| 1. |
- Dahl, Lina, 1981-
(författare)
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Stem cell function and organ development : analysis of Lhx2 function in hematopoietic stem cells and eye development
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- When a multicellular organism suffers damages to tissues/organs it heals itself by either substituting the lost cellular matrix by scar formation or by regenerating the lost tissue. Regeneration likely occurs by a recapitulation of the developmental process that formed the organ. Many processes regulating organ development are based on epithelial-mesenchymal interactions and a strict control of organ specific stem/progenitor cells. Elucidation of the molecular basis of these processes is therefore vital in order to develop novel therapies in regenerative medicine. The LIM homebox gene Lhx2 is interesting in this context since Lhx2 has been shown to be important for the formation of several organs by regulating epithelial-mesenchymal interactions and progenitor cell function. Targeted inactivation of Lhx2 leads to a lethal anemia due to malformed liver and severe neural abnormalities such as hypoplasia of the forebrain and anophtalmia. Thus, elucidation of the mechanisms of the function of Lhx2 in different organ systems would give important insights into the molecular mechanisms regulating epithelial-mesenchymal interactions and stem/progenitor cell function. To elucidate the function of Lhx2 in the hematopoietic system Lhx2 was initially expressed in hematopoietic progenitor cells derived from ES cells differentiated in vitro using retroviral vectors. This approach led to the generation of hematopoietic stem cell (HSC)-like cell lines suggesting that Lhx2 could impact HSC function. However neither the specificity nor the efficiency of the Lhx2-induced phenotype could be determined using this approach. To be able to elucidate the function of Lhx2 in the hematopoietic system, an ES cell line with inducible Lhx2 expression was generated. Lhx2 expression induces self-renewal of a distinct hematopoietic progenitor cell from which HSC-like cell lines were established. Down-regulation of Lhx2 in these HSC-like cell lines leads to a rapid loss of stem cell character, providing a good model to study the molecular function of Lhx2 in hematopoietic stem/progenitor cells. A global gene expression analysis was performed comparing the Lhx2+ stem cell population to the Lhx2- differentiated progeny. This approach identified genes putatively linked to self-renewal/differentiation of HSCs. A considerable proportion of the genes showed an overlapping gene expression pattern with Lhx2 expression in tissue of non-hematopoietic origin suggesting that Lhx2 function in stem/progenitor cells partly overlap with Lhx2 function during organ development. In order to define other Lhx2-dependent progenitor cell populations and to generate a tool to analyze the function of Lhx2 in organ development a new transgenic mouse model was generated. By using a specific part of the Lhx2 promoter to drive expression of Cre recombinase in vivo (Lhx2-Cre mice) we have been able to define the first eye committed progenitor cells in the forebrain. By using the Lhx2-Cre mice it will be possible to distinguish the function of genes during eye development from their function in the patterning of the forebrain e.g. the eye field transcription factors. Conditional inactivation of Lhx2 in these eye specific progenitor cells causes an immediate developmental arrest. The transgene is also active in Lhx2-/- embryonic forebrain, but re-expression of Lhx2 in Lhx2-/- progenitor cells only promote formation of retinal pigment epithelium cells. Analysis of genes expressed by the Lhx2+ stem cell population allowed us to define novel genes putatively linked to Lhx2 function in eye development. Thus, we have defined the progenitor cells in the forebrain committed to eye development and the expansion and patterning of these progenitors are dependent on Lhx2. Although commitment to eye development is Lhx2-independent, Lhx2 might be important for the acquisition of the oligopotent fate of these progenitor cells.
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| 2. |
- Abrahamsson, Pernilla, 1972-
(författare)
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Methodological aspects on microdialysis sampling and measurements
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: The microdialysis (MD) technique is widely spread and used both experimentally and in clinical practice. The MD technique allows continuous collection of small molecules such as glucose, lactate, pyruvate and glycerol. Samples are often analysed using the CMA 600 analyser, an enzymatic and colorimetric analyser. Data evaluating the performance of the CMA 600 analysis system and associated sample handling are sparse. The aim of this work was to identify sources of variability related to handling of microdialysis samples and sources of error associated with use of the CMA 600 analyser. Further, to develop and compare different application techniques of the microdialysis probes both within an organ and on the surface of an organ. Material and Methods: Papers I and II are mainly in vitro studies with the exception of the No Net Flux calibration method in paper I where a pig model (n=7) was used to examine the true concentration of glucose and urea in subcutaneous tissue. Flow rate, sampling time, vial and caps material and performance of the analyser device (CMA 600) were examined. In papers III and IV normoventilated anaesthetised pigs (n=33) were used. In paper III, heart ischemia was used as intervention to compare microdialysis measurements in the myocardium with corresponding measurements on the heart surface. In paper IV, microdialysis measurements in the liver parenchyma were compared with measurements on the liver surface in association with induced liver ischemia. All animal studies were approved by the Animal Experimental Ethics Committee at Umeå University Sweden. Results: In paper I we succeeded to measure true concentrations of glucose (4.4 mmol/L) and Urea (4.1 mmol/L) in subcutaneous tissue. Paper II showed that for a batch analyse of 24 samples it is preferred to store microdialysis samples in glass vials with crimp caps. For reliable results, samples should be centrifuged before analysis. Paper III showed a new application area for microdialysis sampling from the heart, i.e. surface sampling. The surface probe and myocardial probe (in the myocardium) showed a similar pattern for glucose, lactate and glycerol during baseline, short ischemic and long ischemic interventions. In paper IV, a similar pattern was observed as in paper III, i.e. data obtained from the probe on the liver surface showed no differences compared with data from the probe in liver parenchyma for glucose, lactate and glycerol concentrations during baseline, ischemic and reperfusion interventions. Conclusion: The MD technique is adequate for local metabolic monitoring, but requires methodological considerations before starting a new experimental serie. It is important to consider factors such as flow rate, sampling time and handling of samples in association with the analysis device chosen. The main finding in this thesis is that analyses of glucose, lactate and glycerol in samples from the heart surface and liver surface reflect concentrations sampled from the myocardium and liver parenchyma, respectively.
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| 3. |
- Andersson, Emma, 1978-
(författare)
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Human adenoviruses : new bioassays for antiviral screening and CD46 interaction
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Adenoviruses are common pathogens all over the world. The majority of the population has at some point been infected with an adenovirus. Although severe disease can occur in otherwise healthy individuals an adenovirus infection is most commonly self limited in these cases. For immunocompromised individuals however, adenoviruses can be life-threatening pathogens capable of causing disseminated disease and multiple organ failure. Still there is no approved drug specific for treatment of adenovirus infections. We have addressed this using a unique whole cell viral replication reporter gene assay to screen small organic molecules for anti-adenoviral effect. This RCAd11pGFP-vector based assay allowed screening without any preconceived idea of the mechanism for adenovirus inhibition. As a result of the screening campaign 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid turned out to be a potent inhibitor of adenoviral replication. To establish a structure-activity relationship a number of analogs were synthesized and evaluated for their anti-adenoviral effect. The carboxylic acid moiety of the molecule was important for efficient inhibition of adenovirus replication.There are 54 adenovirus types characterized today and these are divided into seven species, A-G. The receptors used by species B and other adenoviruses are not fully characterized. CD46 is a complement regulatory molecule suggested to be used by all species B types and some species D types but this is not established. We have designed a new bioassay for assessment of the interaction between adenoviruses and CD46 and investigated the CD46-binding capacity of adenovirus types indicated to interact with CD46. We concluded that Ad11p, Ad34, Ad35, and Ad50 clearly bind CD46 specifically, whereas Ad3p, Ad7p, Ad14, and Ad37 do not.CD46 is expressed on all human nucleated cells and serves as a receptor for a number of different bacteria and viruses. Downregulation of CD46 on the cell surface occurs upon binding by some of these pathogens. We show that early in infection Ad11p virions downregulate CD46 upon binding to a much higher extent than the complement regulatory molecules CD55 and CD59.These findings may lead to a better understanding of the pathogenesis of adenoviruses in general and species B adenoviruses in particular and hopefully we have discovered a molecule that can be the basis for development of new anti-adenoviral drugs.
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| 4. |
- Andersson, Gustav, 1983-
(författare)
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Influences of paratendinous innervation and non-neuronal substance P in tendinopathy : studies on human tendon tissue and an experimental model of Achilles tendinopathy
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pain of the musculoskeletal system is one of the most common reasons for people seeking medical attention, and is also one of the major factors that prevent patients from working. Chronic tendon pain, tendinopathy, affects millions of workers world-wide, and the Achilles tendon is an important structure often afflicted by this condition. The pathogenesis of tendinopathy is poorly understood, but it is thought to be of multifactoral aetiology. It is known that tendon pain is often accompanied not only by impaired function but also by structural tissue changes, like vascular proliferation, irregular collagen organisation, and hypercellularity, whereby the condition is called tendinosis. In light of the poor knowledge of tendinosis pathophysiology and recent findings of a non-neuronal signalling system in tendon tissue, the contributory role of neuropeptides such as substance P (SP) has gained increased interest. SP, known for afferent pain signalling in the nervous system, also has multiple efferent functions and has been described to be expressed by non-neuronal cells. As pain is the most prominent symptom of tendinopathy, the focus of the studies in this thesis was the innervation patterns of the tissue ventral to the Achilles tendon (i.e. the tissue targeted in many contemporary treatment methods) as well as the distribution of SP and its preferred receptor, the neurokinin-1 receptor (NK-1R), in the tendon tissue itself. It was hereby hypothesised that the source of SP affecting the Achilles tendon might be the main cells of the tendon tissue (the tenocytes) as well as paratendinous nerves, and that SP might be involved in tendinosis- development. The studies were conducted, via morphological staining methods including immunohistochemistry and in situ hybridisation, on tendon biopsies from patients suffering from Achilles tendinosis and on those from healthy volunteers. The hypothesis of the thesis was furthermore tested using an experimental animal model (rabbit) of Achilles tendinopathy, which was first validated. The model was based on a previously established overuse protocol of repetitive exercise. In the human biopsies of the tissue ventral to the Achilles tendon, there was a marked occurrence of sympathetic innervation, but also sensory, SP-containing, nerve fibres. NK-1R was expressed on blood vessels and nerve fascicles of the paratendinous tissue, but also on the tenocytes of the tendon tissue proper itself, and notably more so in patients suffering from tendinosis. Furthermore, the human tenocytes displayed not only NK-1R mRNA but also mRNA for SP. The animal model was shown to produce objectively verified tendinosis-like changes, such as hypercellularity and increased vascularity, in the rabbit Achilles tendons, after a minimum of three weeks of the exercise protocol. The contralateral leg of the animals in the model was found to be an unreliable control, as bilateral changes occured. The model furthermore demonstrated that exogenously administered SP triggers an inflammatory response in the paratendinous tissue and accelerates the intratendinous tendinosis-like changes such that they now occur after only one week of the protocol. Injections of saline as a control showed similar results as SP concerning hypercellularity, but did not lead to vascular changes or pronounced paratendinous inflammation. In summary, this thesis concludes that interactions between the peripheral sympathetic and sensory nervous systems may occur in Achilles tendinosis at the level of the ventral paratendinous tissue, a region thought to be of great importance in chronic tendon pain since many successful treatments are directed toward it. Furthermore, the distribution of NK-1R:s in the Achilles tendon described in these studies gives a basis for SP, whether produced by nerves mainly outside the tendon or by tenocytes within the tendon, to affect blood vessels, nerve structures, and/or tendon cells, especially in tendinosis patients. In light of this and of previously known SP-effects, such as stimulation of angiogenesis, pain signalling, and cell proliferation, the proposed involvement of SP in tendinosis development seems likely. Indeed, the animal model of Achilles tendon overuse confirms that SP does induce vascular proliferation and hypercellularity in tendon tissue, thus strengthening theories of SP playing a role in tendinosis pathology.
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| 5. |
- Andersson, Jonas, 1977-
(författare)
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Inflammation and lifestyle in cardiovascular medicine
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite major advances in the treatment and prevention of atherosclerosis the last several decades, cardiovascular disease still accounts for the majority of deaths in Sweden. With the population getting older, more obese and with rising numbers of diabetics, the cardiovascular disease burden may increase further in the future. The focus in cardiovascular disease has shifted with time from calcification and narrowing of arteries to the biological processes within the atherosclerotic plaque. C-reactive protein (CRP) has emerged as one of many proteins that reflect a low grade systemic inflammation and is suitable for analysis as it is more stable and easily measured than most other inflammatory markers. Several large prospective studies have shown that CRP is not only an inflammatory marker, but even a predictive marker for cardiovascular disease. C-reactive protein is associated with several other risk factors for cardiovascular disease including obesity and the metabolic syndrome. Our study of twenty healthy men during a two week endurance cross country skiing tour demonstrated a decline in already low baseline CRP levels immediately after the tour and six weeks later. In a study of 200 obese individuals with impaired glucose tolerance randomised to a counselling session at their health care centre or a one month stay at a wellness centre, we found decreased levels of CRP in subjects admitted to the wellness centre. The effect remained at one, but not after three years of follow-up. In a prospective, nested, case-referent study with 308 ischemic strokes, 61 intracerebral haemorrhages and 735 matched referents, CRP was associated with ischemic stroke in both uni- and multivariate analyses. No association was found with intracerebral haemorrhages. When classifying ischemic stroke according to TOAST criteria, CRP was associated with small vessel disease. The CRP 1444 (CC/CT vs. TT) polymorphism was associated with plasma levels of CRP, but neither with ischemic stroke nor with intracerebral haemorrhage. A study on 129 patients with atrial fibrillation was used to evaluate whether inflammation sensitive fibrinolytic variables adjusted for CRP could predict recurrence of atrial fibrillation after electrical cardioversion. In multivariate iv models, lower PAI-1 mass was associated with sinus rhythm even after adjusting for CRP and markers of the metabolic syndrome. In conclusion, lifestyle intervention can be used to reduce CRP levels, but it remains a challenge to maintain this effect. CRP is a marker of ischemic stroke, but there are no significant associations between the CRP1444 polymorphism and any stroke subtype, suggesting that the CRP relationship with ischemic stroke is not causal. The fibrinolytic variable, PAI-1, is associated with the risk of recurrence of atrial fibrillation after electrical cardioversion after adjustment for CRP. Our findings suggest a pathophysiological link between atrial fibrillation and PAI-1, but the relation to inflammation remains unclear.
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| 6. |
- Andersson, Therése, 1978-
(författare)
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Estrogen and Glucocorticoid Metabolism
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Cardiovascular disease (CVD) is the leading cause of death among women in Sweden. The risk of CVD increases rapidly after the menopause. A major contributing factor may be the redistribution of adipose tissue, from the peripheral to central depots, associated with menopause. This change in body composition is commonly attributed to declining estrogen levels but may also be affected by tissue-specific alterations in exposure to other steroid hormones, notably glucocorticoids – mainly cortisol in humans. Indeed, adipose tissue-specific overexpression of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) induces central obesity, insulin resistance and hypertension in mice. Interestingly, estrogen may regulate this enzyme. The aim of this thesis was to investigate putative links between estrogen and glucocorticoid activation by 11βHSD1. Materials and Methods: 11βHSD1 expression and/or activity in adipose tissue and liver, and adipose estrogen receptor α and β (ERα and ERβ) gene expression, were investigated in lean pre- and postmenopausal women and ovariectomized rodents with and without estrogen supplementation. In lean women measures of 11βHSD1 were correlated to risk markers for CVD. The association between adipose 11βHSD1 and ER mRNA expression was investigated in both lean women and rats and in an additional cohort of obese premenopausal women. In vitro experiments with adipocyte cell lines were used to explore possible pathways for estrogen regulation of 11βHSD1. Results: Subcutaneous adipose tissue transcript levels and hepatic activity of 11βHSD1 were higher in postmenopausal vs. premenopausal women. In rodents, estrogen treatment to ovariectomized rats decreased visceral adipose tissue 11βHSD1, resulting in a shift towards higher subcutaneous (vs. visceral) 11βHSD1 mRNA expression/activity. Increased adipose and hepatic 11βHSD1 were associated with increased blood pressure and a disadvantageous blood lipid profile in humans. We found significant positive associations between 11βHSD1 and ERβ transcript levels in adipose tissue. The in vitro experiments showed upregulation of 11βHSD1 mRNA expression and activity with estrogen or ERβ-agonist treatment at low (corresponding to physiological) concentrations. Conclusions: Our studies show for the first time increased local tissue glucocorticoid activation with menopause/age in women. This may contribute to an increased risk of CVD. Estrogen treatment in rodents induces a shift in 11βHSD1 activity towards the subcutaneous adipose tissue depots, which may direct fat accumulation to this metabolically “safer” depot. The in vitro studies suggest that low-dose estrogen treatment upregulates 11βHSD1 via ERβ. In summary, estrogen - glucocorticoid metabolism interactions may be key in the development of menopause-related metabolic dysfunction and in part mediate the beneficial effects of postmenopausal estrogen treatment on body fat distribution.
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| 7. |
- Árnadóttir, Guðrún, 1955-
(författare)
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Measuring the impact of body functions on occupational performance : validation of the ADL-focused occupation-based neurobehavioral evaluation (A-ONE)
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Among the instruments commonly used by occupational therapists working in the area of rehabilitation of persons with neurological disorders are evaluations of both occupation, such as activities of daily living (ADL), and body functions. While persons with neurological diagnoses typically have symptoms that represent diminished neurobehavioral functions, the resulting pattern of neurobehavioral impairments affecting ADL performance often differs among diagnostic groups. Usually, neurobehavioral impairments are evaluated in a context that is separate from and not natural for ADL task performance. The A-ONE is a unique instrument that can be used to evaluate both ADL performance (ADL scale) and, in the natural context of the ADL task performance, the underlying neurobehaviors that cause diminished ADL task performance among persons with neurological disorders (Neurobehavioral scale). The scales of the instrument are of ordinal type, and in their existing form, do not have measurement properties. Measurement properties are a requirement of evidence-based and quality assured rehabilitation services.The overall aim of this doctoral study was to further develop and validate the A-ONE. This included (a) internal validation to explore the potential for converting the ordinal scales of the instrument to interval scales, (b) examination of which of the neurobehavioral items would be most beneficial and clinically useful for constructing a new Neurobehavioral Impact (NBI) scale for evaluating persons with different neurological diagnoses, and (c) exploration of whether persons with right and left cerebrovascular accidents (RCVA, LCVA) differ in mean NBI measures. Methods: This thesis is comprised of four studies which all contribute in different ways to the validation of the scales of the A-ONE. In the first three studies, Rasch analyses, a widely accepted modern test theory methodology, was used to examine internal validity of the scales and the reliability of the A-ONE measures. In the fourth study, ANCOVA was used to explore between group differences, and Pearson correlation coefficients were used to explore relations between person measures from the different A-ONE scales.Results: The first study of 209 persons diagnosed with CVA and dementia provided support for converting the ordinal ADL scale to an interval scale that has potential to be used to measure change in ADL performance over time. The second and third studies, including 206 and 422 persons respectively, indicated that it is possible to construct several unidimensional versions of a new NBI scale from the neurobehavioral items of the instrument, each with different item content and hierarchical item structure. Further, some of these NBI scales could be used across different diagnostic groups. When exploring differences between 215 persons with RCVA and LCVA on the NBI scale developed for CVA, results of the ANCOVA (with ADL ability as a covariate) indicated that there is no significant difference between groups in their mean NBI measures, despite known differences in patterns of neurobehavioral impairments.Conclusions: The results of this thesis indicate that the A-ONE, although developed by traditional psychometric methods for the purpose of providing useful information for intervention planning, now also has the potential to be used to measure change and compare diagnostic groups. This additional feature will likely enhance both clinical and research potential of the instrument. In order to make the results of the study accessible for clinicians, conversion tables need to be developed.
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| 8. |
- Arnadottir, Solveig, 1968-
(författare)
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Physical activity, participation and self-rated health among older community-dwelling Icelanders : a population-based study
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: The main objective of this study was to investigate older people’s physical activity, their participation in various life situations, and their perceptions of their own health. This included an exploration of potential influences of urban versus rural residency on these outcomes, an evaluation of the measurement properties of a balance confidence scale, and an examination of the proposed usefulness of the International Classification of Functioning, Disability and Health (ICF) as a conceptual framework to facilitate analysis and understanding of selected outcomes.Methods: The study design was cross-sectional, population-based, with random selection from the national register of one urban and two rural municipalities in Northern Iceland. There were 186 participants, all community-dwelling, aged 65 to 88 years (mean = 73.8), and 48% of the group were women. The participation rate was 79%. Data was collected in 2004, in face-to-face interviews and through various standardized assessments. The main outcomes were total physical activity; leisure-time, household, and work-related physical activity; participation frequency and perceived participation restrictions; and self-rated health. Other assessments represented aspects of the ICF body functions, activities, environmental factors and personal factors. Moreover, Rasch analysis methods were applied to examine and modify the Activities-specific Balance Confidence (ABC) scale and the ICF used as a conceptual framework throughout the study.Results: The total physical activity score was the same for urban and rural people and the largest proportion of the total physical activity behavior was derived from the household domain. Rural females received the highest scores of all in household physical activity and rural males were more physically active than the others in the work-related domain. However, leisure-time physical activity was more common in urban than rural communities. A physically active lifestyle, urban living, a higher level of cognition, younger age, and fewer depressive symptoms were all associated with more frequent participation. Rural living and depressive symptoms were associated with perceived participation restrictions. Moreover, perceived participation restrictions were associated with not being employed and limitations in advanced lower extremity capacity. Both fewer depressive symptoms and advanced lower extremity capacity also increased the likelihood of better self-rated health, as did capacity in upper extremities, older age, and household physical activity. Rasch rating scale analysis indicated a need to modify the ABC to improve its psychometric properties. The modified ABC was then used to measure balance confidence which, however, was found not to play a major role in explaining participation or self-rated health. Finally, the ICF was useful as a conceptual framework for mapping various components of functioning and health and to facilitate analyses of their relationships.Conclusions: The results highlighted the commonalities and differences in factors associated with participation frequency, perceived participation restrictions, and self-rated health in old age. Some of these factors, such as advanced lower extremity capacity, depressive symptoms, and physical activity pattern should be of particular interest for geriatric physical therapy due to their potential for interventions. While the associations between depressive symptoms, participation, and self-rated health are well known, research is needed on the effects of advanced lower extremity capacity on participation and self-rated health in old age. The environment (urban versus rural) also presented itself as an important contextual variable to be aware of when working with older people’s participation and physically active life-style. Greater emphasis should be placed on using Rasch measurement methods for improving the availability of quality scientific measures to evaluate various aspects of functioning and health among older adults. Finally, a coordinated implementation of a conceptual framework such as ICF may further advance interdisciplinary and international studies on aging, functioning, and health.
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| 9. |
- Bergemalm, Daniel, 1977-
(författare)
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Mutant superoxide dismutase-1-caused pathogenesis in amyotrophic lateral sclerosis
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyotrophic lateral sclerosis (ALS) is a devastating disease that affects people in their late mid-life, with fatal outcome usually within a few years. The progressive degeneration of neurons responsible for muscle movement (motor neurons) throughout the central nervous system (CNS) leads to muscle wasting and paralysis, and eventually affects respiratory function. Most cases have no familial background (sporadic) whereas about 10% of cases have relatives affected by the disease. A substantial number of familial cases are caused by mutations in the gene encoding superoxide dismutase-1 (SOD1). Since the initial discovery of this relationship about 17 years ago, numerous workers have tried to identify the pathogenicity of mutant SOD1 but without any final agreement or consensus regarding mechanism. The experiments in this thesis have been aimed at finding common pathogenic mechanisms by analyzing transgenic mouse models expressing mutant SOD1s with widely different properties. Mitochondrial pathology and dysfunction have been reported in both ALS patients and murine models. We used density gradient ultracentrifugation for comparison of mitochondrial partitioning of SOD1 in our transgenic models. It was found that models with high levels of mutant protein, overloaded mitochondria with high levels of SOD1-protein whereas models with wild type-like levels of mutant protein did not. No significant association of the truncation mutant G127X with mitochondria was found. Thus, if mitochondrial dysfunction and pathology are fundamental for ALS pathogenesis this is unlikely to be caused by physical association of mutant SOD1 with mitochondria. Density gradient ultracentrifugation was used to study SOD1 inclusions in tissues from an ALS patient with a mutant SOD1 (G127X). We found large amounts in the ventral horns of the spinal cord but also in the liver and kidney, although at lower levels. This showed that such signs of the disease can also be found outside the CNS. This method was used further to characterize SOD1 inclusions with regard to the properties of mutant SOD1 and the presence of other proteins. The inclusions were found to be complex detergent-sensitive structures with mutant SOD1 reduced at disulfide C57-C146 being the major inclusion protein, constituting at least 50% of the protein content. Ten co-aggregating proteins were isolated, some of which were already known to be present in cellular inclusions. Of great interest was the presence of several proteins that normally reside in the endoplasmic reticulum (ER), which is in accordance with recent data suggesting that the unfolded protein response (UPR) has a role in ALS. To obtain unbiased information on the pathogenesis of mutant SOD1, we performed a total proteome study on spinal cords from ALS transgenic mice. By multivariate analysis of the 1,800 protein spots detected, 420 (23%) were found to significantly contribute to the difference between transgenic and control mice. From 53 proteins finally identified, we found pathways such as mitochondrial function, oxidative stress, and protein degradation to be affected by the disease. We also identified a previously uncharacterized covalent SOD1 dimer. In conclusion, the work described in this thesis suggests that mutant SOD1 affects the function of mitochondria, but not mainly through direct accumulation of SOD1 protein. It also suggests that SOD1 inclusions, present in both the CNS and peripheral tissues, mainly consist of SOD1 but they also trap proteins involved in the UPR. This might be deleterious as motor neurons, unable to renew themselves, are dependent on proper protein folding and degradation.
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| 10. |
- Bjur, Dennis, 1965-
(författare)
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The human Achilles tendon : innervation and intratendinous production of nerve signal substances - of importance in understanding the processes of Achilles tendinosis
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tendinopathies are painful tendon conditions of presumably multifactorial genesis. In tendinosis, as in Achilles tendinosis, there is apart from pain also morphological changes which are described as degenerative with no signs of inflammation. The exact mechanisms behind these conditions are still, to a large extent, unknown. Pain, being the foremost impairing symptom, leads us to the hypothesis that nerves are deeply involved in the symptoms and processes of Achilles tendinosis. Locally produced nerve signal substances may also be involved in the processes. Knowledge of the innervation patterns within the tendon and knowledge on a possible local nerve signal substance production are therefore of utmost importance. There is a lack of information on these aspects. The specific aims of this thesis were 1) to investigate the innervation patterns regarding general, sensory, cholinergic and sympathetic innervations, and 2) to examine for the possible occurrence of a production of nerve signal substances and a presence of receptors related to these in the tendon cells, the tenocytes. Painfree normal and tendinosis Achilles tendons were examined. Immunohistochemistry, using antibodies against the general nerve marker PGP9.5, the synthesizing enzymes for acetylcholine (choline acetyltransferase; ChAT), and catecholamines (tyrosine hydroxylase; TH), the vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), substance P and calcitonin gene-related peptide, was applied. Immunohistochemistry was also used for the delineation of muscarinic (M2R), adrenergic (α1-AR) and NPY-ergic (Y1 and Y2) receptors. To detect mRNA for TH and ChAT, in situ hybridization was used. In normal Achilles tendons, as well as in the tendinosis tendons, there was a very scanty innervation within the tendon tissue proper, the main general, sensory and sympathetic innervations being found in the paratendinous loose connective tissue. Interestingly, the tenocytes showed immunoreactions for ChAT, VAChT, TH, M2R, α1-AR and Y1R. The reactions were clearly more observable in tendons of tendinosis patients than in those of controls. The tenocytes of tendinosis patients also displayed mRNA reactions for ChAT and TH. Nevertheless, all tenocytes in the tendinosis specimens did not show these reactions. Immunoreactions for α1-AR, M2R and Y1R were also seen for blood vessel walls. The present thesis shows that there is a very limited innervation within tendon tissue proper, whilst there is a substantial innervation in the paratendinous loose connective tissue. It also gives evidence for an occurrence of production of catecholamines and acetylcholine in tenocytes, especially for tendinosis tendons. Furthermore, that ACh, catecholamines and NPY can have effects on these, as well as on blood vessels, via the receptors observed. The observations suggest that Achilles tendon tissue, whilst containing a very scarce innervation, exhibits autocrine/paracrine cholinergic/catecholaminergic/NPY-ergic effects that are upregulated in tendinosis. These findings are of great importance as the results of such effects may mimic processes that are known to occur in tendinosis. That includes effects related to proliferation and angiogenesis, and blood vessel and collagen regulating effects. In conclusion, within the Achilles tendon there is a very scarce innervation, whilst there appears to be a marked local production of nerve signal substances in Achilles tendinosis, namely in the tenocytes, the cells also harbouring receptors for these substances. The observations give a new insight into how the tendon tissue of the Achilles tendon is influenced by signal substances and may give options for new treatments of Achilles tendinosis.
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