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  • Public defence: 2017-03-03 09:00 KB.E3.01, Umeå
    Ranjbarian, Farahnaz
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Targets and strategies for drug development against human African sleeping sickness2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Trypanosoma brucei is a causative agent of African sleeping sickness. It is an extracellular parasite which circulates in the blood, lymph and eventually invades the central nervous system. There is a great need for new medicines against the disease and specific properties of nucleoside kinases in the pathogen can be exploited as targets for chemotherapy. 

    T. brucei contains a gene where two thymidine kinase sequences are fused into a single open reading frame. These types of tandem thymidine kinases were found only in different types of parasites, which made us to believe that it might be beneficial for them. Each thymidine kinase sequence in these tandem enzymes are here referred to as a domain. By cloning and expressing each domain from T. brucei separately, we found that domain 1 was inactive and domain 2 was as active as the full-length enzyme. T. brucei thymidine kinase phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine and to some extent purine nucleosides like deoxyinosine and deoxyguanosine. Human thymidine kinase increases the affinity to its substrates when it forms oligomers. Similarly, the T. brucei two thymidine kinase sequences, which can be viewed as a pseudodimer, had a higher affinity to its substrates than domain 2 alone. 

    T. brucei lacks de novo purine biosynthesis and it is therefore dependent on salvaging the required purine nucleotides for RNA and DNA synthesis from the host. Purine salvage is considered as a target for drug development. It has been shown that in the presence of deoxyadenosine in the growth medium, the parasites accumulate high levels of dATP and the extensive phosphorylation of deoxyadenosine leads to depleted ATP pools. Initially, we wondered if deoxyadenosine could be used as a drug against T. brucei. However, we found that T. brucei is partially protected against deoxyadenosine because it was cleaved by the enzyme methylthioadenosine phosphorylase (MTAP) to adenine and ribose-1-phosphate. At higher concentration of deoxyadenosine, 3 the formed adenine was not efficiently salvaged into ATP and started to inhibit MTAP instead. The deoxyadenosine was then instead phosphorylated by adenosine kinase leading to accumulation of dATP. The MTAP reaction makes deoxyadenosine itself useless as a drug and instead we focused on finding analogues of deoxyadenosine or adenosine that were cleavage-resistant and at the same time good substrates of T. brucei adenosine kinase. Our best hit was then 9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine (FANA-A). An additional advantage of FANA-A as a drug was that it was taken up by the P1 nucleoside transporter family, which makes it useful also against multidrug resistant parasites that often have lost the P2 transporter function and take up their purines solely by the P1 transporter. In parallel with our study of nucleoside metabolism in T. brucei, we also have a collaboration project where we screen essential oils from plants which are used in traditional medicine. If the essential oils are active against the trypanosomes, we further analyze the different components in the oils to identify new drugs against African sleeping sickness. One such compound identified from the plant Smyrnium olusatrum is isofuranodiene, which inhibited T. brucei proliferation with an IC50 value of 3 μM.

  • Public defence: 2017-03-03 10:00 Hörsal 1031, Norra beteendevetarhuset, Umeå
    Knekta, Eva
    Umeå University, Faculty of Social Sciences, Department of applied educational science.
    Motivational aspects of test-taking: measuring test-taking motivation in Swedish national test contexts2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The overall aim of the work underlying this thesis was to improve the understanding of students’ test-taking motivation in connection to achievement tests for young adolescents. The thesis includes four studies and a summary. All four studies explore test-taking motivation and are all connected to validity in one way of another. The expectancy-value theory of achievement motivation was used as a theoretical framework in the operationalization and measurement of test-taking motivation and the achievement tests in focus were the Swedish national test in science for grade nine students, a Swedish national test in mathematics for upper secondary students and field trials for both these tests.

    In the first study psychometric properties of an expectancy-value based questionnaire measuring five aspects of test-taking motivation were evaluated. Findings provided support for construct validity, as internal structure of the data corresponded to the theoretical model, partial scalar invariance was obtained and correlations between test-taking motivation and test performance were found. In the second study the students’ test-taking motivation at the field trial and the national test were analysed in more detail. The analyses showed a significant increase in all test-taking motivation aspects between the field trial and the regular national test. Test-taking motivation at the field trial did also differ between school classes and test anxiety and expectancies differed between females and males. Further, effort, expectancies, importance and interest were significant predictors of test score after preliminary grades had been accounted for. The third study was an interview study aiming to examine students’ test-taking motivation at national test as well as the field trial in more depth. The interviews provided a rich understanding of the different aspects of test-taking motivation as described by the expectancy-value theory, for example why students find the national test important and what they mean by giving effort to a test. In the fourth study psychometric properties of an extended and revised test-taking motivation questionnaire were evaluated in a new sample of students. The analysis showed that the questionnaire had sound psychometric properties and supported the assumption that test-taking motivation consists of several distinctly different aspects, but also showed that the different subscales are highly related.

    In conclusion, the results provide support for several aspects of construct validity of the test-taking motivation questionnaire. Further, all studies showed that test-taking motivation differ between test with different stakes as well as between students and classes taking similar tests. Test-taking motivation also seem to affect test results. Thus, all students do not seem to be equally motivated to do their best on all tests and consideration should be taken to how students approach and experience each specific test when planning and interpreting achievement tests. 

  • Public defence: 2017-03-10 13:00 KBC-huset, sal KB.E3.01, Umeå
    Fong, Gloria
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Influence of neuromodulators and mechanical loading on pathological cell and tissue characteristics in tendinosis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Background: Tendinosis is a painful chronic, degenerative condition characterized by objective changes in the tissue structure of a tendon. Hallmark features in tendinosis tendons include increased number of cells (hypercellularity), extracellular matrix (ECM) degradation and disorganized collagen. The progression of these pathological changes seen in tendinosis is neither well characterized nor fully understood.

    Studies have suggested that there are biochemical and mechanical elements involved in tendinosis. From a biochemical perspective, studies have shown that the tendon cells, tenocytes, produce a number of neuronal signal substances/neuromodulators, such as substance P (SP) and acetylcholine (ACh), traditionally thought to be confined to the nervous system. Furthermore, it has been shown that the expression of these neuromodulators is elevated in tendinosis tendons as compared to normal healthy tendons. Interestingly, studies on other tissue types have revealed that both SP and ACh can induce tissue changes seen in tendinosis, such as hypercellularity and collagen disorganization. From a mechanical angle, it has been suggested that overload of tendons, including extensive strain on the primary tendon cells (tenocytes), causes the degenerative processes associated with tendinosis. In vivo studies have shown that in overloaded tendons, the presence of neuromodulators is elevated, not least SP, which also precedes the development of the tissue changes seen in tendinosis. This further supports the importance of combining biochemical factors and mechanical factors in the pathogenesis of tendinosis.

    Hypotheses: In this thesis project, we hypothesize: 1) that neuromodulators, such as SP and ACh when stimulating their preferred receptors, the neurokinin 1 (NK-1 R) and muscarinic receptors (mAChRs), respectively, can cause increased tenocyte proliferation; 2) that the effects of SP and ACh on tenocyte proliferation converge mechanistically via a shared signalling pathway; 3) that mechanical loading of tenocytes results in increased production of SP by the tenocytes; and 4) that SP enhances collagen remodelling by tenocytes via NK-1 R.

    Model system: In vitro studies offer insight into the function of healthy tendon matrix and the etiology of tendinopathy. Using a cell culture model of human primary tendon cells, highly controlled experiments were performed in this thesis project to study a subset of biological and mechanical parameters that are implicated in tendinosis. The FlexCell® Tension System was used to study the influence of mechanical loading on tenocytes. As well, a collagen gel contraction assay was used to examine the intrinsic ability of tenocytes to reorganise type I collagen matrices under the influence of the neuromodulator SP.

    Results: The studies showed that exogenous administration of SP and ACh results in increased tenocyte proliferation that is mediated via activation of the ERK1/2 mitogenic pathway when the preferred receptors of SP and ACh, the NK-1 R and mAChRs, respectively, are stimulated. Furthermore, the studies resulted in the novel finding that SP and ACh both converge mechanistically via transforming growth factor (TGF)-β1 and that a negative feedback mechanism is present in which TGF-β1 downregulates the expression of mAChRs and NK-1 R. The studies also showed that SP can increase collagen remodelling and upregulate expression of genes related to tendinosis. Finally, it was established that tenocytes are mechanoresponsive by showing that cyclic mechanical loading increases the expression of SP by human tenocytes.

    Conclusions: This thesis work concludes that stimulation of NK-1 R and mAChRs results in proliferation of human tenocytes, which both involve the ERK1/2 signalling pathway. It also shows that SP and ACh converge mechanistically via TGF-β1 in their contribution to tenocyte proliferation. The role of hypercellularity in tendinosis tissue is unknown. Possibly, it has different roles at different stages of the disease. The findings also show that SP increases collagen remodelling, suggesting that increased SP not only results in hypercellularity but also contributes to the collagen morphology in tendinosis.

  • Public defence: 2017-03-17 09:00 Hörsal E04, Byggnad 6A, Umeå
    Nordstrand, Annika
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Prostate cancer and bone cell interactions: implications for metastatic growth and therapy2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The skeleton is the most common site of prostate cancer bone metastasis, and at present, there are no curable treatments for these patients. To further understand what stimulates tumor cell growth in the bone microenvironment and to find suitable therapies, reliable model systems are needed. For this purpose, we have developed an in vitro co-culture system that can be used to study interactions between tumor cells and murine calvarial bones. To validate the model, we measured the release of collagen fragments and monitored changes in expression levels of genes normally expressed during active bone remodeling.

    One of the major reasons why prostate cancer cells colonize bone is the abundance of tumor-stimulating factors, such as insulin-like growth factors (IGFs), present in this milieu. We found that the IGF-1 receptor (IGF-1R) was one of the most highly activated receptor tyrosine kinases in tumor cell lines stimulated with bone conditioned media. Since IGF-1 is known to be a strong survival factor for tumor cells, we hypothesized, that concurrent inhibition of IGF-1R signaling can enhance the effects of apoptosis-inducing therapies, such as castration. We used our co-culture model to target human prostate cancer cell lines, PC-3 and 22Rv1, with simvastatin (an inhibitor of the mevalonate pathway and an inducer of apoptosis), in combination with anti-IGF-1R therapy. Tumor cell viability declined with either one of the therapies used alone, and the effect was even more pronounced with the combined treatment. The hypothesis was also tested in rats that had been inoculated with rat prostate cancer cells, Dunning R3327-G, into the tibial bone, and treated with either anti-IGF-1R therapy, castration, or a combination of both therapies. Immunohistochemistry was used to evaluate therapeutic effects on tumor cell proliferation and apoptosis, as well as tumor cell effects on bone remodeling. The tumor cells were found to induce an osteoblastic response, both in vivo in rats, and in vitro using the co-culture model. Interestingly, the therapeutic response differed depending on whether tumor cells were located within the bone marrow cavity or if they had leaked out into the knee joint cavity, highlighting the role of the microenvironment on metastatic growth and therapeutic response. Therapies targeting the IGF-1R have been tested in clinical trials, unfortunately with disappointing results. By immunohistochemical evaluation of bone metastases from patients with castration-resistant prostate cancer, we found a large variance in IGF-1R staining within this group of patients. Hence, we postulate that the effects of anti-IGF-1R therapies could be more beneficial in patients with high tumoral IGF-1R-activity than in IGF-1R negative cases. We also believe that side effects, such as hyperglycemia, associated with anti-IGF-1R therapy, could be reduced if this treatment is administered only to selected patients and for shorter time periods.

    In a separate study, using whole-genome expression data from bone metastases obtained from prostate cancer patients, we present evidence that a high activity of osteoblasts is coupled to a high activity of osteoclast. Moreover, we found that high bone remodeling activity is inversely related to tumor cell androgen receptor (AR) activity. The results from this study may be of importance when selecting therapy for patients with bone metastatic cancer, especially when bone-targeting therapies are considered, and could aid in the search for novel therapeutic targets.

    In summary, we present an in vitro model for studies of the bidirectional interplay between prostate cancer cells and the bone microenvironment. We also demonstrate the importance of IGF-1 in prostate cancer bone metastases and suggest that inhibition of IGF-1R signaling can be used to treat prostate cancer as well as to enhance effects of other treatments such as androgen deprivation therapy. Furthermore, we emphasize the possibility of molecular tumor characterization when designing treatment plans for individual patients, thereby maximizing the therapeutic effects.

  • Public defence: 2017-03-17 10:00 Lilla hörsalen, Umeå
    Nilsson, Lina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kinetic stabilization of transthyretin and its role as an inhibitor of Aβ amyloid formation2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Amyloid formation occurs when normally soluble proteins and peptides misfold and aggregate into intractable threadlike structures called fibrils. There are currently more than 30 proteins associated with this aberrant structure, including the Aβ peptide in Alzheimer’s disease (AD) and transthyretin (TTR) in TTR amyloidosis. TTR is a homotetrameric transporter protein present in both cerebrospinal fluid and plasma. Dissociation of its tetrameric structure is required for the formation of amyloid fibrils. Small molecule ligands able to bind and stabilize the tetrameric structure of TTR thus represent a potential therapeutic intervention. Interestingly, apart from TTR’s role as a toxic agent in TTR amyloidosis, it also has a role as an inhibitor of the Aβ toxicity associated with AD. The work presented in this thesis focused on small molecules that have the potential ability to prevent TTR amyloidosis. We also sought to gain a greater understanding of the interaction between TTR and the Aβ peptide with respect to Aβ fibril formation.

    The ability of a drug to stabilize TTR is directly correlated to its binding affinity. However, since TTR is a plasma protein, it is of great importance that the drug binds selectively to TTR. In paper I, we used a newly developed urea denaturation assay, in combination with isothermal titration calorimetry, to show that, in a complex environment such as plasma, the enthalpy of binding correlates better with a drug’s ability to stabilize TTR than the binding affinity. In paper II, we modified the highly selective but rapidly degraded TTR ligand luteolin in order to increase its resistance against biotransformation. Using a liver-based microsome assay, in combination with HPLC, we show how the luteolin analogues have gained increased stability. However, using the urea assay, we also show that the analogues have lost much of luteolin’s selectivity. In paper III, we show that tetrabromobisphenol A is a highly selective binder of TTR in plasma and is able to rescue cells from TTR-induced toxicity. In paper IV, we studied the interaction of TTR with Aβ and its effect on Aβ fibril formation. We used a ThT fluorescence-based assay and dot blotting to show that TTR inhibits Aβ amyloid formation and promotes the formation of high molecular weight assemblies with an open N-terminus. Using surface plasmon resonance, we further show how TTR is unable to inhibit fibril elongation and instead targets the nucleation processes, both primary and fibril-catalyzed secondary nucleation. To conclude, we present new molecules with the ability to selectively stabilize TTR that can serve as scaffolds in drug design. We also elucidate TTR’s inhibiting effects on toxic Aβ amyloid formation.