No description available.

No description available.

No description available.

No description available.

Tuberous sclerosis complex (TSC) is characterised by the development of benign growths across the body and is caused by mutations in TSC1 or TSC2. The TSC gene products have an established role in the regulation of mammalian target of Rapamycin (mTOR) signalling. Clinical trials are underway for the treatment of TSC- associated tumours using mTOR inhibitors. Here, we show that many of the earliest renal lesions from Tsc1+, and Tsc2+/' mice (cysts) do not exhibit mTOR activation, suggesting alternative pathways should be targeted to prevent tumour formation. Patients with TSC often develop renal cysts (derived from dilated tubules) and those with inherited co-deletions of the autosomal dominant polycystic kidney disease (ADPKD) gene 1 (PKD1) develop severe, early onset polycystic kidneys. Using mouse models, we have shown that the Tsc and Pkd1 gene products are required for correct cell polarisation during renal tubule and bile duct elongation. When polarity is disrupted in Tsc1+I', Tsc2+, and Pkd1+' mice, we found significant alterations in the length of primary cilia projecting from pre-cystic tubule and duct cells (consistent with the highly polar nature of this organelle). The primary cilium is proposed to facilitate many signalling events and provides a mechanosensory input into renal tubule cells. Despite widespread defects in cell polarity and primary cilia in the developing kidney of a Tsc1+I , Tsc2+/ or Pkd1+I' mouse, we found no evidence of tubule dilation, occlusion or cyst formation until around 3-6 months of age. On the basis of this delay period, combined with our data showing significantly higher levels of cleaved caspase-3 in pre-cystic renal tubules from these mice, we suggest that apoptosis destroys these misaligned cells to protect against cyst formation. We found that almost all cysts without mTOR-activation failed to stain for cleaved caspase-3, and therefore sought activation of a pro-survival pathway. There was strong upregulation of Bcl2 in mTOR inactive cysts that were not undergoing apoptosis, suggesting this was the mediator of survival in our cysts. In cysts without activation of mTOR or apoptosis, we found significant activation of Jak2 and its downstream target Stat3. We finally sought gain-of-function mutations in this pathway, and found several somatic Jak2 mutations with likely oncogenic potential in Tsc-associated cysts. These data suggest that defective cell polarity in the context of abnormal Jak2 signalling can drive Tsc-associated cystogenesis in the absence of mTOR dysregulation and targeting of this pathway may be of key therapeutic benefit.

The data presented shows that IL-1beta has a biphasic effect on PTC TGF-beta signalling, with early NF-kappaB-mediated inhibition and delayed sensitization via an autocrine IL-6 loop, and possibly also via an autocrine IL-6 loop, and possibly also via a switch from NF Kappa B p52/p50 heterodimer to p50/p50 homodimer formation. Secondly, the data indicates that BMP-7 prevents TGF-beta1-mediated loss of the transcriptional repressor SnoN and hence specifically limits Smad3 DNA binding, altering the balance of transcriptional responses to TGF-beta1 in PTC.

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by germline mutations in either TSC1 or TSC2 and characterised by the development of benign hamartomatous growths in multiple organs and tissues. Clinical trials are underway for the treatment of TSC-associated tumours using mammalian target of rapamycin (mTOR) inhibitors. Here, we show that many of the earliest renal lesions from Tsc1+/ and Tsc2+/ mice do not exhibit mTOR activation, suggesting that pharmacological targeting of an alternative pathway may be necessary to prevent tumour formation. Patients with TSC often develop renal cysts and those with inherited co- deletions of the autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) develop severe, early onset, polycystic kidneys. Using mouse models, we crossed Tsc1+, and Tsc2+I mice with Pkd1+/ mice to generate double heterozygotes. We found that Tsc1+lPkd1+, and Tsc2+l Pkd1+, mice had significantly more renal lesions than their corresponding single heterozygote littermates indicating a genetic interaction between Tsd and Tsc2 with Pkd1. In agreement with our findings from Tsc1+/ and Tsc2+/ mice, we found that a large proportion of cysts from Tsc1+l Pkd1+, and Tsc2+l Pkd1+, mice failed to stain for pS6, suggesting that initiation of renal cystogenesis in these animals may occur independently of mTOR activation. We analysed primary cilia in phenotypically normal renal tubule epithelial cells by scanning electron microscopy (SEM) and found that those from Tsc1+, and Tsc2+I mice were significantly shorter than those from wild-type littermates (2.122pm and 2.016pm vs. 2.233pm, respectively, P<0.001). Primary cilia from epithelial cells lining renal cysts of Tsc1+' and Tsc2+I' mice were consistently longer (5.157pm and 5.091pm respectively). Interestingly, we found that Pkd1- deficiency coupled with either Tsd or 7sc2-deficiency altered the length of the primary cilia from both normal renal tubule cells (restored to 'wild-type' length) and epithelial cells lining cysts (Tsc1+tPkd1+, Mean 3.38pm and Tsc2+,Pkd1+l Mean 3.09pm). These novel data demonstrate that the Tsc and Pkd1 gene products help regulate primary cilia length which may prevent renal cystogenesis. Consistent with the observation that primary cilia modulate the planar cell polarity (POP) pathway, we found that many dividing pre-cystic renal tubule epithelial cells from Tsc1+/ , Tsc2+/ and Pkd1+/ mice were highly misorientated along the tubule axis. This could potentially lead to tubule dilation and subsequent cyst formation. We therefore propose that defects in cell polarity underlie both TSC and ADPKD-associated renal cystic disease and targeting of this pathway may be of key therapeutic benefit.

This thesis examines the role of microRNAs in renal fibrosis. MicroRNAs constitute a large family of approximately 22-nucleotide-long non-coding RNAs, that in animal cells regulate gene expression posttranscriptionally. At the start of the project, microRNAs were emerging as potentially important factors in various physiological and pathological processes however, there was very little known about their expression and function in the kidney, in particular in tubulointerstitial fibrosis. In this thesis, global microRNA expression has been analysed in vitro in proximal tubular epithelial cells, and in vivo in formalin-fixed, paraffin-embedded kidney biopsy samples from patients with diabetic nephropathy. Among microRNAs altered by profibrotic stimuli, the greatest difference has been found in expression ofmiR-192, which is downregulated in severe diabetic nephropathy. Further examination of miR-192 in kidney biopsy samples has revealed that its expression correlates well with renal function and inversely correlates with fibrosis. A possible function of miR-192 has been then studied in vitro in proximal tubular epithelial cells. It has been found that treatment of the cells with the profibrotic cytokine TGF-β1 downregulates miR-192. Moreover, manipulation of miR-192 expression has shown that miR-192 is involved in E-cadherin regulation. The mechanism of that regulation has been investigated, pointing to two transcriptional repressors of E-cadherin, ZEB1 and ZEB2, as direct targets of miR-192. The presented data suggest that, in the kidney, miR-192 may prevent epithelial-to-mesenchymal transition, known to contribute to renal fibrosis. In parallel, global microRNA downregulation in proximal tubular epithelial cells has been attempted. However, knockdown of Dicer or TRBP, proteins involved in microRNA processing, has not been sufficient to induce changes in microRNA expression. The possible explanations have been discussed. Finally, microRNA role in direct regulation of TGF-β1 synthesis has been investigated. In particular, human microRNAs similar to viral hsv-miR-LAT, reported to directly target TGF-β1 mRNA, have been tested.

Urine samples were collected from 71 urostomy patients over a period 2 years. E. coli was the most stable (stability index 88%) followed by Pr. Mirabilis (72%), Ent. faecalis (55%) and Kleb. pneumoniae (52.5%). Perianal swabs were obtained from 6 patients with Pr. mirabilis urinary infections and 3 of these produced Pr. mirabilis on culture. Urostomy strains of Pr. mirabilis had higher protease, urease and swarming abilities than strains from other sources. The mannose resistant haemagglutination assay revealed that all the 20 urostomy isolates and 15 of the environmental isolates of Pr. mirabilis were positive for MR/P fimbriae. Examination of the sensitivity of Pr. mirabilis isolates to antiseptics revealed that the minimum inhibitory concentration of chlorhexidine ranged from 50-800microg/ml compared to 0.1-0.3microg/ml for triclosan. All urostomy isolates were sensitive to ampicillin, amoxicillin, gentamicin, ciprofloxacin and cefadroxyl. Resistance was confined to trimethoprim (17/30) and nitrofurantoin (30/30). Analysis of the sensitivity of all the Pr. mirabilis isolates revealed an association between resistance to chlorhexidine and the multiplicity of drug resistance. Isolates of Pr. mirabilis from four sources, typed using Dienes typing and pulsed field gel electrophoresis (PFGE), resulted in many different types. Both methods showed that urostomy isolates of Pr. mirabilis were all distinct strains. Cross-infection, therefore, between urostomy patients is rare. Antibody-coated bacteria were found in all the urine samples tested. Serum antibodies to all the isolates tested were high (typically =1:1024). The results of the endotoxin assays indicated that particularly in patients infected with Pr. mirabilis, bacterial lysis and release of endotoxin is occurring in conduit urine.

This thesis provides an insight into the problems of urinary tract infection (UTI) through the eyes of women sufferers, non-sufferers, and health professionals. It describes the use of language and metaphor in women’s descriptions. It investigates current ideas and knowledge published in academic journals, in books, and on the Internet, and assesses the quality of currently available web-based information. The thesis is based almost entirely on qualitative methodologies. I used grounded theory for the studies of lay and professional ideas. Focus groups preceded one-to-one interviews. The study of language and metaphor is derived from lay interviews and uses discourse analysis. I based the studies of Internet information on two surveys, one year apart, of popular websites drawn from four commonly used search engines. I rank ordered popular websites and assessed information in the ‘top twenty’ using content analysis and a simple, predominantly binary, scoring system based on an internationally recognised set of criteria. Folklore and myths, often passed down the generations, and sometimes shared by doctors, are important factors in women’s health beliefs. Early learning experiences during medical training may contribute disproportionately to doctors’ beliefs. UTIs cause embarrassment, and women rarely discuss their illness with male friends and relatives. They are also happier to discuss their problems with female health professionals, though they more commonly cite shared experience rather than embarrassment as the reason for this choice. Since these studies were completed, a major project concluded that delayed prescriptions should be used for UTI. The natural history of this illness and women’s prior use of self-management prior to attendance suggest that this strategy may not be readily accepted. Nurses and pharmacists are keen to manage UTI. As UTI lends itself to management by algorithm, delegation to professionals other than doctors may be effective. Easy access to antibiotics increases resistance; fear of this inhibits the implementation of devolved care. The quality of information on the Internet is variable and some of the most popular sites score poorly when compared against recognised criteria. Better quality sites are becoming more prominent when searching the Internet through popular search engines, and efforts to improve this source of information are important. Future research is probably best directed at information transfer and new models of delivery of care.