<p>Drugs and other foreign compounds must often be metabolised before they can be excreted from the body. One enzyme system that is responsible for this is the cytochrome P450 gene family (CYP). In this thesis, new sensitive molecular techniques have been used to study the human gene expression of some CYP enzymes, as well as the P-glycoprotein transporter (P-gp). The aim was to evaluate whether tissues other than the liver, e.g. the blood, could be used to assess an individual's drug metabolic capacity. Another aim was to investigate the gene expression in relation to the liver transplant process and a third aim was to evaluate the expression in gastrointestinal mucosa in both normal and inflamed mucosa.</p><p>We evaluated the CYP gene expression in paired specimens of liver and blood but found no correlation in the expression patterns of these two tissues. Instead, we found the opposite pattern, where, for example, CYP1B1 had the highest expression in the blood but the lowest in the liver and CYP2E1 was the enzyme with the highest expression in the liver. In an investigation of the expression of four different CYP enzymes and P-gp in liver transplants before and during the first year after transplantation, we found that the levels of all the CYP enzymes but not P-gp increased with time. We also found that the expression of CYP3A4 was inversely related to the normalised plasma levels of the immunosuppressive drugs cyclosporine and tacrolimus.</p><p>In the gastrointestinal tract, CYP2E1 was the enzyme with the highest mRNA expression compared with CYP3A4, CYP3A5 and the transporter P-gp. CYP3A4 has its highest expression in the duodenum compared with the expression in the stomach and the colon. CYP3A5 is expressed at a higher level than CYP3A4 in the colon. P-gp expression levels increase through the gastrointestinal tract to the left colon. Gene expression levels of CYP2E1 and CYP3A4 decrease in severely inflamed rectal mucosa. </p><p>In conclusion, this is a sensitive method for studying gene activity in a clinical situation, even though at this point we are not able to use blood or gastrointestinal mucosa as “surrogate” tissue to estimate an individual’s drug metabolic capacity. The studies in liver transplants and gastrointestinal mucosa are unique in that the gene expression is investigated during a clinical course of events.</p>

In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. Different in vitro systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans. In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low in vitro but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.

Drugs and other foreign compounds must often be metabolised before they can be excreted from the body. One enzyme system that is responsible for this is the cytochrome P450 gene family (CYP). In this thesis, new sensitive molecular techniques have been used to study the human gene expression of some CYP enzymes, as well as the P-glycoprotein transporter (P-gp). The aim was to evaluate whether tissues other than the liver, e.g. the blood, could be used to assess an individual's drug metabolic capacity. Another aim was to investigate the gene expression in relation to the liver transplant process and a third aim was to evaluate the expression in gastrointestinal mucosa in both normal and inflamed mucosa. We evaluated the CYP gene expression in paired specimens of liver and blood but found no correlation in the expression patterns of these two tissues. Instead, we found the opposite pattern, where, for example, CYP1B1 had the highest expression in the blood but the lowest in the liver and CYP2E1 was the enzyme with the highest expression in the liver. In an investigation of the expression of four different CYP enzymes and P-gp in liver transplants before and during the first year after transplantation, we found that the levels of all the CYP enzymes but not P-gp increased with time. We also found that the expression of CYP3A4 was inversely related to the normalised plasma levels of the immunosuppressive drugs cyclosporine and tacrolimus. In the gastrointestinal tract, CYP2E1 was the enzyme with the highest mRNA expression compared with CYP3A4, CYP3A5 and the transporter P-gp. CYP3A4 has its highest expression in the duodenum compared with the expression in the stomach and the colon. CYP3A5 is expressed at a higher level than CYP3A4 in the colon. P-gp expression levels increase through the gastrointestinal tract to the left colon. Gene expression levels of CYP2E1 and CYP3A4 decrease in severely inflamed rectal mucosa. In conclusion, this is a sensitive method for studying gene activity in a clinical situation, even though at this point we are not able to use blood or gastrointestinal mucosa as “surrogate” tissue to estimate an individual’s drug metabolic capacity. The studies in liver transplants and gastrointestinal mucosa are unique in that the gene expression is investigated during a clinical course of events.

Hypoxia Inducible Factor-1α in Renal Cell Carcinoma Departments of Surgical and Perioperative Sciences, Urology and Andrology; Radiation Sciences, Oncology; Medical Biosciences, Pathology; and Medical Biosciences, Clinical Chemistry, Umeå University, Umeå, Sweden Background: Renal cell carcinoma (RCC) accounts for approximately 2-3% of all human cancers. A distinguished feature of RCC is vascularisation and among the three dominating RCC types conventional RCC (cRCC) generally is more vascularised than papillary RCC (pRCC) and chromophobe RCC (chRCC). Angiogenesis is a critical step in tumour progression controlled by a balance involving molecules that have positive and negative regulatory activity. A balance distorted by metabolic stress such as hypoxia, acidosis, and inflammation. Hypoxia-Inducible Factor 1α (HIF-1α) is a key transcription factor in angiogenesis and tumour progression, targeting more than a 100 genes involved in vascular growth and regulation, iron metabolism and erythropoesis, collagen matrix formation, regulation of extracellular pH, glucose uptake and metabolism, proliferation, apoptosis, differentiation, and cell viability. Methods: Tumour tissue and corresponding kidney cortex from nephrectomised RCC patients was used in order to characterize HIF-1α expression and one of its target genes, Glucose Transporter 1 (GLUT-1). All tumour samples were thoroughly described regarding tumour type, TNM stage, nuclear grade, tumour size, vein invasion, and patient survival. Utilizing RT-PCR, Westen Blot and Tissue micro array (TMA) we studied HIF-1α mRNA and protein expression as well as GLUT-1 protein expression, correlating them to each other and clinicopathological parameters. Results: Using Western Blot, HIF-1α protein expression differed significantly between the different RCC types and kidney cortex. In cRCC, high expression of HIF-1α was an independent prognostic factor for favourable prognosis. TMA is a useful method to analyze HIF-1α protein expression in RCC. HIF-1α levels were significantly lower in locally aggressive cRCC and patients with high levels of HIF-1 tended to have a better prognosis. GLUT-1 levels were higher in cRCC than in other RCC types and for cRCC a correlation to HIF-1α was seen. HIF-1α mRNA levels were significantly lower in cRCC compared to other RCC types and kidney cortex. An inverse correlation between HIF-1α protein expression and mRNA levels was observed. Summary: These results demonstrate a discrepancy between RCC types, highlighting the need to separately evaluate biological events in different RCC types. Overexpression of HIF-1α protein is not necessarily all bad and translational regulation appears more critical than anticipated. Further studies are encouraged to clarify angiogenic pathways in RCC.

Artificial divergent selection of chickens for high and low body weight at 8 weeks of age has produced two lines: the high (HWS) and low (LWS) body weight chicken lines. In addition to the difference in body weight, the lines show extreme differences in feeding behaviour and body composition. The aim of this study was to uncover the genetic and molecular factors that contribute to and determine these differences, especially regarding body energy regulation and appetite. In papers I and II, genome-wide gene expression in a brain sample containing hypothalamus and in dissected hypothalamus was analysed using DNA microarray and qRT-PCR. We found that levels of differential expression were generally moderate, which was consistent with the idea that polygenic factors were involved in the establishment of the chicken lines. Genes associated with neural plasticity, lipid metabolism and body energy regulation were differentially expressed. This result indicated that the neural systems regulating feeding behaviour and body weight were altered in the chicken lines. However, genes that were involved in the central melanocortin system were not systematically differentially expressed. Interestingly, the biggest differences in expression between the lines found in endogenous retrovirus sequences of the ALV subgroup E. Thus, in paper III, we characterized the number of integrations, the expression of ALVE retroviral elements and their effects on body weight. A significant correlation between low body weight and high ALVE expression was observed in female F9 birds from an HWS x LWS advanced intercross line. This implied that ev-loci contributing to increased ALVE expression levels were genetically linked to loci influencing the low body weight of the pullets. In paper IV, the carnitine palmitoyltransferase-1b gene (CPT1B), which was highly differentially expressed in the hypothalami, was investigated. We mapped chicken CPT1B to the distal tip of chromosome 1p. The levels of CPT1B mRNA in the HWS line were higher in the hypothalamus and lower in muscle than in the LWS line. This pattern of differential expression indicates that this gene could contribute to the remarkable phenotypic differences between HWS and LWS chickens. However, comparison with quantitative trait loci data showed that the expression of CPT1B is a trans effect, rather than a direct causative locus. In conclusion, the data suggested that the long-term selection for body weight resulted in differential gene expression in the brains of the selected chicken lines. These results may have relevance for the poultry industry and will also contribute to increasing knowledge about human diseases such as obesity and anorexia.

One-lung ventilation (OLV) as an established procedure during thoracic surgery may be injurious in terms of increased mechanical stress characterised by alveolar cell stretch and overdistension, increased cyclic tidal recruitment of alveolar units, compression of alveolar vessels and increased pulmonary vascular resistance. This may result in ventilation-induced lung injury with pro-inflammatory cytokine production, leukocyte recruitment and neutrophil-dependent tissue destruction. Despite the consequences of delivering the whole tidal volume (VT) to only a single lung, relatively high VT are used during OLV to maintain arterial oxygenation and carbon dioxide elimination. However, this may increase mechanical stress in the dependent lung and may aggravate alveolar injury. There is a lack of data on the alveolar immune consequences of OLV. Therefore, the present studies investigate the epithelial damage and pro-inflammatory response induced by mechanical ventilation and OLV. OLV induced pulmonary injury, but alveolar damage in the ventilated lung decreased by reduction of the tidal volume in patients scheduled for thoracic surgery (study I). The use of the volatile anaesthetic desflurane in OLV patients attenuated the OLV-induced alveolar immune response (study II). Furthermore, an experimental model of thoracic surgery was established to investigate the systemic and pulmonary consequences of OLV and thoracic surgery in comparison with the effects of conventional two-lung ventilation and spontaneous breathing. The experimental data indicate that beside the pulmonary immune response volatile anaesthetics have also modulated the plasma concentrations of cytokines during and after OLV (study III). In contrast, OLV and thoracic surgery increased the expression of pro-inflammatory mRNA in BAL cells and lung tissue samples. General anaesthesia did not affect this response (study 4). The results of the present studies indicate that OLV and thoracic surgery may be injurious to the lung tissue to a similar degree. The recruitment and activation of alveolar granulocytes characterise the alveolar damage. The administration of different anaesthetics modulates the activation of alveolar cells, specified by decreased inflammatory mediator release in subjects that receive desflurane anaesthesia, which does not affect the expression of cytokine mRNA in alveolar cells and lung tissue samples.

Gene expression differences between patients and controls can be used to find susceptibility genes and drug targets for a disease. High-resolution strategies are required because the differences between the investigated groups may be small and numerous factors may affect the mRNA quantity. This thesis is based on the use of real-time RT-PCR combined with a new statistical approach, developed to detect small differences between patients and controls and differences due to patient subgroups. Comparisons between human brain biopsy and autopsy samples showed that post-mortem tissue can be used to make conclusions on the relative mRNA levels in the living brain. Power analysis based on human brain mRNA expression from 14 genes adjusted with two reference genes, revealed that a sample size of 50 patients and 50 controls was required to detect a 2-fold difference with a power and a confidence of 95%. A similar study in rats revealed that approximately the same sample size was required for rat brain mRNA expression studies. The mRNA levels of several genes were studied in 55 schizophrenia and 55 control prefrontal brain autopsies, using a novel and more powerful statistical analysis. The serotonin receptor 2C gene (HTR2C) showed a significant 1.5-fold decrease in the patients as compared to controls, and the monoamine oxidase B gene (MAOB) a 1.2-fold increase. The mechanism behind the decrease of HTR2C mRNA levels was investigated by studying the correlation of drug treatment and HTR2C promoter polymorphisms to the HTR2C expression levels. The observed decrease was present in untreated patients, suggesting that the HTR2C mRNA decrease is correlated with the disease and not the treatment. There was no association between promoter polymorphisms and HTR2C expression levels. Thus, the molecular mechanism for the decreased expression remains unclear. Nevertheless, the results support a role for monoaminergic synapses in schizophrenia.

Insulin-like growth factor-1 (IGF-1), a known secretory product of intestinal subepithelial myofibroblasts (ISEMF), is essential for the intestinotrophic effects of glucagon-like peptide-2(GLP-2). I hypothesized that GLP-2 increases the production of IGF-1 by primary murine ISEMF in culture. Immunocytochemistry showed that the ISEMF stained appropriately for α smooth muscle actin and vimentin but not for desmin. The ISEMF also expressed GLP-2 receptor and IGF-1 mRNA transcripts. ISEMF treated with GLP-2 revealed a maximal increase in IGF-1 mRNA transcript levels at 10-8 M GLP-2 and 2hr. Interestingly, immunoblotting revealed an increase in P-AKT/T-AKT with GLP-2, but no changes in cAMP, P-ERK/T-ERK or calcium were detected. PI3K inhibition and kinase-dead AKT over-expression abrogated GLP-2-induction of IGF-1 mRNA, and ISEMF from GLP-2R null mice demonstrated reductions in IGF-1 mRNA and cellular IGF-1, but not in media IGF-1, vs. wild-type ISEMF. These findings suggest a possible mechanism by which GLP-2 increases intestinal growth in-vivo.

Insulin-like growth factor-1 (IGF-1), a known secretory product of intestinal subepithelial myofibroblasts (ISEMF), is essential for the intestinotrophic effects of glucagon-like peptide-2(GLP-2). I hypothesized that GLP-2 increases the production of IGF-1 by primary murine ISEMF in culture. Immunocytochemistry showed that the ISEMF stained appropriately for α smooth muscle actin and vimentin but not for desmin. The ISEMF also expressed GLP-2 receptor and IGF-1 mRNA transcripts. ISEMF treated with GLP-2 revealed a maximal increase in IGF-1 mRNA transcript levels at 10-8 M GLP-2 and 2hr. Interestingly, immunoblotting revealed an increase in P-AKT/T-AKT with GLP-2, but no changes in cAMP, P-ERK/T-ERK or calcium were detected. PI3K inhibition and kinase-dead AKT over-expression abrogated GLP-2-induction of IGF-1 mRNA, and ISEMF from GLP-2R null mice demonstrated reductions in IGF-1 mRNA and cellular IGF-1, but not in media IGF-1, vs. wild-type ISEMF. These findings suggest a possible mechanism by which GLP-2 increases intestinal growth in-vivo.

Prostate cancer is one of the most common malignant tumors in solid organs of old men. However, the patients are nearly 100 percent survivable if detected early. Prostate-specific antigen (PSA) is a valuable prostate cancer biomarker that is now wildly used for population screening, diagnosis, and monitoring of patients with prostate cancer. But PSA is not good enough for a biomarker because it can not distinguish benign prostate hyperplasia (BPH) from prostate tumor . Recently, there are some tumor marker still in study, for example: free prostate specific antigen¡]fPSA), complexed prostate specific antigen (C-PSA), kallikrein,prostate specific membrane antigen (PSMA). From previously study,we had found many different protein expressions between serum of normal and prostate patients. Mayven is one of the novel proteins that had been identified. The mRNA expression of Mayven in prostate cancer tissue is determined by quantitative RT-PCR. The result shows that the mRNA expression of Mayven in Benign Prostatic Hyperplasia (BPH) is about 5.0-11.3 fold than normal tissue , 12.7 fold in Prostate Cancer (PCa) stage T1 and 0.1- 3.7 fold after cancer stage T2. The Mayven gene expression is predominate in tumor stage T1, decrease after T2 stage. However the expressed pattern of mayven in BPH remains further investigation due to the limited sample size. Furthermore, with 2 dimensional electrophoresis (2¡VDE), we have found 7 differentially expressed proteins between tissue of normal and prostate patients, and these proteins are identified by MALDI-TOF mass spectrometry and MS-Fit. These identified proteins are Keratin 8¡]KRT8¡^, MAPKkinase5¡]MAP2K5¡^, Acid phosphatase (ACPP), Annexin A3¡]ANXA3¡^, Phosphoglycerate mutase 1 (PGAM1), Spindlin-like protein 2 (SPIN2) and Transgelin 2¡]TAGLN2¡^.