<p>Due to its close proximity to the gastrointestinal tract, the human urinary tract is</p><p>subjected to constant barrage by gut-­associated bacteria. However, for the most part, this tract has resisted infection by various microbes. The impregnability of the urinary tract to microbial colonization is attributable to the ability of the bladder to promptly sense and mount robust responses to microbial challenge. A powerful mechanism for the elimination of invading bacteria was recently described in bladder epithelial cells, involving non-­lytic ejection of intracellular bacteria back into the extracellular milieu. In spite of the effectiveness of this defense strategy, much of the underlying mechanisms surrounding how this powerful cellular defense activity detects intracellular UPEC and shuttles them from their intracellular location to the plasma membrane of BECs to be exported remains largely a mystery.</p><p> Here, we describe uropathogenic E.coli (UPEC) expelled from infected bladder</p><p>epithelium cells (BECs) within membrane-­bound vesicles as a distinct cellular defense</p><p>response. Examination of the intracellular UPEC revealed that intracellular bacteria were</p><p>initially processed via autophagy, the conventional degradative pathway, then delivered</p><p>into multivesicular bodies (MVBs) and encapsulated in nascent intraluminal vesicle membrane. We further show the bacterial expulsion is triggered when intracellular UPEC follow the natural degradative trafficking pathway and reach lysosomes and attempt to neutralize its pH to avoid degradation. This pathogen-­mediated activity is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized on lysosomes, which spontaneously initiates lysosome exocytosis resulting in expulsion of exosome-­encased bacteria. These studies reveal a cellular default system for lysosome homeostasis and also, how it is coopted by the autonomous defense program to clear recalcitrant pathogens.</p> / Dissertation

Urinary tract infection (UTI) is a common bacterial infection in women of all ages but the incidence and prevalence increase with age. Despite the high incidence of UTI, little is known about its impact on morale or subjective wellbeing and daily life in old women. UTI in older people can be a complex problem in terms of approach to diagnosis, treatment and prevention because in these patients it frequently presents with a range of atypical symptoms such as delirium, gastrointestinal signs and falls. Even if UTI has been shown to be associated with delirium it has frequently been questioned whether UTI can cause delirium or if it is only accidentally detected when people with delirium are assessed. The main purpose of this thesis was to describe the prevalence of UTI, to identify factors associated with UTI among very old women and to illuminate the impact of a UTI on old women’s health and wellbeing.  This thesis is based on two main studies, the GErontological Regional DAtabase (GERDA) a cross-sectional, population-based study carried out in the northern parts of Sweden and Finland during 2005-2007 and a qualitative interview study in western Sweden 2008-2009. Data were collected from structured interviews and assessments made during home visits, from medical records, care givers and relatives. UTI was diagnosed if the person had a documented symptomatic UTI, with either short- or long-term ongoing treatment with antibiotics, or symptoms and laboratory tests judged to indicate the presence of UTI by the responsible physician or the assessor. One hundred and seventeen out of 395 women (29.6%) were diagnosed as having suffered from at least one UTI during the preceding year and 233 of these 395 (60%) had had at least one diagnosed UTI during the preceding 5 years. These old women with UTI were more dependent in their activities of daily living, and had poorer cognition and nutrition. In these women, UTI during the preceding year was associated with vertebral fractures, urinary incontinence, inflammatory rheumatic disease and multi-infarct dementia. Eighty-seven of 504 women (17.3%), were diagnosed as having a UTI with or without ongoing treatment when they were assessed, and almost half (44.8%) were diagnosed as delirious or having had episodes of delirium during the past month. In all, 137 of the 504 women (27.2%) were delirious or had had episodes of delirium during the past month and 39 (28.5%) of them were diagnosed as having a UTI. Delirium was associated with Alzheimer’s disease, multi-infarct dementia, depression, heart failure and UTI. Forty-six out of 319 women (14.4%) were diagnosed as having had a UTI with or without ongoing treatment and these had a significantly lower score on the Philadelphia Geriatric Center Morale Scale (PGCMS), (10.4 vs 11.9, p=0.003) than those without UTI, indicating a significant impact on morale or subjective wellbeing among very old women. The medical diagnoses significantly and independently associated with low morale were depression, UTI and constipation. The experience of suffering from repeated UTI was described in interviews conducted with 20 old women. The interviews were analysed using qualitative content analysis. The participants described living with repeated UTI as being in a state of manageable suffering and being dependent on alleviation. Being in a state of manageable suffering was described in terms of experiencing physical and psychological inconveniences, struggling to deal with the illness and being restricted regarding daily life. Being dependent on alleviation was illustrated in terms of having access to relief but also experiencing receiving inadequate care. In conclusion, UTI is very common among old and very old women and is a serious health problem. UTI seems to be associated with delirium and to have a significant impact on the morale or subjective wellbeing of old women and those affected suffer both physically and psychologically and their social life is limited. UTI was also associated with vertebral fractures, urinary incontinence, inflammatory rheumatic disease and multi-infarct dementia which might raise the suspicion that UTI can have serious medical effects on health in old women. / Embargo t o m 2011-11-11

Proteome analysis techonologies are commonly employed for discovery-based biomarker identification studies. This thesis aims to help bridge the gap between analytical technology development and clinical application by improving and appling a proteomics workflow for biomarker discovery in congenital urinary tract obstruction (UTO). By accentuating the importance of experimental design, and evaluating the biological relevance of quantitative proteome analyses, the results of this research provide confidence in a number of identified candidate biomarkers of UTO. A sensitive method for quantification of proteome samples was developed using temperature controlled reversed-phase liquid chromatography (TPLC). The TPLC system provides high recovery (> 90 %), as well as high accuracy and precision in estimating the concentration across a number of protein sample types (CV < 10 %). The need for extensive fractionation strategies coupled with LC-MS analysis challenges the throughput of the overall experiment. Development of a dual column LC-MS interface reduced the total analysis time by a factor of 2 over conventional single column LC-MS systems. The system was applied to a quantitative proteome analysis of proximal tubule cells exposed to mechanical stretch, mimicking the conditions they experience during UTO and a urinary exosomal proteome analysis for candidate biomarker identification of this disease. A total of 1636 proteins were identified in the whole cell proteome analysis, of which 317 were found to be significantly altered in abundance. Analysis of the urinary exosomal proteome yielded 318 proteins, of which 189 were found to be altered in abundance due to obstruction. Western blot confirmation of a few select proteins provided backing to the quantitative proteome analysis, while gene ontology and KEGG pathway analysis yielded functional information. The results from the quantitative analyses of the urinary exosomes and proximal tubule cells identified candidates for both diagnosis and prognosis of UTO. In addition, activation of a novel pathway was identified, presenting a potential drug target which could be exploited to improve recovery of children following relief of UTO. This thesis therefore contributes useful technological and methodological advancements towards routine proteome analysis, as well as providing candidate biomarker identification for the leading cause of renal functional loss in children.

<p>The mammalian urinary bladder is a highly specialized organ that must be able to withstand considerable amounts of osmotic pressure at its mucosal surface, in addition to maintaining an impenetrable barrier against potential pathogens. The lower urinary tract's virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected with uropathogenic E.coli (UPEC) in combination with clinical observations that antibody responses following bladder infections are not detectable, suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells. This mast cell-dependent phenomenon involves localized production of IL-10 and results in suppressed humoral and cell-mediated responses and bacterial persistence. Therefore, in addition to the previously described role of mast cells orchestrating the early innate immune responses in the bladder during infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of mast cell-maintained immune privilege.</p><p> Interestingly, though the bladder is not capable of initiating an effective adaptive immune response during bladder infections, we have generated data showing that it was possible to circumvent the immune limitations of the bladder to provoke a strong adaptive and protective immune response by vaccinating against UPEC at an alternate mucosal site. We reasoned that by immunizing the nasal regions of mice with a vaccine formulation comprising of FimH adhesin, a highly conserved adhesive moiety of type 1 fimbriae expressed on UPEC, and an effective mucosal adjuvant we would evoke protective immunity against UPEC infections. We found that a FimH vaccine coupled with either a mast cell activating adjuvant c48/80 or CpG oligodeoxynucleotide, a TLR9 agonist, evoked high levels of FimH specific IgG antibody in the serum and IgA in the urine of immunized mice. We also observed that following UPEC challenge, these FimH/adjuvant immunized mice exhibited significantly reduced bacterial load in the bladders compared to mice challenged with just FimH. These studies reveal that immunization of nasal regions with a FimH vaccine is an effective strategy to overcome the limitation in adaptive immunity observed in the bladder.</p> / Dissertation

Urine culture, the current gold standard for urinary tract infection (UTI) diagnosis, does not produce results in an acceptable length of time. An ultra-sensitive, cost-effective electrochemical biosensing platform with nanostructured microelectrodes was designed to address the need for a rapid, point-of-care (PoC) test that could achieve a sample-to-answer time in less than an hour. Printed circuit boards and metallized glass slides were processed using various techniques and then tested for their ability to form nanostructured microelectrodes. Peptide nucleic acid probes for the bacteria and yeast as well as ten probes for antibiotic resistance genes were designed and synthesized for use with the new platform. Validation of the sensor's specificity was performed using high concentrations (100nM) of synthetic DNA oligomers. Furthermore, a clinically relevant sensitivity of 103 cfu/mL was demonstrated by detecting 4 pathogen lysates (Staphylococcus saprophyticus, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae) in a buffered solution.

Urine culture, the current gold standard for urinary tract infection (UTI) diagnosis, does not produce results in an acceptable length of time. An ultra-sensitive, cost-effective electrochemical biosensing platform with nanostructured microelectrodes was designed to address the need for a rapid, point-of-care (PoC) test that could achieve a sample-to-answer time in less than an hour. Printed circuit boards and metallized glass slides were processed using various techniques and then tested for their ability to form nanostructured microelectrodes. Peptide nucleic acid probes for the bacteria and yeast as well as ten probes for antibiotic resistance genes were designed and synthesized for use with the new platform. Validation of the sensor's specificity was performed using high concentrations (100nM) of synthetic DNA oligomers. Furthermore, a clinically relevant sensitivity of 103 cfu/mL was demonstrated by detecting 4 pathogen lysates (Staphylococcus saprophyticus, Pseudomonas aeruginosa, Enterococcus faecalis and Klebsiella pneumoniae) in a buffered solution.

Antibiotic-resistant bacteria are increasingly a major healthcare problem but very few new classes of antibiotics have been discovered or launched in recent decades. Approaches to dealing with the problem include learning how bacteria evolve to resistance and improving dosing regimens with current antibiotics so as to reduce the selection of resistant bacteria. This thesis presents studies examining whether antibiotic dosing at high levels can prevent the selection of fluoroquinolone-resistant mutants in Escherichia coli. It also addresses the genetics of fluoroquinolone resistance in E. coli in relation to fitness costs for the resistant bacteria, and the evolution of E. coli to reduce the costs of resistance. The mutant prevention concentration (MPC) of ciprofloxacin was measured for a set of clinical urinary tract infection E. coli strains showing that MPC could not be predicted from the minimum inhibitory concentration (MIC). Results from an in vitro kinetic model showed that an AUC/MPC &gt;22 for ciprofloxacin was the single best pharmacodynamic index that predicted prevention of resistance emergence in the wild-type. Simulating currently approved dosing regimens for three different fluoroquinolones it was found that only a few were effective in preventing the selection of a small sub-population of pre-existing mutants. Step-wise selection of fluoroquinolone resistance showed that the accumulation of mutations usually reduced bacterial fitness in vitro and in vivo. Systematic construction of isogenic resistant strains confirmed this result and revealed that some combinations of resistance mutations mutually compensate and increase both resistance and fitness. It was discovered that mutations altering RNA polymerase could ameliorate the fitness costs of fluoroquinolone resistance. Thus, the major fitness cost of fluoroquinolone resistance is due to defective transcription. The finding that fluoroquinolone resistance mutations can increase resistance while mutually compensating their fitness costs, shows that resistance to fluoroquinolones can continue to evolve in the absence of antibiotic selection.

Doctor of Medicine / Publications 1-49 represent studies that I have undertaken myself or conjointly over a 34 year period to investigate a variety of issues relating to diseases of the kidney and urinary tract in children. The studies were carried out at the Royal Alexandra Hospital for Children, Camperdown when I was Clinical Superintendent from 1968 - 1970; The Department of Paediatrics, University of Minnesota, Minneapolis, USA when I was Overseas Research Fellow of the Post Graduate Foundation in Medicine, University of Sydney, 1970 - 1972, then as Staff Physician in Nephrology at the Royal Alexandra Hospital for Children, Camperdown, 1972 - 1977, and then Head of that Department at the Hospital until 1995 and then as an Honorary Staff Specialist at that hospital. Some of the studies were done conjointly with members of the Renal Unit of Royal Prince Alfred Hospital where I hold an Honorary appointment and others conjointly with members of the Renal Unit of Prince Henry Hospital, Little Bay. I was appointed Clinical Associate Professor to the Department of Paediatrics and Child Health, University of Sydney in 1993. In 1966 paediatric nephrology was in the early phase of development as a medical subspecialty. There was no definitive textbook, the first was published in 1975 (Pediatric Nephrology, Ed. Mitchell I. Rubin. Williams and Wilkins.). In the preface to the 2nd edition of Renal Disease (Blackwell) in 1967 the editor D.A.K. Black noted that he had included a chapter on paediatric aspects which had been planned for the 1st edition in 1962 but ”it could not be arranged”. In the chapter on Renal Disease in Children the author, D.Macauly, comments that the mortality rate of acute renal failure in children was 50%. When I joined the resident staff of the Royal Alexandra Hospital for Children in 1966, children with renal disease were managed by general paediatricians. There was no active program for the treatment of children with acute or chronic renal failure. A small number of kidney biopsies had been performed by Dr Trefor Morgan who, together with Dr Denis Wade, had taught me the technique while I was a resident medical officer at the Royal Prince Alfred Hospital in the preceding year. With the guidance and support of Dr S.E.J. Robertson and Dr C. Lee, Honorary Medical Officers, and Dr R.D.K. Reye, Head of the Department of Pathology, I began performing kidney biopsies on children at the request of the paediatrician in charge. In the same year, encouraged again by Doctors Robertson and Lee, and by J.C.M. Friend and J. Brown, I introduced peritoneal dialysis for the treatment of children with acute renal failure, a technique which I had also been taught by Dr Trefor Morgan whilst I was a resident at Royal Prince Alfred Hospital. Dr Robertson encouraged me to present my experience in percutaneous renal biopsy in children at the Annual Meeting of the Australian Paediatric Association in 1968 and this study became the first paper I published in relation to disease of the urinary tract in children (1). In 1970 I was granted an Overseas Research Fellowship by the Post Graduate Foundation in Medicine, University of Sydney, to enable me to undertake a fellowship in the Department of Paediatrics at the University of Minnesota. I had the great fortune in undertaking studies in the new discipline of paediatric nephrology and related research under the guidance of Dr A. F. Michael, Dr R.L.Vernier and Dr A. Fish. I acquired the techniques of immunopathology and electron microscopy. On my return to Australia I established a Department of Nephrology at the Royal Alexandra Hospital for Children. I introduced immunofluorescent and electron microscopic studies for the kidney biopsies that I continued to perform and, with the support of Dr R.D.K. Reye, I provided the official reports of these studies until 1990. As a result these studies became part of the histopathologic service provided by the hospital. I continue to be consulted concerning the interpretation of some electron microscopic findings in renal tissue. With the assistance of Dr J.D. Harley I set up a laboratory in the Children’s Medical Research Foundation to continue and expand the studies I had commenced during my Fellowship. Establishing a dialysis and transplant program for children with end stage renal disease (ESRD) was extremely time consuming. At that time most children with ESRD died. The program was initially established jointly with the Renal Unit at Royal Prince Alfred Hospital in 1972 and eventually dialysis facilities were established at the Children’s Hospital using predominantly peritoneal dialysis. By 1978 the existence of the Unit was well known in the general community and articles appeared in the press. One prompted the late Sir Lorimer Dods, the first Professor of Paediatrics in Australia to write to me congratulating me on what I had achieved. He remarked “I have just read with special interest Shaun’s review in the SMH of some of your recent achievements in the field of renal failure in infancy and childhood and want to offer you my personal congratulations on all that you have achieved and are achieving in this area of paediatrics which, in my little world of yesterday, meant nothing more than progressive and unrelenting fatal illness”. Taking part in the development of a relatively new discipline led me to study a number of areas. I encouraged trainees to write reports concerning clinical observations and eventually I was joined by Fellows whom I encouraged and supported to study a number of different areas to ensure that children were being cared for in an environment of strong and open enquiry. This led to studies on investigations of chronic renal failure which Dr Elisabeth Hodson pursued and studies on urinary tract infection in small children for which Dr Jonathon Craig was awarded a PhD. As I had been a contributor and co-author in a number of these studies they have been included in my list of publications. As a result of this diversity I have listed the publications in 9 sections. The overall theme is to study diseases of the renal tract in children and treatments used to understand the processes and ensure the most effective treatment. Some published abstracts of papers presented at scientific meetings have been included to clarify invitations I received to prepare reviews and chapters on various subjects and my involvement in some conjoint studies. I was author or coauthor of several book chapters, reviews, editorials and certain published studies to which I was invited to contribute as a result of my primary studies and these I have included as “Derivative References”numbered 50-76.

Published papers (M.D.)--Dept. of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, 2005. / Title from title screen (viewed June 28, 2007). Submitted in fulfilment of the requirements for the degree of Doctor of Medicine to the Dept. of Paediatrics and Child Health, Faculty of Medicine. Includes bibliographical references. Also issued in print.

Tez (Tıpta Uzmanlık) - Süleyman Demirel Üniversitesi, Tıp Fakültesi, Klinik Mikrobiyoloji ve Enfeksiyon Hastalıkları Anabilim Dalı, 2003. / Bibliyografya var.