criopreservação dos estágios foliculares mais iniciais (I e II) foi possível identificar alterações morfológicas em todos os grupos avaliados. Dentre as principais alterações identificadas estão a aglutinação do citoplasma e enrugamento e ruptura da membrana do envelope celular. Ao avaliar os resultados pode-se concluir que apesar do uso da capsula de metal em associação com as soluções SC4 e SC2 apresentarem os melhores resultados, as soluções SC1 e SC3 também foram eficientes na manutenção da integridade morfológica de folículos imaturos, e portanto essa metodologia pode ser utilizada com sucesso na criopreservação de folículos imaturos. / The appeal for environmental conservation and the significant increase in the number of farmed organisms with high genetic value demand technologies to allow preserving their genetics, even after the death. Cryopreservation of gametes allows the preservation of genetics of the endangered and commercial species, prolonging their reproductive life. Furthermore, this technology prevents the loss of genetic material caused by diseases, disasters, transfer of animals or loss of natural habitat. Cryopreservation has been applied to the conservation of ovaries and ovarian tissue, however, there are many controversies regarding what would be the best protocol to use. Thus, the aim of this study was to evaluate the morphology of cryopreserved zebrafish ovarian tissue using a metal capsule with four different cryoprotectant solutions. The cryoprotectant solutions used were: 1.5M methanol + 4.5 M propylene glycol (CS1); 1.5M methanol + 5.5 M Me2SO (CS2); 1.5M methanol + 4.5 M propylene glycol + 0.5 M sucrose (CS3); Methanol + 1.5 M 5.5 M 0.5 M sucrose + Me2SO (CS4). After heating the integrity of the five stages of follicular development was assessed in each group. Cell morphology was observed by histological analysis. The thermal gradient inside the capsule and the sample was verified by a thermistor Pt500, model Keithley 2001A. The data analysis shows that the follicles at stage I and II were better cryopreserved among all experimental groups. The treatments CS4 and CS2 showed the best results, respectively with 88.26% and 84.2% of follicles without morphological changes in stage I. The most advanced follicular development stages (stages IV and V) showed changes in all treatments. Therefore, despite the successful cryopreservation of earlier follicular stages (I and II), it was possible to identify morphological changes in all the groups. Among the main changes identified, agglutination of cytoplasm and rupture of cell and wrinkling of the egg envelope could be observed. Despite the use of the metal capsule in association with the CS4 and CS2 solutions showed the best results, CS1 and CS3 solutions were also effective in maintaining the morphological integrity of immature follicles, therefore this method can be successfully used in the cryopreservation of immature follicles.

O zebrafish (Danio rerio) tem se destacado na pesquisa biomédica por sua homologia fisiológica e genética aos humanos. No entanto, há poucos relatos sobre a criopreservação ovariana desta espécie. Assim, pesquisamos a utilização de um recipiente de metal na vitrificação de tecido ovariano de zebrafish. O objetivo foi avaliar a sobrevivência e o desenvolvimento in vitro de folículos de zebrafish após a vitrificação de fragmentos ou ovários inteiros usando a cápsula de metal. Primeiro, testamos quatro soluções de vitrificação (VS1 – 1,5 M metanol + 4,5 M propilenoglicol; VS2 – 1,5 M metanol + 5,5 M Me2SO; VS3 – 1,5 M metanol + 4,5 M propilenoglicol + 0,5 M sacarose; VS4 – 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose) e cinco estágios de desenvolvimento folicular utilizando o teste de coloração supravital iodeto de propídio combinado com diacetato de fluoresceína. Estes resultados mostraram que os folículos em estágio I, imaturos, apresentaram as maiores taxas de sobrevivência celular e que VS1 foi a melhor solução em termos de viabiidade. No Experimento 2, utilizou-se VS1 para vitrificar o tecido ovariano em diferentes dimensões (fragmentos ou ovários inteiros) e em dois diferentes recipientes (palheta de plástico ou cápsula de metal). Para avaliar a sobrevivência e o crescimento folicular dos folículos em estádio I, o diâmetro dos folículos foi mensurado antes e depois de cultivo in vitro por 24 horas. A morfologia folicular foi analisada por microscopia de luz após vitrificação utilizando a cápsula de metal. Os dados mostraram que a morfologia de folículos imaturos foi bem preservada após a criopreservação. A taxa de sobrevivência folicular foi maior (P <0,05) em fragmentos vitrificados, quando comparados com a vitrificação de ovários inteiros. Não houve diferenças significativas na sobrevivência e crescimento folicular entre os dois recipientes de vitrificação, palheta de plástico ou cápsula de metal. No entanto, a cápsula de metal diminui os riscos de contaminação, pois é hermeticamente fechada evitando contato com nitrogênio líquido e poder ser esterilizada, em vista que é manufaturada em aço inoxidável. Por essas razões, acreditamos que a cápsula de metal tem um uso potencial em reprodução humana para a vitrificação em grau clínico de tecido ovariano. / Zebrafish (Danio rerio) has excelled in biomedical research for its physiological and genetic homology to humans. However, there are few reports on ovarian cryopreservation of this specie. Thus, we studied the use of a metal capsule to vitrify zebrafish ovarian tissue. The aim of this study was to assess the survival and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the metal capsule. First, we tested four vitrification solutions (VS1 - 1.5 M methanol + 4.5 M propylene glycol; VS2 - 1.5 M methanol + 5.5 M Me2SO; VS3 - 1.5 M methanol + 4.5 M propylene glycol + 0.5 M sucrose; VS4 - 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose) and five follicular developmental stages using fluorescein diacetate and propidium iodide supravital staining test. These results showed that immature follicles, stage one, presented the highest survival rates and VS1 the best vitrification solution in terms of viability. In Experiment 2, we used VS1 to vitrify ovarian tissue in different dimensions (fragments or whole ovaries) and tested two different carriers (plastic straw or metal capsule). To evaluate follicular survival and growth of stage I, we measured follicle diameter before and after twenty-four-hour in vitro culture. The follicular morphology was analyzed by light microscopy after vitrification using the metal capsule. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P<0.05) on vitrified fragments, when compared to whole ovaries. There were no significant differences on follicular survival and growth between the two vitrification devices, plastic straw or metal capsule. However, the metal capsule being tightly sealed and manufactured in stainless steel avoids contact with liquid nitrogen and can be sterilized reducing contamination risk. These reasons lead us to believe that the metal capsule has a potential use in human reproduction for the clinical grade vitrification of ovarian tissue.

O apelo pela conservação ambiental e o significativo aumento no número de organismos cultivados de alto valor genético demandam tecnologias que permitam conservar sua genética, mesmo após a morte do animal. A criopreservação de gametas possibilita a preservação da genética de espécies ameaçadas e de interesse comercial, prolongando sua vida reprodutiva evitando assim a perda de material genético por doenças, catástrofes, transferência de animais ou perda do habitat natural. A criopreservação tem sido aplicada à conservação de ovários e tecido ovariano, no entanto, há muitas controvérsias acerca de qual seria o melhor protocolo a ser utilizado. Tendo isso em vista, o presente estudo teve como objetivo avaliar a morfologia do tecido ovariano de zebrafish criopreservado em cápsula de metal com o uso de diferentes soluções crioprotetoras. As soluções crioprotetoras utilizadas foram: 1,5 M metanol + 4,5 M propileno glicol (SC1); 1,5 M metanol + 5,5 M Me2SO (SC2); 1,5 M metanol + 4,5 M propileno glicol + 0,5 M sacarose (SC3); 1,5 M metanol + 5,5 M Me2SO + 0,5 M sacarose (SC4). Após o descongelamento a integridade de cinco estágios de desenvolvimento folicular foi avaliada em cada grupo. A morfologia celular foi observada através de análise histológica. A análise dos dados mostrou que os folículos em estágio I e II foram os melhores criopreservados em todos os grupos experimentais. Sendo que, os grupos SC4 e SC2 foram os que apresentaram os melhores resultados, respectivamente com 88,26% e 84,2% de folículos sem alterações morfológicas. Já os estágios foliculares mais avançados de desenvolvimento (estágios IV e V) apresentaram-se com alterações em todos os grupos. Portanto, apesar do sucesso na criopreservação dos estágios foliculares mais iniciais (I e II) foi possível identificar alterações morfológicas em todos os grupos avaliados. Dentre as principais alterações identificadas estão a aglutinação do citoplasma e enrugamento e ruptura da membrana do envelope celular. Ao avaliar os resultados pode-se concluir que apesar do uso da capsula de metal em associação com as soluções SC4 e SC2 apresentarem os melhores resultados, as soluções SC1 e SC3 também foram eficientes na manutenção da integridade morfológica de folículos imaturos, e portanto essa metodologia pode ser utilizada com sucesso na criopreservação de folículos imaturos. / The appeal for environmental conservation and the significant increase in the number of farmed organisms with high genetic value demand technologies to allow preserving their genetics, even after the death. Cryopreservation of gametes allows the preservation of genetics of the endangered and commercial species, prolonging their reproductive life. Furthermore, this technology prevents the loss of genetic material caused by diseases, disasters, transfer of animals or loss of natural habitat. Cryopreservation has been applied to the conservation of ovaries and ovarian tissue, however, there are many controversies regarding what would be the best protocol to use. Thus, the aim of this study was to evaluate the morphology of cryopreserved zebrafish ovarian tissue using a metal capsule with four different cryoprotectant solutions. The cryoprotectant solutions used were: 1.5M methanol + 4.5 M propylene glycol (CS1); 1.5M methanol + 5.5 M Me2SO (CS2); 1.5M methanol + 4.5 M propylene glycol + 0.5 M sucrose (CS3); Methanol + 1.5 M 5.5 M 0.5 M sucrose + Me2SO (CS4). After heating the integrity of the five stages of follicular development was assessed in each group. Cell morphology was observed by histological analysis. The thermal gradient inside the capsule and the sample was verified by a thermistor Pt500, model Keithley 2001A. The data analysis shows that the follicles at stage I and II were better cryopreserved among all experimental groups. The treatments CS4 and CS2 showed the best results, respectively with 88.26% and 84.2% of follicles without morphological changes in stage I. The most advanced follicular development stages (stages IV and V) showed changes in all treatments. Therefore, despite the successful cryopreservation of earlier follicular stages (I and II), it was possible to identify morphological changes in all the groups. Among the main changes identified, agglutination of cytoplasm and rupture of cell and wrinkling of the egg envelope could be observed. Despite the use of the metal capsule in association with the CS4 and CS2 solutions showed the best results, CS1 and CS3 solutions were also effective in maintaining the morphological integrity of immature follicles, therefore this method can be successfully used in the cryopreservation of immature follicles.