| 11. |
- Ahl, Ing-Marie, 1974-
(författare)
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Protein Engineering of Extracellular Superoxide Dismutase : Characterization of Binding to Heparin and Cellular Surfaces
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Accumulating evidence indicates that oxygen free radicals are involved in many diseases and pathological conditions, such as aging, inflammation, reperfusion damage of ischemic tissue and various cardiovascular diseases. Extracellular superoxide dismutase (ECSOD) thus plays a major role in the maintenance of cells by providing protection against these toxic substances in the extracellular space. Various animal studies have shown that ECSOD has the ability to protect against many of these disorders, and interest has therefore evolved in the potential therapeutic use of the enzyme.However, despite strenuous efforts, large-scale production of the enzyme has not been achieved. To overcome this problem, a mimic of the enzyme, PseudoECSOD, has previouslybeen constructed. This chimera is easy to produce in large amounts and has all the structural, enzymatic and heparin-binding characteristics of ECSOD, making it a potential substitute for ECSOD in therapeutic situations. However, the copper content of PseudoECSOD has been shown to be rather low, and since the copper ion is very important for the catalytic function of the enzyme, a production system that utilizes a copper chaperone for proper insertion of copper into the active site of the enzyme was constructed. The results show that the copper content of PseudoECSOD produced by this system is close to 100 %.In order to use PseudoECSOD therapeutically, further investigations of its binding capability and protective properties are needed. Therefore, the binding of ECSOD and PseudoECSOD to heparin was investigated using isothermal titration calorimetry. The results show that although some purely ionic interactions are important for the binding between ECSOD and heparin, there is also a substantial contribution from non-ionic interactions. The investigation also showed that the C-terminal domain is the only part of ECSOD that contributes to productive binding, and that the binding of PseudoECSOD and ECSOD to heparin is similar.In addition, analysis of mutant proteins strongly indicated that the amino acids R210, K211 and R214 are important for optimal binding of ECSOD to heparin, accounting for about 30 % of the total binding energy. The structural placement of these amino acids in an α-helix also confirms the hypothesis postulated by Margalit et al., that a common structural motif for heparin-binding proteins may be two positively charged amino acids at a distance of approximately 20 Å in the 3D-structure, facing opposite directions of a α-helix. The importance of these residues was also confirmed by analysis of a phage display library of the C-terminal domain of ECSOD.The binding of PseudoECSOD to heparan sulfate on cell surfaces of two different cell types, HepG2 and endothelial cells, was also investigated. The results clearly show that PseudoECSOD binds to these cells in a very similar manner to ECSOD. To investigate the protective properties of PseudoECSOD against ischemia-reperfusion injuries, an isolated rabbit heart model was used. The results indicate that the enzyme has a protective effect. However, more experiments using the rabbit heart and other animal models are needed to identify the optimal dose for protective purposes. The protective properties of PseudoECSOD in human tissue should also be thoroughly investigated.In summary, the findings in these studies, together with earlier results showing the close resemblance of PseudoECSOD to ECSOD in structural, enzymatic and heparin-binding properties, further support the proposition that PseudoECSOD may be a good substitute for ECSOD to use in therapeutic interventions.
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| 12. |
- Aili Fagerholm, Siri, 1980-
(författare)
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Insulin signaling in primary adipocytes in insulin sensitive and insulin resistant states
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Increasing numbers of people world-wide develops the disease type 2 diabetes. Development of type 2 diabetes is characterized by a shift from an insulin sensitive state to an insulin resistant state in peripheral insulin responding organs, which originates from the development of insulin resistance in the adipose tissue. Insulin resistance in combination with reduced pancreatic insulin secretion lead to overt type 2 diabetes.In this thesis, the insulin signaling network in primary adipocytes was analyzed. Key proteins and mechanisms were studied to gain deeper knowledge of signaling both in the insulin sensitive state and in the insulin resistant state produced by rapid weight gain as well as in type 2 diabetes.The surface of the adipocyte is dotted with invaginations in the cell membrane called caveolae that act as important metabolic and signaling platforms in adipocytes, and also harbor the insulin receptor. In paper I we show that insulin stimulation of primary adipocytes results in a rapid phosphorylation of the insulin receptor and caveolin-1, and that internalization of the proteins is mediated by endocytosis of caveolae.Weight gain due to overfeeding and obesity has been associated with the development of insulin resistance in insulin sensitive tissues such as the adipose tissue. In paper II we show that short-term overfeeding for one month of lean subjects results in an insulin resistant state. At the end of the study, the subjects had developed a mild systemic insulin resistance. Moreover, in isolated subcutaneous adipocytes we found several alterations of the insulin signaling pathway that mimicked alterations found in isolated subcutaneous adipocytes from subjects with type 2 diabetes.In paper III we present a first dynamic mathematical model of the insulin signaling network in human adipocytes that are based on experimental data acquired in a consistent fashion. The model takes account of insulin signaling in both the healthy, insulin sensitive state and in the insulin resistant state of type 2 diabetes. We show that attenuated mTORC1-mediated positive feedback to control of phosphorylation of IRS1 at Ser307 is an essential component of the insulin resistant state of type 2 diabetes. A future application of the model is the identification and evaluation of drug targets for the treatment of insulin resistance and type 2 diabetes.In paper IV we examine the protein kinase that catalyzes the insulin stimulated mTORC1- mediated feedback to IRS1. We find that the phosphorylation of IRS1 at Ser307 is not likely to be catalyzed by the kinases S6K1, mTOR or PKB. However, a catalyzing protein kinase for the in vitro phosphorylation of IRS1 at Ser307 was found to be associated with the complex mTORC1.In conclusion, this thesis provide new insights and characterize mechanisms of the intrinsically complex insulin signaling network of primary adipocytes, both in insulin sensitive and insulin resistant states.
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| 13. |
- Alkhori, Liza
(författare)
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Mechanisms of sensory neuron diversification during development and in the adult Drosophila : How to make a difference
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The nervous system contains a vast number of neurons and displays a great diversity in cell types and classes. Even though this has been known for a long time, the exact mechanism of cell specification is still poorly understood. How does a cell know what type of neuron to which it should be specified? It is important to understand cellular specification, not only for our general understanding of biological processes, but also to allow us to develop treatments for patients with destructive diseases, such as Alzheimer’s, Parkinson, cancer or stroke. To address how neuronal specification and thereby diversification is evolved, we have chosen to study a complex but defined set of neurons, the Drosophila olfactory system. Olfactory sensory neurons (OSNs) detect an enormous variety of small volatile molecules with extremely high specificity and sensitivity. The adult Drosophila olfactory system contains 34 OSN classes each defined by their expression of a specific odorant receptor (OR). In both insects and vertebrates, each OSN expresses only one OR. In mouse there are approximately 1200 and in Drosophila 60 different OR genes. Despite the range of mechanisms known to determine cell identity and that the olfactory system is remarkably conserved across the phyla, it is still unclear how an OSN chooses to express a particular OR from a large genomic repertoire. In this thesis, the specification and diversification of the final steps establishing an OSN identity is addressed. We find seven transcription factors that are continuously required in different combinations for the expression of all ORs. The TFs can in different gene context both activate and repress OR expression, making the regulation more economical and indicating that repression is crucial for correct gene expression. We further identified a repressor complex that is able to segregate OR expression between OSN classes and propose a mechanism on how one single co-repressor can specify a large number of neuron classes.Exploring the OSN we found the developmental Hh signalling pathway is expressed in the postmitotic neuron. We show several fundamental similarities between the canonical Drosophila Hh pathway and the cilia mediated Hh transduction in component function. Further investigation revealed a function of cilia mediated Hh signalling in sensory neuron modulator. The results generated here will create a greater in vivo understanding of how postmitotic processes generate neurons with different fates and contribute to the maintaining of neuron function.
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| 14. |
- Alwin, Jenny, 1978-
(författare)
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Assessment of Support Interventions in Dementia : Methodological and Empirical Studies
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Dementia means a continuous deterioration of abilities and has a large impact on the persons affected as well as on family members and others close to the persons with dementia. Interventions aimed at this population in order to support and alleviate the often straining situation may be of great importance. There is a need to increase the knowledge and understanding of the impact and consequences of these interventions, both at an individual level and at a societal level. The demand for information concerning, e.g., economic and medical impact of technologies and interventions within health care is increasing. Assessment of technologies and interventions in dementia is related to certain methodological issues. The overall aim of this thesis is to contribute to the development of methodological knowledge concerning assessment studies and understanding of support interventions in dementia.This thesis work includes four studies that examine methodological aspects of assessment of support interventions in dementia and also report empirical research on the perception of support interventions from the perspectives of family caregivers of persons with dementia. In study I, an assessment model for assistive technology (AT) interventions in dementia was developed. In study II, part of the assessment model is applied, and a process evaluation of the AT intervention is presented. Study III examines family caregivers’ perception of the importance of different types of support and services. In the final study, study IV, a well‐acknowledged methodological challenge within the field of assessment in dementia is addressed: measuring health‐related quality of life (HRQoL). The studies are based on different data materials.An assessment model of AT interventions was developed that includes health economic evaluation from a societal perspective as well as evaluation of the intervention process. The perspectives of both persons with dementia and their relatives were incorporated in the model. The process evaluation was applied in study II in order to study the relatives’ perception of an AT intervention process. Assistive technology interventions in dementia may be of great significance for the relatives. By performing the process evaluation, several important aspects that need to be acknowledged in AT intervention processes in dementia were identified.Family caregivers of persons with dementia perceived different types of support/services within the comprehensive areas of counselling, relief and information as very important. Knowledge of the caregivers’ preferences is significant so that more directed support interventions may be provided.One specific methodological issue was addressed in study IV. The results of study IV showed that there were large differences in the results of cost‐effectiveness analyses depending on whether patient self‐ratings or proxy ratings (ratings of persons close to the patient) of patient HRQoL were used for the same analysis. These differences in the results could ultimately have an important impact on decision making and resource allocation.Support interventions aimed at persons with dementia and their relatives may be of great value. Conducting assessment studies of technologies and interventions within health care is important in order to increase the knowledge concerning, e.g., economic and medical impact. Assessment studies of support interventions dementia entails methodological issues that need to be addressed. There is a need for increased knowledge within this field and suggestions on methodology are made.
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| 15. |
- Andersson, Sten-Ove, 1953-
(författare)
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Militär akutsjukvård i fält : när den övade verkligheten blir verklig
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aims: Governing for the Armed Forces health care is respect for human dignity and the view of each individual as irreplaceable. The quality of the military care will be at a level equivalent to that of today's civil peace healthcare, which requires that the training is of high quality. Casualty care in the military environment is a complex, challenging and dangerous task. Today there is a lack of knowledge about how to learn these skills. The purpose of the research was to identify the knowledge area pre-hospital care in the military environment by examining what doctors, nurses, paramedics and officers learn about military health care during preparation and exercise.Method: A phenomenographic approach was used in all sub-studies, and empirical data were collected by means of semi-structured interviews. Study I‐III are cross-sectional studies and study IV is a longitudinal study. Twelve registered nurses who had served in Bosnia were interviewed for study I, and 24 conscript paramedics were interviewed for study II. Study III included 20 officers in their pre-deployment training for service in Afghanistan and Liberia, and Study IV included 7 doctors and twenty nurses who previously had served in Afghanistan, Bosnia, Kosovo and Liberia.Results: Learning military health care by training and gaining experience can be seen as different abilities; interaction, action and reflection. In-depth analysis at a meta--‐level revealed that the integration between the military and military medical fields of knowledge was missing. The results indicate that participants' understanding of their respective fields of knowledge is inadequate and needs to be integrated in education in a clearer way.Implications: To create opportunities for better education and integration in these fields of knowledge are proposed (1) that the findings of the thesis could be utilised to develop the design of the curriculum (2) the inter-professional learning is introduced as a part of creating a safer and more effective pre-hospital care, (3) the clinical competence becomes part of the development of pre-hospital emergency care in the field, and (4) that the training is built according to the principle of "train as you fight" with emphasis on the requirements in the combat zone.
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| 16. |
- Annerbäck, Eva-Maria, 1948-
(författare)
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Child Physical Abuse : Characteristics, Prevalence, Health and Risk‐taking
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The home is supposed to provide support and safety for children but can also be the place where children suffer abuse and other adverse treatment by their parents. Violence against children in homes has been banned in Sweden for more than 30 years but it is still a considerable problem in the society and a threat to public health. The overall aim of this thesis was to create comprehensive knowledge of the phenomenon Child Physical Abuse (CPA) in Sweden after the ban on corporal punishment. The focus has been on examining the characteristics of cases reported to the police as well as self-reported CPA, prevalence of CPA and finally associations between CPA and health-problems/risk-taking behaviors among adolescents.Two samples are used in this thesis. The first comprises cases reported to the police during 11 years (n=142) in a Swedish police-district and the second is a population-based youth survey of the total number of pupils in three different school grades (13, 15 and 17 years old) in Södermanland County, Sweden.Cases of severe abuse constituted 14 % of the total number of cases reported to the police. The main difference between the group of severe cases and the remaining was the higher occurrence of convictions in court in the severe cases and the pattern of reporting to the police. The severe cases were reported by agencies to a greater degree than minor cases. Cases of severe abuse were characterized by an accumulation of risk factors in different areas as perpetrator factors, stress- and strain factors, factors of insufficient social network and finally child-related factors.In the cross-sectional study a prevalence of 15 % was found for self-reported CPA (n=8 494). There were associations between risk factors in different areas and abuse and there was a dos-response relationship between risks and reported abuse. It was shown that children who reported parental intimate-partner violence were at considerably higher risk for CPA than other children and that only 7 % of the children exposed to violence had disclosed this to authorities.The study of associations between health and risk-taking behaviors, were performed among the 15 and 17 years old pupils (n=5 933). Associations with health-problems and risk-taking behaviors were shown and the associations became stronger when the pupils reported repeated abuse. Finally there was a cumulative effect of multiple abuse in the form of being exposed to child physical abuse plus other types of abuse (parental intimate partner violence, bullying and being forced to engage in sexual acts) and the associations increased with the number of concurrent abuse.
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| 17. |
- Ansell, Anna, 1983-
(författare)
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Identification of Tumor Cell- and Stroma Derived Biomarkers of Treatment Response in Head and Neck Cancer
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Head and neck squamous cell carcinoma (HNSCC) poses a major health problem in the world with approximately 600 000 new cases yearly. Treatment resistance is a major problem within this patient group and despite advances in treatment strategies the overall survival rate has unfortunately not increased.One of the major components of the tumor microenvironment is the cancer associated fibroblasts (CAFs) which can modulate the treatment sensitivity, tumor growth, and the invasive potential of tumor cells.The aim of this thesis was to identify predictive markers for treatment response in HNSCC and to study the crosstalk between tumor cells and CAFs that may underlie treatment resistance.In paper I, we identified gene expression differences between one cisplatin sensitive cell line and two cisplatin resistant cell lines, by microarray analysis, and found that a high expression of matrix metalloproteinase (MMP) -7 was associated with resistance to cisplatin. In paper II, the epidermal growth factor (EGF) receptor ligands EGF, amphiregulin, and epiregulin were evaluated regarding their potential use as predictive biomarkers for cetuximab treatment response in tongue cancer cell lines and it was shown that EGF may serve as a marker for poor cetuximab response. In paper III and IV, we investigated the influence of CAFs on the proliferation, migration, gene expression, and cetuximab response of tumor cells. It was found that CAFs induced resistance to cetuximab in a MMP-dependent manner. In addition, a microarray analysis, comparing tumor cells co-cultured with CAFs and tumor cells cultured alone, revealed that CAFs induced multiple gene expression changes in tumor cells some of which are related to epithelial to mesenchymal transition. Some of these changes were found to be dependent on cell-cell contact.Taken together, we here suggest MMP-7 and EGF to be predictive markers of cisplatin and cetuximab response, respectively. We also show that CAFs protect HNSCC cells from cetuximab treatment; however, the factor responsible for the protective effect is yet to be discovered.
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| 18. |
- Appelqvist, Hanna, 1981-
(författare)
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Lysosomal Membrane Stability and Cathepsins in Cell Death
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lysosomes are acidic organelles that are critically involved in a number of physiological processes, including macromolecule degradation, endocytosis, autophagy, exocytosis and cholesterol homeostasis. Several pathological conditions, such as cancer, neurodegenerative disorders and lysosomal storage diseases, involve lysosomal disturbances, indicating the importance of the organelle for correct cellular function. The aim of this thesis was to investigate the role of lysosomes in cell death signaling.Previous studies have shown that permeabilization of the lysosomal membrane and release of hydrolytic enzymes such as cathepsin D to the cytosol occurs during apoptosis. We identified Bid and 14-3-3 proteins as cytosolic targets of cathepsin D in human fibroblasts. Truncated Bid, generated by cathepsin D proteolytic cleavage, stimulates Bax-mediated release of pro-apoptotic factors from the mitochondria, thereby engaging the intrinsic pathway to apoptosis.Since the presence of cathepsins in the cytosol is sufficient to induce apoptosis, the permeability of the lysosomal membrane influences the fate of the cell. In this thesis, we demonstrated that the stability of the lysosomal membrane can be manipulated by altering the lysosomal cholesterol content. Cells with high lysosomal cholesterol content were less prone to undergo apoptosis when challenged with stimuli known to induce lysosome-mediated cell death. In addition, cholesterol accumulation was associated with increased expression of lysosome-associated membrane proteins and storage of other lipids; however, these factors did not contribute to lysosomal stabilization.Lysosomal membrane permeabilization and cathepsins contribute to ultraviolet (UV) irradiation-induced apoptosis. We demonstrate plasma membrane damage induced by UVA irradiation to be rapidly repaired by lysosomal exocytosis in human keratinocytes. Despite efficient plasma membrane resealing, the cells underwent apoptosis, which was dependent on early activation of caspase-8. The activation of caspase-8 was lysosome-dependent and occurred in vesicles positive for lysosomal markers.This thesis demonstrates the importance of lysosomal stability for apoptosis regulation and that this stability can be influenced by drug intervention. Modulation of the lysosomal membrane permeability may have potential for use as a therapeutic strategy in conditions associated with accelerated or repressed apoptosis.
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| 19. |
- Arvidsson, Eva
(författare)
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Priority Setting and Rationing in Primary Health Care
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Studies on priority setting in primary health care are rare. Priority setting and rationing in primary health care is important because outcomes from primary health care have significant implications for health care costs and outcomes in the health system as a whole.Aims: The general aim of this thesis has been to study and analyse the prerequisites for priority setting in primary health care in Sweden. This was done by exploring strategies to handle scarce resources in Swedish routine primary health care (Paper I); analysing patients’ attitudes towards priority setting and rationing and patients’ satisfaction with the outcome of their contact with primary health care (Paper II); describing and analysing how general practitioners, nurses, and patients prioritised individual patients in routine primary health care, studying the association between three key priority setting criteria (severity of the health condition, patient benefit, and cost-effectiveness of the medical intervention) and the overall priority assigned by the general practitioners and nurses to individual patients (Paper III); and analysing how the staff, in their clinical practise, perceived the application of the three key priority setting criteria (Paper IV).Methods: Both qualitative (Paper I and IV) and quantitative (Paper II and III) methods were used. Paper I was an interview study with medical staff at 17 primary health care centres. The data for Paper II and Paper III were collected through questionnaires to patients and staff at four purposely selected health care centres during a 2-week period. Paper IV was a focus group study conducted with staff members who practiced priority setting in day-to-day care.Results: The process of coping with scarce resources was categorised as efforts aimed to avoid rationing, ad hoc rationing, or planned rationing. Patients had little understanding of the need for priority setting. Most of them did not experience any kind of rationing and most of those who did were satisfied with the outcome of their contact with primary health care. Patients, compared to medical staff, gave relatively higher priority to acute/minor conditions than to preventive check-ups for chronic conditions when prioritising individual patients in day-today primary health care. When applying the three priority setting criteria in day-to-day primary health care, the criteria largely influenced the overall prioritisation of each patient. General practitioners were most influenced by the expected cost-effectiveness of the intervention and nurses were most influenced by the severity of the condition. Staff perceived the criteria as relevant, but not sufficient. Three additional aspects to consider in priority setting in primary health care were identified, namely viewpoint (medical or patient’s), timeframe (now or later) and evidence level (group or individual).Conclusion: There appears to be a need for, and the potential to, introduce more consistent priority setting in primary health care. The characteristics of primary health care, such as the vast array of health problems, the large number of patients with vague symptoms, early stages of diseases, and combinations of diseases, induce both special possibilities and challenges.
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| 20. |
- Axelsson, Stina, 1981-
(författare)
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GAD65 An Immunomodulator in Type 1 Diabetes
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type 1 diabetes (T1D) is caused by a deficiency of insulin as a result of an autoimmune destruction of the pancreatic ² -cells. A possibility to preserve remaining ² -cells in children with newly diagnosed T1D is of great importance since sustained ² -cell function is recognized to result in reduced end-organ complications. Glutamic acid decarboxylase 65 (GAD65) is one of the major antigens targeted by self-reactive T cells in T1D, and immunomodulation with GAD65 formulated in aluminum (GAD-alum) has been considered both in prevention and treatment of T1D. Results from a Phase II trial have shown clinical effect of subcutaneous injections with GAD-alum, this was unfortunately not fully confirmed in the following larger Phase III trial which therefore was closed after 15 months. The general aim of this thesis was to study the immunomodulatory effect of GAD-alum-treatment in children with T1D participating in the Phase II and Phase III trials. We hypothesized that treatment with GAD-alum contributes to the preservation of residual insulin secretion through deviation of the GAD65-specific immune response from a destructive to a protective process, accompanied by a shift from T helper (Th) 1 towards a predominant Th2 profile. In the Phase II trial, GAD-alum-treated patients responded with an early GAD65-specific Th2 skewed cytokine secretion, with highest IL-5 and IL-13 secretion in clinical responders. Also, the CCR4/CCR5 ratio indicating balance between Th2/Tc2 and Th1/Tc1 responses, increased in treated patients. The recall response to GAD65 was characterized by a wide range of cytokines, but the relative contribution of each cytokine suggests a shift towards a more pronounced Th2-associated profile over time. Induction of a CD4+ cell subset upon GAD65-stimulation 4 years after treatment, suggesting clonal expansion of the memory T-cell compartment upon antigen re-challenge, was seen in parallel to a persistent GAD65-specific cytokine response. Finally, even if the phase III trial failed to reach the primary endpoint at 15 months, a subgroup analysis showed that the treatment had an effect on preservation of residual insulin secretion, but the effect was not seen until after 30 months. Taken together, these results suggest that GAD-alum treatment might exert its effect through induction of an early Th2 skewed immune response which tends to deviate away from a destructive Th1/Tc1 response upon GAD65 re-challenge, and generation of GAD65-specific memory T cells that produce cytokines and exert effector responses which may be important for regulating GAD65 immunity. Continued research to better understand how immunomodulation with autoantigen modifies T-cell responses and also which patients are suitable for treatment, is crucial for optimizing future intervention trials using ² -cell antigens.
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