Abstract not available

[Truncated abstract] For this thesis, the molecular biology of cancer was approached from two directions. Firstly, an investigation was conducted on the role of growth factor receptor-bound protein 7 (Grb7) in breast cancer. Grb7 is an adapter molecule that binds to a variety of proteins, including the growth factor receptor and proto-oncogene, ErbB2, and mediates signalling to downstream pathways. It has been linked to cell migration and an invasive phenotype, and is of interest as a therapeutic target. To investigate the role of Grb7 in breast cancer, preliminary experiments were performed that, firstly, determined the expression of wild-type Grb7 and a splice variant, Grb7V, in a range of cell lines, and secondly, aided the development of a protocol for treating cells with short interfering RNA (siRNA) against Grb7 and the ErbB ligand, heregulin (HRG), in a cell system appropriate for measuring the functional outcomes. Using this protocol in conjunction with CellTitre (CT) proliferation assays, it was demonstrated that Grb7 does not play a role in the proliferation of either unstimulated or HRG-stimulated SK-BR-3 breast cancer cells. Furthermore, using the protocol in conjunction with Boyden chamber migration assays, it was shown that inhibition of Grb7 expression has a slight stimulatory effect on HRG-stimulated SK-BR-3 cell migration. Thus, Grb7 was found to play only a minor role in the migration of SK-BR-3 cells, suggesting that it is not an ideal anti-cancer target for breast cancers modelled by this cell system. Concurrently, a second investigation was conducted, which similarly sought insight into the molecular biology of cancer, but adopted a more strategic approach. ... These results provide evidence for a biologically significant role for the miR-7-mediated regulation of EGFR expression. A microarray experiment was also performed to identify genes that were down-regulated following treatment with miR-7 compared to NS precursor. Of 248 down-regulated genes, including EGFR, 37 promising new miR-7 target candidates were identified. Functional clustering of down-regulated genes and promising target candidates suggested that miR-7 may have functionally-related targets involved in processes including cell motility and brain-associated functions. This investigation thus yielded a program capable of accurately predicting a miRNA target not predicted by any other target prediction program, verified a previously unknown miRNA:target interaction with functional consequences in cancer cells and provided the first steps towards investigating miR-7-mediated regulation in greater depth. Furthermore, EGFR was, to our knowledge, the first example of a verified miRNA target with target sites that are not conserved across mammals, an observation with important implications for computational target prediction and the evolution of miRNA regulatory systems. In addition, the demonstrated growth inhibitory and cytotoxic effects of miR-7 on lung cancer cells raise the possibility of a miR-7-based therapeutic for the treatment of EGFR-overexpressing tumours.

<p>In this thesis, the importance of the growth hormone (GH)/insulin-like growth factor (IGF) system and sex steroids for male bone metabolism has been investigated, and the effects of continuous low dose GH replacement in GH deficient (GHD) adults. In a population-based sample of men, positive correlations were found between bone mineral density (BMD) and IGF-I, IGF-II, IGF binding protein (IGFBP)-3 and the testosterone/sex hormone binding globulin (SHBG) ratio. Serum IGFBP-3 and testosterone levels and weight accounted for 34% to 48% of the variation in BMD at different sites. Compared to healthy age matched controls, men with idiopathic osteoporosis had lower estradiol/SHBG ratio and higher SHBG levels. There were no differences between the groups in serum levels of IGF-I, IGFBP-3, 24 hour cumulated GH secretion or peak GH secretion. In the patients, there was a positive correlation between the estradiol/SHBG ratio and BMD in femoral neck. Treatment of patients and controls with GH 0.8 mg/day for one week resulted in similar increases in serum markers for bone turnover in both groups. Several positive correlations between indices of GH secretion and markers for bone turnover were found in the patients. Men with idiopathic osteoporosis were treated with GH, continuously (0.4 mg/day) or intermittently (0.8 mg/day for two weeks every third month), for two years followed by one year of follow-up. After two years, the BMD and bone mineral content in lumbar spine and total body and serum osteocalcin levels were increased in both groups. This increase was sustained one year post treatment. Treatment of GHD adults with a low fixed dose of GH (0.17 mg/day) for three months, resulted in increases in serum IGF-I and IGFBP-3 levels and lean body mass, and a reduction in fat mass and total and low-density lipoprotein cholesterol levels. These beneficial effects were accomplished without serious side effects. These findings indicate that: i) the sex hormone and GH/IGF systems are important in male bone metabolism, ii) a combination of subtle disturbances in these two systems could contribute to the development of male idiopathic osteoporosis, iii) GH treatment could be considered as a treatment option in this condition.</p>

<p>Ormarsdóttir, S. 2001. Osteoporosis in Chronic Liver Disease. Acta Universitatis Upsaliensis. <i>Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine</i> 1037. 60 pp. Uppsala. ISBN 91-554-5021-0. </p><p>Osteoporosis is a well-known and frequently reported complication of chronic liver disease (CLD) with a high fracture rate contributing to significant morbidity after liver transplantation. The pathogenesis is unknown and controversy exists about many risk factors for osteoporosis in CLD. </p><p>In the present thesis, bone mineral density (BMD) was found to be significantly lower at the lumbar spine (<i>p</i><0.01) in a cohort of patients with CLD compared with age- and gender -matched individuals. Osteoporosis was found in 30% of the patients and 15% of the controls, respectively. Low body mass index (BMI), corticosteroid treatment, prothrombin time, age and female gender were independent risk factors for osteoporosis in the patients. </p><p>In a follow-up study, 43 of 72 patients were available for a second BMD measurement 25 months (median) after the first. Bone loss at the femoral neck was 1.5 ± 2.4% in females and 2.9 ± 2.0% in males with a significant decrease in BMD Z-score over time (<i>p</i>=0.005 and <i>p</i>=0.02 for females and males, respectively), indicating increased bone loss at this site. Hyperbilirubinaemia and low circulating levels of 25-hydroxy vitamin D₃ predicted increased bone loss at the femoral neck. These findings suggest that cortical bone, in addition to trabecular bone, may be affected in CLD and bilirubin and vitamin D₃ may be involved in the pathophysiology of osteoporosis in CLD. </p><p>In order to elucidate the suggested role of insulin-like growth factors (IGFs) and leptin in the pathophysiology of osteoporosis in CLD, we studied the relationship between these factors and BMD. Levels of IGFs were extremely low (<i>p</i><0.0001 compared with the controls) and related to liver function but no correlation was found between the IGFs and BMD. Serum leptin adjusted for BMI correlated negatively with BMD in female patients (<i>p</i>=0.003 and <i>p</i>=0.04 at the lumbar spine and the femoral neck, respectively) and in male patients at the femoral neck (<i>p</i>=0.04). Thus, the IGFs appear not to be involved in the pathophysiology of osteoporosis in CLD but a role of circulating leptin is possible. </p>

Ormarsdóttir, S. 2001. Osteoporosis in Chronic Liver Disease. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1037. 60 pp. Uppsala. ISBN 91-554-5021-0. Osteoporosis is a well-known and frequently reported complication of chronic liver disease (CLD) with a high fracture rate contributing to significant morbidity after liver transplantation. The pathogenesis is unknown and controversy exists about many risk factors for osteoporosis in CLD. In the present thesis, bone mineral density (BMD) was found to be significantly lower at the lumbar spine (p&lt;0.01) in a cohort of patients with CLD compared with age- and gender -matched individuals. Osteoporosis was found in 30% of the patients and 15% of the controls, respectively. Low body mass index (BMI), corticosteroid treatment, prothrombin time, age and female gender were independent risk factors for osteoporosis in the patients. In a follow-up study, 43 of 72 patients were available for a second BMD measurement 25 months (median) after the first. Bone loss at the femoral neck was 1.5 ± 2.4% in females and 2.9 ± 2.0% in males with a significant decrease in BMD Z-score over time (p=0.005 and p=0.02 for females and males, respectively), indicating increased bone loss at this site. Hyperbilirubinaemia and low circulating levels of 25-hydroxy vitamin D3 predicted increased bone loss at the femoral neck. These findings suggest that cortical bone, in addition to trabecular bone, may be affected in CLD and bilirubin and vitamin D3 may be involved in the pathophysiology of osteoporosis in CLD. In order to elucidate the suggested role of insulin-like growth factors (IGFs) and leptin in the pathophysiology of osteoporosis in CLD, we studied the relationship between these factors and BMD. Levels of IGFs were extremely low (p&lt;0.0001 compared with the controls) and related to liver function but no correlation was found between the IGFs and BMD. Serum leptin adjusted for BMI correlated negatively with BMD in female patients (p=0.003 and p=0.04 at the lumbar spine and the femoral neck, respectively) and in male patients at the femoral neck (p=0.04). Thus, the IGFs appear not to be involved in the pathophysiology of osteoporosis in CLD but a role of circulating leptin is possible.

In this thesis, the importance of the growth hormone (GH)/insulin-like growth factor (IGF) system and sex steroids for male bone metabolism has been investigated, and the effects of continuous low dose GH replacement in GH deficient (GHD) adults. In a population-based sample of men, positive correlations were found between bone mineral density (BMD) and IGF-I, IGF-II, IGF binding protein (IGFBP)-3 and the testosterone/sex hormone binding globulin (SHBG) ratio. Serum IGFBP-3 and testosterone levels and weight accounted for 34% to 48% of the variation in BMD at different sites. Compared to healthy age matched controls, men with idiopathic osteoporosis had lower estradiol/SHBG ratio and higher SHBG levels. There were no differences between the groups in serum levels of IGF-I, IGFBP-3, 24 hour cumulated GH secretion or peak GH secretion. In the patients, there was a positive correlation between the estradiol/SHBG ratio and BMD in femoral neck. Treatment of patients and controls with GH 0.8 mg/day for one week resulted in similar increases in serum markers for bone turnover in both groups. Several positive correlations between indices of GH secretion and markers for bone turnover were found in the patients. Men with idiopathic osteoporosis were treated with GH, continuously (0.4 mg/day) or intermittently (0.8 mg/day for two weeks every third month), for two years followed by one year of follow-up. After two years, the BMD and bone mineral content in lumbar spine and total body and serum osteocalcin levels were increased in both groups. This increase was sustained one year post treatment. Treatment of GHD adults with a low fixed dose of GH (0.17 mg/day) for three months, resulted in increases in serum IGF-I and IGFBP-3 levels and lean body mass, and a reduction in fat mass and total and low-density lipoprotein cholesterol levels. These beneficial effects were accomplished without serious side effects. These findings indicate that: i) the sex hormone and GH/IGF systems are important in male bone metabolism, ii) a combination of subtle disturbances in these two systems could contribute to the development of male idiopathic osteoporosis, iii) GH treatment could be considered as a treatment option in this condition.

The overall objective of this study was to investigate how NS0 cell proliferation is regulated in protein-free media. The hypothesis was that during the adaptation to growth factor-free media, animal cell lines start to produce their own autocrine growth factors to support proliferation, and after some time in a culture the effects of these factors are lost which results in cessation of proliferation. A chemically defined, protein-free and animal component-free medium was developed for the NS0 cells. This medium was comprised of a basal hybridoma medium to which phosphatidyl¬choline, cholesterol, β-cyclodextrin, ferric citrate and amino acids were added. A fed-batch process was then developed in this medium. The feed profile was optimised in a step-wise manner with a final feed solution containing glucose, glutamine, lipids, amino acids, vitamins, sodium selenite and ethanolamine. Specifically, supplementation with lipids (cholesterol) had a drastic effect on cell growth. Calcium, magnesium and potassium were not depleted during culture and a feed containing also iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield further. More than 8 x 106 viable cells mL-1 and 600 mg antibody L-1 was obtained in the final fed-batch. This corresponded to a 4.3-fold increase in viable cell yield and an 11.4-fold increase in product yield compared to bioreactor batch culture when the dilution of the fed-batch culture was also accounted for. The presence of autocrine growth factors in NS0 cell cultures was initially investigated by studying the effects of conditioned medium (CM). Concentrated CM had a significant positive effect on cell growth and part of this effect could be attributed to factor(s) eluting from a gel-filtration column at 20-25 kDa. In the search for cell-derived factors affecting cell growth the following proteins were identified as released/secreted by the NS0 cells; cyclophilin A, cyclophilin B, cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerise, macrophage migration inhibitory factor (MIF), β2-microglobulin, niemann pick type C2, secretory leukocyte protease inhibitor (SLPI), thioredoxin-1, TNF-α, tumour protein translationally controlled-1 and ubiquitin. Zymogram electrophoresis further identified aspartic acid, papain-like cysteine (including cathepsin L) and serine protease activity in the CM. Pro/cathepsin L, CypB, EGF, IFN-α/β/γ, IGF-I/II, leukaemia inhibitory factor, IL-6, IL-11, IL-25, MIF, oncostatin M, TGF-β and TNF-α were excluded as involved in autocrine regulation of NS0 cell proliferation. The serine protease activity was suggested to affect the cells negatively and since the serine protease inhibitor SLPI is also present in NS0 CM, a balance in serine protease activity may be crucial for optimal cell growth. Further, the receptor gp130, known to be associated with myeloma cell growth, was shown to be essential for NS0 cell proliferation as demonstrated by siRNA gene silencing. The results suggested that autocrine regulation of proliferation in NS0 cell cultures involves the receptor subunit gp130. / QC 20100920

Regional development discourses and theories have significantly changed since the born of regional science. Focus of regional development theories has shifted from industrialization efforts via large-scale enterprises and transfer of central government funds to disadvantaged regions to endogenous capabilities and potentials of innovation and knowledge creation. Endogenous factors and self-development capabilities are highly emphasized by recent development literature. Changes in the regional development discourses also affected regional policies, policy tools and actors / endogenous factors and self-development discourses became dominant in regional development policies and implementations. However, source of regional development for less developed regions that do not have endogenous potentials and self-development capability have not clearly defined and have not empirically tested. The thesis attempted to empirically define regional growth factors and the usefulness of theoretical frameworks. In the thesis, econometric model of Turkey is used for the empirical study. The theoretical framework discussed in the thesis is both economic theories and regional development models. The study shows that all theoretical models offer only partial explanations of regional growth. While study shows that factors emphasized by traditional theories support regional growth, the study has no evidence supporting that soft factors emphasized by recent theories support regional growth. The main findings of this study contribute to theoretical and empirical field by reintroducing role of government and interventions. Factors highlighted by recent regional development theories are not sufficient for explaining growth, since the regional policies at the national level continue to be important therefore factors emphasized by traditional theories still have significant contributions to growth.

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. In particular, Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, and it has been shown to play a role in inner ear development and morphogenesis. However, there is no understanding of the cellular effects of BMP4 in prosensory progenitors, and about its role in the process of sensory fate specification. The present thesis project was aimed at exploring the effects of BMP-signaling on the development of hair-cells, using the chick inner ear as a model.The specific aims proposed were:1- Analyze the cellular effects caused by addition of BMP4 in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.2- Analyze the cellular effects caused by inhibition of BMP4 signaling in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.3- Analyze the expression in the innear ear of downstream targets of BMP signalling, in particular, analyse the members of Id gene family.4- Analyze the regulation of Id genes by BMP signalling in the inner ear.5- Analyze the expression of genes involved in the process of terminal differentiation, in particular, Btg1 and Btg2 genes6- Analyze the regulation of Btg1 and Btg2 gene by BMP signalling in the inner ear

Die im Rahmen dieser Arbeit durchgeführten Untersuchungen zum Einfluss der Wachstumsfaktoren IGF-I, EGF und dem Zytokin IL-6 auf den Androgenrezeptor-Signalweg zeigten in verschiedenen Prostatakarzinom-zelllinien schon nach zwei Stunden eine deutliche Degradation des Androgenrezeptor-Proteins. Die ausschließlich auf Protein-Ebene stattfindende, Wachstumsfaktor-induzierte negative Regulation des Androgenrezeptors konnte durch einen schnellen Androgeneffekt wieder aufgehoben werden. Mittels Luziferase-Reportergen-Assays wurde kein Einfluss der Wachstums-faktorwirkung auf die transkriptionelle Aktivität des Androgenrezeptors nachgewiesen. Darüber hinaus konnte eine signifikant reprimierende Wirkung durch IGF-I und EGF in Kombination mit geringen Mengen DHT beobachtet werden. Weitere Resultate dieser Arbeit deuten auf einen, durch den PI3-Kinase-Signalweg vermittelten, proteasomalen Abbauprozess des Rezeptors hin. Da die Suppression der downstream gelegenen Proteinkinase Akt keine Veränderung hinsichtlich der Degradation aufwies, konzentrierte sich die weiterführende Arbeit auf eine mögliche direkte Regulation des Androgen-rezeptors durch die PI3-Kinase. Unter Verwendung von rekombinanten GST-Fusionsproteinen konnte in Interaktionsstudien unter in vitro Bedingungen eine Phosphotyrosin-unabhängige Bindung zwischen der C-SH2-Domäne der p85-Untereinheit der PI3-Kinase und dem N- und C-Terminus des Androgenrezeptors nachgewiesen werden. Durch die nähere Charakterisierung dieser Bindungsbereiche mit Hilfe von Peptidarrays und anschließenden Alanin-Substitutionen war es möglich, für den N-Terminus 18, für den C-Terminus des Androgenrezeptors 6 und für die p85-C-SH2-Domäne der PI3-Kinase 11 Aminosäuren zu identifizieren. Die durch gezielte Punktmutagenese an diesen Aminosäurepositionen hergestellten Androgenrezeptor-Einzel- und -Mehrfachmutanten wiesen in Bindungsstudien dennoch Interaktion zur PI3-Kinase auf. Eine von Anderson und Kollegen postulierte Phosphotyrosin-unabhängige Bindung der SH2-Domänen der p85-Untereinheit der PI3-Kinase durch sogenannte „basic-X-basic“-Motive wurde ebenfalls in Interaktionstests zwischen der PI3-Kinase und dem Androgenrezeptor überprüft. Aufgrund der Tatsache, dass einige der identifizierten Aminosäuren auf dem Androgenrezeptor Teil eines „basic-X-basic“-Bindungsmotives sind, wurden Kombinationsmutanten generiert, die sowohl im N-Terminus als auch im C﷓Terminus des Androgenrezeptors ein bzw. zwei zerstörte „basic-X-basic“-Motive enthielten. Untersuchungen zum Bindungsverhalten dieser Mutanten zeigten zwar weiterhin Interaktion zur p85-C-SH2-Domäne der PI3-Kinase, jedoch der durch Western-blot-Analyse überprüfte IGF-I-induzierte Degradationseffekt des Androgenrezeptor-Proteins konnte mit zwei der verwendeten Androgenrezeptor-Kombinationsmutanten nicht mehr beobachtet werden.