Several epidemiological studies have revealed an association between particulate matter (PM) pollution and various health effects. Importantly, there is evidence to suggest that individuals with pre-existing respiratory disease, such as asthma, are more sensitive to elevated ground levels of particulate matter as compared to healthy subjects. Among the various sources of PM pollution, diesel powered vehicles have been identified as important contributors. The aim of this thesis was to investigate the airway effects of experimental chamber exposure to diesel exhaust (DE) in healthy and asthmatic subjects, focusing on airway responsiveness, airway inflammation and lung function. To achieve a comprehensive picture of the airway responses to DE, a number of different methods were used, including lung function measurements, methacholine inhalation tests, induced sputum and bronchoscopy. Each subject acted as his/her own control by being exposed both to filtered air and DE in a crossover design. Short term exposure to DE, at a particle concentration (PMi0) of 300 ug/m3, was associated with a clinically significant increase in bronchial hyperresponsiveness in asthmatic subjects. In accordance with the epidemiological data suggesting a 1-4 day lag effect for most health outcomes to PM pollution, the increase was detected one day after DE exposure, indicating a long lasting response to DE in asthmatic airways. Diesel exhaust induced a range of airway inflammatory changes as reflected in induced sputum, bronchoalveolar lavage and bronchial mucosal biopsies. In healthy subjects, DE exposure was associated with an increase in neutrophils and IL-6 in sputum, elevated levels of IL-8 and IL-6 in bronchial wash (BW), enhanced expression of IL-8 and GRO-a in the bronchial epithelium and with increases in P-selectin and VCAM-1 in the airway mucosa. In contrast, asthmatics responded with an increase in IL-6 in sputum and an enhanced expression of IL-10 in the bronchial epithelium following exposure DE. Thus, clear differences were identified between healthy and asthmatic subjects in the inflammatory response to DE. Airway epithelial cells constitute the first line of cellular defence towards inhaled air pollutants and increasing evidence suggests that these cells contribute markedly to the initiation of airway inflammatory responses. The bronchial epithelium was identified to have an important regulatory role in response to diesel exhaust, including the capacity to produce chemoattractant and immunoregulatory proteins associated with development of airway inflammation and bronchial hyperresponsiveness. Lung function measurements revealed that short-term exposure to DE induces an immediate bronchoconstrictive response in both healthy and asthmatic individuals, with significant increases in airway resistance (Raw) following DE exposure. This thesis also investigated the effects of a lower concentration of DE (PMio 100 ug/m3) than previously studied. It was shown that exposure to DE at a concentration corresponding to a PM level that may be encountered in busy traffic situations, was still associated with potentially adverse airway responses in healthy and asthmatic subjects. In summary, the results presented here indicate that short term exposure to diesel exhaust, at high ambient concentrations, has the potential to induce a range of biological events in the airways of healthy and asthmatic subjects. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 2002, härtill 4 uppsatser.</p> / digitalisering@umu

This thesis have explored the functions of the protein Tissue Factor (TF), which together with its ligand coagulation factor VII/VIIa (FVII/FVIIa) forms a proteolytic complex that functions in initiation of blood coagulation and activation of cell signaling. In paper I, the mechanisms behind the observation that TF/FVIIa signaling protects cells from apoptosis were further investigated. Using cell culture models, we found that antiapoptotic signaling by TF/FVIIa requires signaling by the Insulin-like growth factor I receptor (IGF-1R), as synthetic IGF-1R inhibitors and IGF1-R siRNA knock-down abolished the antiapoptotic effect of FVIIa. Furthermore, the IGF-1R translocated to the cell nucleus after FVIIa stimulation, implying a role in regulation of gene expression. Papers II and III describe the discovery that the Eph tyrosine kinase receptors EphB2 and EphA2 are proteolytically cleaved directly by TF/FVIIa. By using mass spectrometry and N-terminal Edman sequencing, the exact cleavage site was identified after a conserved arginine residue in the EphA2/EphB2 ligand binding domains, in agreement with the cleavage preferences of FVIIa. TF and EphA2/EphB2 co-localized in cancer cell lines and FVIIa potentiated ligand-dependent Eph signaling by increasing cytoskeletal remodeling and cell repulsion, demonstrating a novel proteolytical event that modulates Eph receptor signaling. In paper IV, expression of TF was investigated in colorectal cancer in both the stromal and tumor cell compartments by immunohistochemistry using an anti-TF-antibody developed and validated by the Human Protein Atlas project. In normal large intestine, TF was strongly expressed in the innermost pericryptal sheath cell layer lining the epithelium, in a cell population distinct from intestinal pericryptal myofibroblasts. We evaluated TF expression in two colorectal cancer materials, and found that TF was variably present in both the stromal and tumor cell compartments. TF expressed by pericryptal sheath cells was progressively lost after the adenoma-to-carcinoma transition and was a strong predictor of survival in rectal but not colon cancer patients independently of disease stage, histological tumor grade and age. In summary, this thesis demonstrates novel signaling mechanisms for the TF/FVIIa complex, and provides evidence of a hitherto unknown role of TF expressed by a specific population of stromal cells in colorectal cancer.

Androgens are the main regulators of human hair growth stimulating larger, terminal hair development e.g. beard and causing scalp balding, androgenetic alopecia. Hair disorders cause psychological distress but are poorly controlled. Androgens probably act by altering regulatory paracrine factors produced by the mesenchyme-derived dermal papilla. This study aimed to investigate paracrine factors involved in androgen-regulated alopecia, particularly hepatocyte growth factor (HGF) family members, by investigating their in vivo status. Balding and non-balding scalp hair follicles and their component tissues were isolated and analysed by molecular biological methods (reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative PCR and DNA microarray analysis), cell culture and immunohistochemistry. Scalp follicles expressed a range of paracrine messenger genes. The dermal papilla, cultured dermal papilla cells and dermal sheath expressed several HGF family genes, while matrix cells only produced the receptor RON suggesting autocrine roles for HGF and MSP, but a paracrine route only for MSP. Comparing balding and non-balding follicles from the same individuals revealed the expected reduction in several keratin and keratin-related protein genes supporting this approach's validity. There were also significant differences in paracrine factors previously implicated in androgen action by in vitro studies. Several factors believed to increase during androgen stimulation of larger, darker follicles, e.g. IGF-I and SCF, were lowered in balding follicles, while putative inhibitory factors, e.g. TGFß-1, were increased. HGF and MSP and their receptors, c-Met and RON, were significantly reduced. These results increase our understanding of androgen action in human hair follicles; this could lead to better treatments for hair disorders.

This series of experiments investigated dystrophin localization in the normal cerebellum and examined Purkinje neuron function in normal and dystrophin-deficient mice to better understand the physiological basis for cognitive deficits associated with Duchenne muscular dystrophy (DMD), a common genetic disorder among children. Cognitive impairments are consistently reported in DMD, yet precise mechanisms for their occurrence are unknown. Dystrophin protein, which is absent in DMD, is normally localized to muscles and specific neurons in the brain. Purkinje neurons are rich in dystrophin, specifically in somatic and dendritic membranes. Studies demonstrate perturbed cerebellar function in the absence of dystrophin, suggesting that DMD should be regarded as a cerebellar disorder in addition to being considered a neuromuscular disorder. However, theory and evidence are not generated from overlapping information: research investigating cerebellar involvement in DMD has focused on the vermal region, associated with motor function. The lateral region, implicated in cognition, has not been explicitly examined in DMD. The first experiment revisited the issue of dystrophin distribution in the mouse cerebellum using immunohistochemistry to investigate qualitative and quantitative differences between cerebellar regions. Both regions showed dystrophin localized to Purkinje neuron somatic and dendritic membranes, but dystrophin density was 30% greater in the lateral than the vermal region. The second experiment examined intrinsic electrophysiological properties of vermal and lateral Purkinje neurons from wild-type (WT) mice and from the mdx mouse model of DMD which lack dystrophin. Significant differences in action potential firing frequency, regularity, and shape were found between cerebellar regions. Purkinje neurons from mdx mouse cerebellum exhibited membrane hyperpolarization and irregular action potential firing, regardless of region. Spontaneous action potential firing frequency was reduced in Purkinje neurons from lateral cerebellum in mdx mice relative to controls, demonstrating that a loss of dystrophin causes a potent dysregulation of Purkinje neuron function in the region associated with cognition. This research extends our understanding of cerebellar pathology in DMD and its potential relevance to cognitive deficits in the disorder. Moreover, this research further supports the role of the cerebellum as a structure important for cognition and contributes to our understanding of dystrophin’s role in the brain.

Ovarian cancer is a significant concern for women’s health as it is the most lethal of all gynaecological malignancies. One of the reasons for the high mortality of this disease is that traditionally used chemotherapeutic treatments tend to have poor initial or sustained efficacy against ovarian tumours. Resistance to such treatments may in part be mediated by autophagy, a cell survival process in which unnecessary or damaged components of the cytoplasm are engulfed within a double-membraned vesicle known as an autophagosome and ultimately degraded upon fusion of the autophagosome with a lysosome. Autophagy has been shown to be employed by cells to aid in their survival under stresses such as nutrient deprivation, hypoxia, chemotherapy treatment, and growth factor withdrawal. As these stresses are commonly encountered by ovarian cancer cells, it is possible that autophagy promotes ovarian cancer cell survival. This thesis aims to investigate which stimuli induce autophagy in ovarian cancer cells and whether or not this induction can promote cell survival. In addition, there is a particular focus on the comparison of autophagy utilization between subtypes of ovarian cancer, as the subtypes are in fact considered different diseases and may vary in their usage of autophagy. The first chapter of this thesis provides relevant background information on autophagy as well as ovarian cancer and its subtypes. In the second chapter, I describe studies in which tumours from a large cohort of patients with ovarian cancer are assessed for LC3A, a marker of autophagy, in addition to markers of other cellular processes including hypoxia. Here I found that LC3A was significantly associated with poor patient survival in patients with the clear cell subtype of ovarian cancer, but not other subtypes. I also found that LC3A expression was associated with markers of hypoxia in the clear cell patient tumours and that clear cell carcinoma cell lines preferentially induced autophagy in response to hypoxia in vitro as compared to cell lines of the high-grade serous subtype. These results indicate that clear cell ovarian tumours are uniquely dependent upon autophagy in response to hypoxia. In the third chapter, I investigated the autophagic response to treatment with the standard ovarian cancer chemotherapy drugs carboplatin and paclitaxel in a syngeneic mouse model of ovarian cancer. I found that these drugs did indeed induce autophagy and that the cancer cells utilized autophagy to promote resistance to these chemotherapeutics. In addition, when the tumour cells were grown in syngeneic mice, treatment with the autophagy inhibitor hydroxychloroquine resulted in a significant suppression of tumour growth. Together, my findings indicate that further investigation into the use of autophagy inhibitors in ovarian cancer patients is warranted and that different specific rational drug combinations for each subtype will likely yield optimal results. / Graduate

ABSTRACT MULTIDRUG RESISTANCE IN LOCALLY ADVANCED BREAST CANCER ATALAY, Mustafa Can Ph. D., Department of Biotechnology Supervisor: Prof. Dr. Ufuk G&Uuml / ND&Uuml / Z June 2004, 70 pages Breast cancer is the most frequently detected cancer among women. Early diagnosis leads to long term survival when the patients are treated with surgery, radiotherapy, chemotherapy, and hormone therapy. Unfortunately, advanced disease could still be encountered in some patients resulting in a poorer prognosis. The primary treatment modality is chemotherapy for this group of patients. Drug resistance is a serious problem resulting in the use of different drugs during chemotherapy and knowing the possibility of resistance before initiating first line chemotherapy may save time and money, and most importantly, may increase patient&rsquo / s survival. Therefore in this study, multidrug resistance is studied in locally advanced breast cancer patients. The breast tissues obtained from 25 patients both before and after chemotherapy were examined for drug resistance. Reverse transcriptase polymerase chain reaction was used for the detection of mdr1 and mrp1 gene expression. In addition, immunohistochemistry technique was used for P-glycoprotein and MRP1 detection. JSB-1 and QCRL-1 monoclonal antibodies were utilized to detect P-glycoprotein and MRP1, respectively. Five patients were unresponsive to chemotherapy. In four of these patients mdr1 gene expression was induced by chemotherapy where as the fifth patient initially had mdr1 gene expression. In addition, Pgp positivity was detected in 9 patients after chemotherapy. Both the induction of mdr1 gene expression (p&lt / 0.001) and Pgp positivity (p&lt / 0.001) during chemotherapy were significantly related with clinical response. On the other hand, mrp1 gene expression and MRP1 positivity were detected in 68% of the patients before the therapy. After chemotherapy, mrp1 expression increased to 84%. Although 80% of the clinically unresponsive patients had mrp1 gene expression, the relation between mrp1 expression and clinical drug response was not strong. Thus, it can be concluded that in locally advanced breast cancer mdr1 gene expression during chemotherapy contributed to clinical unresponsiveness. However, mrp1 gene expression did not correlate strongly with the clinical response. When RT-PCR and immunohistochemistry methods are compared in terms of detection of drug resistance, it seems that both methods gave similar and reliable results.

Specific antibodies are invaluable tools for proteomics research. The availability of thoroughly validated antibodies will help to improve our understanding of protein expression, localization and function; fundamental processes and features of all living organisms. The objectives of the studies in this thesis were to develop high-throughput methods to facilitate the generation and purification of monospecific antibodies, and to address problems associated with antigen selection for difficult target proteins and subsequent validation issues. In the first of the studies, it was demonstrated that antibodies specific to human proteins could be generated in a high-throughput manner using protein epitope signature tags (PrESTs) as both antigens and affinity ligands. A previously developed purification process was adapted to a high-throughput format and this, in combination with the development of a protein microarray assay, resulted in monospecific antibodies that were used for profiling protein expression in 48 human tissues. Data obtained in these analyses suggest that a complete Human Protein Atlas should be attainable within the next ten years. In order to reduce the number of animals needed for such a massive project, and improve the cost-efficiency of antibody generation, a multiplex immunization strategy was developed in a further study. Antisera from rabbits immunized with mixtures of two, three, five and up to ten different PrESTs were successfully purified and analyzed for specificity using protein arrays. Almost 80% of the animals immunized with up to three PrESTs yielded antibodies towards all the PrESTs administered, and they yielded comparable immunohistochemical staining patterns (of consecutive human tissue sections) to those of antibodies obtained from traditional single PrEST immunizations. Proteins with highly similar sequences to other proteins present a major challenge for the proteome-wide generation of antibodies. In another study, Cytokeratin-17 which displays high sequence similarity to closely related members of the intermediate filament family, was used as a model and the specificity and cross-reactivity of antibodies generated against this target were investigated using epitope mapping in combination with comparative IHC analyses. Antibodies identified by epitope mapping as binding to the most unique parts of the Cytokeratin-17 PrESTs also showed the most Cytokeratin-17-like staining pattern, thus further supporting the strategy of using sequence identity scores as the main criteria for PrEST design. An alternative antigen design strategy was investigated for use in raising antibodies towards G-proteincoupled receptors (GPCRs). The extracellular loops and N-terminus of each of three selected GPCRs were assembled to form single antigens and the resulting antibodies were analyzed by flow cytometric and confocal microscopic analyses of cell lines over-expressing the respective receptors. The results from both flow cytometric and immunofluorescence analyses showed that the antibodies were able to bind to their targets. In addition, the antibodies were used successfully for the in situ analysis of human brain and pancreatic islet cells. / QC 20100727

The management of significant bone defects, delayed and non-union of fractures can be extremely challenging. Development of specific treatment is hindered by an absence of information regarding the molecular events which regulate these processes. In this thesis, a bilateral cancellous bone defect model of the femur and tibia was developed in a rodent and the spatiotemporal profile of TGF-β, BMP 2 and 7, Smads 1, 4 and 5 characterised. Next, the capability of acid solution to augment healing was tested in both a bone defect and in a closed femoral fracture model. Finally, a long term oestrogen deficiency (OVX) rat model of postmenopausal osteoporosis was characterised and the spatiotemporal profiles of IGF-1, IGFR-1, MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, BMP-2, BMP-4, BMP-7, TGF-β, Smad4, Smad7, VEGF, Flt-1, Ihh and FGF-2 were compared in femoral osteotomies between OVX and Sham groups. The bilateral cancellous defect model was successfully created with a number of advantages with which to recommend its use in future studies. TGF-β, BMP 2 and 7, Smads 1, 4 and 5 had characteristic spatiotemporal profiles during cancellous bone defect healing suggesting that they have a regulatory role. The results of the acid study were inconclusive and problems with substance delivery and maintenance at the desired site need to be addressed in the future to fully test this hypothesis. No significant differences were detected on histology or three-point mechanical testing between the fracture calluses of acid and control groups. In the final study, OVX rats after six months had significantly increased weight and decreased bone mineral density compared to their sham counterparts. A histological delay in osteotomy healing was observed in the OVX group but no significant differences on tensile testing were seen between OVX and Sham groups up to six weeks. Immunohistochemistry revealed that delayed healing may be due to the down-regulation of IGF-1, BMP-2, 4, and 7 and the up-regulation of MMP-3 in OVX compared to Sham groups. In conclusion, the results of this thesis give some insight into the molecular biology of bone defects and osteoporotic fractures. This information may also be useful in the development of specific treatments aimed at augmenting healing in bone defects and osteoporotic fractures.

The aim of this study was to evaluate new methods to improve detection and investigation of the effects of chronic or subclinical infection with Trypanosoma evansi in various mammalian species. Some of the more resistant host species, including pigs and buffaloes, are present in large feral populations in the northern parts of Australia, the area where T. evansi is most likely to gain entry to the country. Existing tests are not sufficiently reliable to detect all cases of disease and they cannot distinguish acute from chronic infections. Furthermore, the tests have different sensitivities in different host species. Surveillance for trypanosomiasis in Australia is problematic because of the need to work in remote parts of northern Australia where provision of a cold-chain for traditional blood and serum storage is difficult. An existing dried blood storage system was modified by treating cotton lint filter paper (Whatman #903) with a commercial post coating buffer (TropBio, Queensland). This treatment increased the longevity of antibodies to T. evansi in serum and blood stored on the paper (detected using an antibody-detection ELISA) compared to samples stored on plain paper, especially when the papers were stored under humid conditions and at high ambient temperatures. Attempts were made to improve the diagnostic utility and repeatability of antibody-ELISAs through the use of 2 recombinant T. brucei antigens (PFRA and GM6) and to optimize a competitive ELISA using RoTat 1.2 variable surface antigen and its monoclonal antibody. Antibody-detection using the two recombinant proteins was not sufficiently specific to enable their use for the detection of T. evansi. The RoTat 1.2 cELISA had good sensitivity and specificity (75% and 98% respectively) when used to test serum from cattle and buffaloes experimentally infected with T. evansi and uninfected animals. However, the test was not able to detect anti-T. evansi antibodies in serum from wallabies, pigs, a dog or a horse that were experimentally infected with T. evansi. The inability of the cELISA to detect anti-T. evansi antibodies may be due to the small number of samples tested or the lack of RoTat 1.2 specific antibodies in the animals tested. The feasibility of using an enzymatic test to detect trypanosome aminotransferase or antibodies to this enzyme was evaluated. Prior publications suggested that the detection of TAT was an appropriate diagnostic tool for the detection of T. evansi infection in camels. However, the results from this study did not support the use of this test for the detection of T. evansi infection in cattle or buffaloes with low to moderate parasitaemia. Trypanosomiasis is an immunological disease that affects most of the body’s organs, with more severe disease developing over time. Attempts were made to determine key cytokine and biochemical patterns that would distinguish infected from uninfected animals and acute from chronic infections. The results from this study showed that there was no specific pattern in serum cytokines or serum biochemistry that could be used to distinguish infected from uninfected animals, or different stages of disease. Immunohistochemistry was used on tissues from buffaloes and mice experimentally infected with T. evansi and T. brucei gambiense respectively to characterise the cellular immune response that was present. The immune response was predominantly cell mediated, with CD3+ T lymphocyte and macrophage infiltration occurring in most tissues. In end stage disease there was often suppression of the immune system with disruption of the architecture of the spleen and a decrease in B lymphocytes in the circulation. Trypanosomes were rarely visible in the tissues and were only seen in those animals with high parasitaemia. Lesions generally became more severe over time, but there was a large variation between animals, which suggests that immunohistochemistry is unsuitable as a diagnostic tool.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.