Abstract Hypericum perforatum L. (St. John’s wort) is a medicinal plant widely utilized for the treatment of depression. The antidepressant activity is mainly attributed to the phenolic compounds hypericins and hyperforins, which also have a wide range of other pharmacologically interesting properties. The biosynthetic routes leading to hypericins and hyperforins are poorly understood, although a polyketide pathway including type III polyketide synthases (PKSs) has been suggested to be involved. Furthermore, a gene called hyp-1 is assumed to attend to the final stages of the hypericin biosynthesis. In the present work, the biosynthesis of hypericins and hyperforins in H. perforatum was further studied by focusing on the elucidation of the precursors and genes involved. The incorporation of isotopically labelled branched-chain amino acids into hyperforins was investigated as well as the possibilities to enhance the production of hyperforins in H. perforatum in vitro cultures by feeding them with amino acid precursors. Furthermore, two novel cDNAs encoding for type III PKSs were isolated from H. perforatum. The functions of these new genes, designated HpPKS1 and HpPKS2, as well as the role of hyp-1 were elucidated by comparing their expression with the levels of hypericins and hyperforins in H. perforatum tissues. The enzymatic activity of the recombinant HpPKS2 protein was also analyzed. To study Hyp-1 at a protein level, a protein extraction method was optimized for tissues of Hypericum species. The results show the incorporation of valine and isoleucine into the acyl side chain of hyperforin and adhyperforin, respectively. Through the biotransformation of the amino acid precursors, it is possible to enhance the levels of adhyperforin, but not hyperforin, in H. perforatum shoot cultures, which demonstrates the tight regulation of the hyperforin biosynthesis. A correlation between HpPKS1 expression and hyperforins was detected in H. perforatum tissues. The localization of HpPKS2 mRNA in dark glands in which hypericins accumulate as well as the octaketide synthase activity of the recombinant HpPKS2 suggest that HpPKS2 is associated with possible co-operating tailoring enzymes in the biosynthesis of hypericins. The presence of both hyp-1 mRNA and Hyp-1 protein in distinct places compared with hypericins in H. perforatum tissues does not support the idea that Hyp-1 would be involved in the biosynthesis of hypericins in dark glands, although mobility of the Hyp-1 protein was shown to be possible. The present thesis extends knowledge about the biosynthesis of hypericins and hyperforins in H. perforatum by providing new candidate genes for their biosynthesis and by identifying precursors for hyperforins. Moreover, new information was obtained about the role of hyp-1 in H. perforatum.

Abstract The Achilles tendon is the largest tendon in the human body and is affected by many diseases and is vulnerable to many forms of damage due to the heavy loads it must bear. Rupture of the Achilles tendon has become more common in recent times, with an almost four-fold increase in prevalence from 1979–1990 to 1991–2000 and a peak incidence of 19 ruptures per 100 000 of population in 1999 in our epidemiological assessment. The incidences of major complications, re-rupture and deep infection, increased along with primary ruptures, peaking in 1999. The results after successful primary repair are good in over 90% of cases, as we have shown in a randomized study and in a review of the literature, and the result after re-rupture is still good in about 70% of cases, but achieving good performance after deep infection is a highly random matter. Our retrospective survey did not identify any good results, but the deep infection cases in our randomized study showed good performance due to prompt action taken for their treatment. The best method for treating a ruptured Achilles tendon has been under debate for almost 100 years, with surgery and conservative methods advocated to equal extents. We have advocated surgical treatment as the primary choice and conservative treatment is given for selected high risk patients, for example patients with diabetes, skin problems, systemic use of corticosteroids or severe other illness. The type of surgery technique is not a straightforward choice, either, and various forms of open surgery and percutaneous techniques exist. We compared an end-to-end simple suture with the same suture augmented with one central gastrocnemius turn-over flap in a randomized series of 60 patients and found no differences with respect to subjective complaints, calf muscle strength or tendon elongation with time. The end-to-end technique is simpler and is therefore justified as the primary method of choice for the surgical repair of fresh complete Achilles tendon ruptures. The tissue composition has been shown to alter not only with time but also after repeated tearing of the tendon collagen fibres. A normal tendon is mainly composed of type I collagen, but the rupture areas express more type III collagen, which is thinner and withstands loads less effectively. Type III collagen accumulates slowly in the tendon, since its production does not increase very much, a situation that is indicative of microtrauma. Crosslinking of the fibres is important for collagen matrix properties, and we found that there is a change in the quality of crosslinking with age and that this may have role in the observed changes in tendon stiffness, as also noted in other studies. We also studied the appearance of tenascin-C at the rupture site in the Achilles tendon and at two other sites in the same tendon, but found no difference in its expression. It has been proposed that tenascin-C may take part in the tendon’s reaction to loading, but its exact function remains unknown.

Doctor of Philosophy / Department of Plant Pathology / Xiaoyan Tang / P. syringae is a group of bacterial phytopathogens that can infect a wide variety of plants. These bacteria rely on the type III secretion system (TTSS) to deliver effectors into plant cells for infection. The TTSS genes, that encode the TTSS apparatus and the effectors, are repressed when bacteria grow in nutrient rich media but are strongly induced in the plants and in minimal medium (MM). Plant cutin monomers appear to negatively regulate the P. syringae TTSS genes. It is poorly understood how bacteria sense the environmental signals to regulate the TTSS genes. By genetic screen, four sets of transposon insertion mutants displaying aberrant TTSS gene expression were isolated: KB and fin mutants derepress the TTSS genes in rich medium KB and in the presence of a cutin monomer precursor in MM, respectively; min and pin mutants are defective in induction of TTSS genes in MM and in plants, respectively. A putative two-component sensor histidine kinase, RohS, is identified to be required for the induction of avrPto-LUC in MM and in plants. The rohS gene is in an operon containing a two-component response regulator gene rohR. Mutation of rohS in P. s. phaseolicola and P. s. tomato reduced the bacterial pathogenicity on hosts and HR-inducing activity on non-hosts. Our results suggested that RohS acts upstream of HrpR/HrpS. The phosphorylated RohR represses TTSS genes. It is likely that RohS acts as phosphatase of RohR in the TTSS-inducing conditions, and subsequently derepresses TTSS genes. Simple sugars such as glucose, sucrose and fructose are known to be inducers of the TTSS genes. Isolation of four min mutants defective in fructose-uptake enabled us to study if sugars serve as extracellular signals or as essential nutrients. Our results suggest that fructose acts as an essential nutrient for the activation of type III genes. These mutants slightly compromised induction of avrPto promoter in Arabidopsis and pathogenicity on the host bean plant, but displayed normal HR elicitation on non-host plant tobacco. The reduced pathogenicity suggested that exploitation of fructose from the host tissue is an important means for pathogenesis of P. s. phaseolicola.

pccenturion@gmail.com / Presentamos una innovadora técnica quirúrgica para el tratamiento de la región abdominal, en la cual combinamos lipoescultura y abdominoplastia con incisiones reducidas, sin neoonfaloplastia, en pacientes tipo III según Clasificación de Matarasso. Realizamos un estudio retrospectivo sobre pacientes intervenidos con esta nueva propuesta quirúrgica, entre los años 2002 y 2008. La lipoescultura corporal superficial y profunda incluyó también la pared abdominal anterior. La técnica quirúrgica se basa en el uso de incisiones reducidas a nivel suprapúbico, máximo de 18 cm. de longitud, con disección de un túnel de aproximadamente 10 cm. en plano supraponeurótico, desinserción del ombligo, plicatura de la diástasis de músculos rectos abdominales y posterior reinserción del ombligo inferiormente. Usamos una férula de yeso durante el postoperatorio. Evaluamos 64 lipoabdominoplastias, todas realizadas en mujeres con edad media de 41,9 años. El tiempo quirúrgico promedio fue de 3 horas y media, el volumen aspirado de la pared abdominal anterior tuvo un promedio de 1000 ml. El 14,1% de las pacientes presentaron seroma como complicación. Como conclusión creemos que esta técnica permite buenos resultados estéticos corporales totales al obtener una cicatriz corta, un colgajo abdominal anterior adelgazado y ausencia de cicatriz umbilical. / Revisión por pares

Orientador: Rodrigo Tavanelli Hernandes / Resumo: O principal mecanismo de virulência de Escherichia coli enteropatogênica (EPEC) é a capacidade de causar uma lesão histopatológica na mucosa intestinal denominada attaching and effacing (AE), caracterizada pela aderência íntima das bactérias, destruição das microvilosidades e formação de estruturas semelhantes a pedestais, ricos em F-actina, nos enterócitos infectados. Genes do locus of enterocyte effacement (região LEE) codificam todas as proteínas necessárias para a formação da lesão AE. As EPEC são divididas em típicas (tEPEC) e atípicas (aEPEC), com base na presença do EPEC adherence factor plasmid no primeiro grupo. A partir de um conjunto de sete amostras de aEPEC pertencentes ao sorotipo O2:H16, obtidos de surtos e casos esporádicos de diarreia, mostramos que cinco deles produziram uma adesão híbrida localizada/difusa (AL/AD) em células HeLa. Neste estudo, uma amostra de aEPEC deste sorotipo (282/14), que produziu o padrão AL/AD, foi selecionada para investigar as estruturas bacterianas envolvidas em seu fenótipo adesivo. Para este propósito, a amostra de aEPEC 282/14 foi mutagenizada usando o kit EZ::TN <R6Kyori/KAN-2> Tnp transposome kit, gerando uma biblioteca de inserções Tn5. Esses mutantes de inserção Tn5 foram testados quanto a perda ou redução da capacidade aderente, em ensaios realizados em 6 h de incubação com células HeLa. Dentre 320 clones pesquisados, nove foram considerados deficientes em sua capacidade de interagir com células epiteliais e quatro deles a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main virulence mechanism of enteropathogenic Escherichia coli (EPEC) is the capacity to cause a histopathological lesion on the intestinal mucosa, termed Attaching and Effacing (AE); characterized by intimate bacterial adherence, microvillus destruction and formation of F-actin rich pedestal-like structures, in infected enterocytes. Genes of the locus of enterocyte effacement (LEE region) encode all proteins necessary for AE lesion formation. EPEC are divided in typical (tEPEC) and atypical (aEPEC), based on the presence of the EPEC adherence factor plasmid in the former group. From a collection of seven aEPEC O2:H16, obtained from outbreak and sporadic cases of diarrhea, we showed that five of them produced a hybrid localized/diffuse adherence (LA/DA) in HeLa cells. In this study, we selected one aEPEC isolate of this serotype (282/14) that produced the LA/DA pattern, to investigate the bacterial structures involved in its adhesive phenotype. For this purpose, aEPEC 282/14 was mutagenized using the EZ::TN < R6Kyori/KAN-2 > Tnp transposome kit, generating a library of Tn5 insertions. These Tn5 insertion mutants were screened for non-adherent or less adherent mutants, in assays performed in 6 h of incubation with HeLa cells. Among the 320 clones screened, nine were considered deficient in their ability to interact with epithelial cells, and four of them presented the Tn5 insertion in genes within the LEE region, such as tir, escV, and grlR. In order to confirm the role of ... (Complete abstract click electronic access below) / Doutor

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative anaerobe that induces severe inflammation resulting in gastroenteritis. In the case of S. Typhimurium infection, induction of an inflammatory response has been linked to its primary virulence mechanism, the type III secretion system (T3SS). The T3SS secretes protein effectors that exploit the host’s cell biology to facilitate bacterial entry and intracellular survival, and to modulate the host immune response. One such effector, SifA, is a bi-functional T3SS effector protein that plays an important role in Salmonella virulence. The N-terminal domain of SifA binds SifA-Kinesin-Interacting-Protein (SKIP), and via an interaction with kinesin, forms tubular membrane extensions called Sif filaments (Sifs) that emanate from the Salmonella Containing Vacuole (SCV). The C-terminal domain of SifA harbors a WxxxE motif that functions to mimic active host cell GTPases. Taken together, SifA functions in inducing endosomal tubulation in order to maintain the integrity of the SCV and promote bacterial dissemination. Since SifA performs multiple, unrelated functions, the objective of this study was to determine how each functional domain of SifA becomes processed. In the present study, we demonstrate that a linker region containing a caspase-3 cleavage motif separates the two functional domains of SifA. To test the hypothesis that processing of SifA by caspase-3 at this particular site is required for function and proper localization of the effector protein domains, we developed two tracking methods to analyze the intracellular localization of SifA. We first adapted a fluorescent tag called phiLOV that allowed for T3SS mediated delivery of SifA and observation of its intracellular colocalization with caspase-3. Additionally, we created a dual-tagging strategy that permitted tracking of each of the SifA functional domains following caspase-3 cleavage to different subcellular locations. The results of this study reveal that caspase-3 cleavage of SifA is required for the proper localization of functional domains and bacterial dissemination. Considering the importance of these events in Salmonella pathogenesis, we conclude that caspase-3 cleavage of effector proteins is a more broadly applicable effector processing mechanism utilized by Salmonella to invade and persist during infection.

Indiana University-Purdue University Indianapolis (IUPUI) / Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in S. maltophilia, isogenic deletion of phosphoglycerate mutase (gpmA) and two chaperone-usher pilin subunits, S. maltophilia fimbrae-1 (smf-1) and cblA, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δsmf-1 and ΔcblA showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in P. aeruginosa, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when polB, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in S. maltophilia and genetic switches during the acute infection lifestyle in P. aeruginosa.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第21459号 / 人健博第66号 / 新制||人健||5(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 青山 朋樹, 教授 岡 昌吾, 教授 髙橋 良輔 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, microbial aspects related to Salmonella as a food pathogen are investigated. A bioluminescent reporting system was developed for Salmonella to monitor the attachment and growth of the pathogen on food products. Twelve and eleven Salmonella strains from the broiler production continuum were tagged with bioluminescence by plasmid and integration of the lux operon into the chromosome, respectively. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, an attachment model using chicken skin was developed. Variables including washing and temperature were tested in the attachment model to determine the effects on attachment of Salmonella strains to chicken skin, a characteristic that enhances persistence during processing. Additionally, the invasion process for two serovars of Salmonella with differing host tropism was examined with emphasis on the initial establishment of the bacterium in the host. The major facilitator for invasion, type III secretion system, was inactivated through deletion mutation to evaluate invasion of human epithelial cell line by additional means. The difference in host tropism between the two subspecies of Salmonella was also taken into account when evaluating invasion. Results showed that invasion of human epithelial cells can be initiated despite inactivation of the type III secretion system. A serovar of Salmonella that is not typically associated with human illness was also shown to initiate invasion of human epithelial cells, a result that carries public health implication as this serovar has recently been shown to be multi-drug resistant.

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