Death pathways activated in the neurotrophic factor-deprived neurons

Programed cell death (PCD) is a fundamental biological process that is as essential for the development and tissue homeostasis as cell proliferation, differentiation and adaptation. The main mode of PCD - apoptosis - occurs via specifi c pathways, such as mitochondrial or death receptor pathway. In the developing nervous system, programed death broadly occurs, mainly triggered by the defi ciency of different survival-promoting neurotrophic factors, but the respective death pathways are poorly studied. In one of the best-characterized models, sympathetic neurons deprived of nerve growth factor (NGF) die via the classical mitochondrial apoptotic pathway. The main aim of this study was to describe the death programs activated in these and other neuronal populations by using neuronal cultures deprived of other neurotrophic factors. First, this study showed that the cultured sympathetic neurons deprived of glial cell line-derived neurotrophic factor (GDNF) die via a novel non-classical death pathway, in which mitochondria and death receptors are not involved. Indeed, cytochrome c was not released into the cytosol, Bax, caspase-9, and caspase-3 were not involved, and Bcl-xL overexpression did not prevent the death. This pathway involved activation of mixed lineage kinases and c-jun, and crucially requires caspase-2 and -7. Second, it was shown that deprivation of neurotrophin-3 (NT-3) from cultured sensory neurons of the dorsal root ganglia kills them via a dependence receptor pathway, including cleavage of the NT- 3 receptor TrkC and liberation of a pro-apoptotic dependence domain. Indeed, death of NT-3-deprived neurons was blocked by a dominant-negative construct interfering with TrkC cleavage. Also, the uncleavable mutant of TrkC, replacing the siRNA-silenced endogeneous TrkC, was not able to trigger death upon NT-3 removal. Such a pathway was not activated in another subpopulation of sensory neurons deprived of NGF. Third, it was shown that cultured midbrain dopaminergic neurons deprived of GDNF or brainderived neurotrophic factor (BDNF) kills them by still a different pathway, in which death receptors and caspases, but not mitochondria, are activated. Indeed, cytochrome c was not released into the cytosol, Bax was not activated, and Bcl-xL did not block the death, but caspases were necessary for the death of these neurons. Blocking the components of the death receptor pathway - caspase-8, FADD, or Fas - blocked the death, whereas activation of Fas accelerated it. The activity of Fas in the dopaminergic neurons could be controlled by the apoptosis inhibitory molecule FAIML. For these studies we developed a novel assay to study apoptosis in the transfected dopaminergic neurons. Thus, a novel death pathway, characteristic for the dopaminergic neurons was described. The study suggests death receptors as possible targets for the treatment of Parkinson s disease, which is caused by the degeneration of dopaminergic neurons.

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time.

Genotype-Phenotype Relationships in Long QT Syndrome: Role of Mental Stress, Adrenergic Activity and a Common KCNH2 Polymorphism

Long QT syndrome is a congenital or acquired arrhythmic disorder which manifests as a prolonged QT-interval on the electrocardiogram and as a tendency to develop ventricular arrhythmias which can lead to sudden death. Arrhythmias often occur during intense exercise and/or emotional stress. The two most common subtypes of LQTS are LQT1, caused by mutations in the KCNQ1 gene and LQT2, caused by mutations in the KCNH2 gene. LQT1 and LQT2 patients exhibit arrhythmias in different types of situations: in LQT1 the trigger is usually vigorous exercise whereas in LQT2 arrhythmia results from the patient being startled from rest. It is not clear why trigger factors and clinical outcome differ from each other in the different LQTS subtypes. It is possible that stress hormones such as catecholamines may show different effects depending on the exact nature of the genetic defect, or sensitivity to catecholamines varies from subject to subject. Furthermore, it is possible that subtle genetic variants of putative modifier genes, including those coding for ion channels and hormone receptors, play a role as determinants of individual sensitivity to life-threatening arrhythmias. The present study was designed to identify some of these risk modifiers. It was found that LQT1 and LQT2 patients show an abnormal QT-adaptation to both mental and physical stress. Furthermore, as studied with epinephrine infusion experiments while the heart was paced and action potentials were measured from the right ventricular septum, LQT1 patients showed repolarization abnormalities which were related to their propensity to develop arrhythmia during intense, prolonged sympathetic tone, such as exercise. In LQT2 patients, this repolarization abnormality was noted already at rest corresponding to their arrhythmic episodes as a result of intense, sudden surges in adrenergic tone, such as fright or rage. A common KCNH2 polymorphism was found to affect KCNH2 channel function as demonstrated by in vitro experiments utilizing mammalian cells transfected with the KCNH2 potassium channel as well as QT-dynamics in vivo. Finally, the present study identified a common β-1-adrenergic receptor genotype that is related a shorter QT-interval in LQT1 patients. Also, it was discovered that compound homozygosity for two common β-adrenergic polymorphisms was related to the occurrence of symptoms in the LQT1 type of long QT syndrome. The studies demonstrate important genotype-phenotype differences between different LQTS subtypes and suggest that common modifier gene polymorphisms may affect cardiac repolarization in LQTS. It will be important in the future to prospectively study whether variant gene polymorphisms will assist in clinical risk profiling of LQTS patients.

Crosstalk between the sympathetic nervous system, inflammation and coagulation in gestational diabetes; a therapeutic approach in postmenopausal hypertension

The occurrence of gestational diabetes (GDM) during pregnancy is a powerful sign of a risk of later type 2 diabetes (T2D) and cardiovascular diseases (CVDs). The physiological basis for this disease progression is not yet fully understood, but increasing evidence exists on interplay of insulin resistance, subclinical inflammation, and more recently, on unbalance of the autonomic nervous system. Since the delay in development of T2D and CVD after GDM ranges from years to decades, better understanding of the pathophysiology of GDM could give us new tools for primary prevention. The present study was aimed at investigating the role of the sympathetic nervous system (SNS) in GDM and its associations with insulin and a variety of inflammatory cytokines and coagulation and fibrinolysis markers. This thesis covers two separate study lines. Firstly, we investigated 41 women with GDM and 22 healthy pregnant and 14 non-pregnant controls during the night in hospital. Blood samples were drawn at 24:00, 4:00 and 7:00 h to determine the concentrations of plasma glucose, insulin, noradrenaline (NA) and adrenomedullin, markers of subclinical inflammation, coagulation and fibrinolysis variables and platelet function. Overnight holter ECG recording was performed for analysis of heart rate variability (HRV). Secondly, we studied 87 overweight hypertensive women with natural menopause. They were randomised to use a central sympatholytic agent, moxonidine (0.3mg twice daily), the β-blocking agent atenolol (50 mg once daily+blacebo once daily) for 8 weeks. Inflammatory markers and adiponectin were analysed at the beginning and after 8 weeks. Activation of the SNS (increase in NA, decreased HRV) was seen in pregnant vs. non-pregnant women, but no difference existed between GDM and normal pregnancy. However, modulation (internal rhythm) of HRV was attenuated in GDM. Insulin and inflammatory cytokine levels were comparable in all pregnant women but nocturnal variation of concentrations of C-reactive protein, serum amyloid A and insulin were reduced in GDM. Levels of coagulation factor VIII were lower in GDM compared with normal pregnancy, whereas no other differences were seen in coagulation and fibrinolysis markers. No significant associations were seen between NA and the studied parameters. In the study of postmenopausal women, moxonidine treatment was associated with favourable changes in the inflammatory profile, seen as a decrease in TNFα concentrations (increase in atenolol group) and preservation of adiponectin levels (decrease in atenolol group). In conclusion, our results did not support our hypotheses of increased SNS activity in GDM or a marked association between NA and inflammatory and coagulation markers. Reduced biological variation of HRV, insulin and inflammatory cytokines suggests disturbance of autonomic and hormonal regulatory mechanisms in GDM. This is a novel finding. Further understanding of the regulatory mechanisms could allow earlier detection of risk women and the possibility of prevention. In addition, our results support consideration of the SNS as one of the therapeutic targets in the battle against metabolic diseases, including T2D and CVD.

Oscillatory brain activity in memory disorders

Neuronal oscillations are thought to underlie interactions between distinct brain regions required for normal memory functioning. This study aimed at elucidating the neuronal basis of memory abnormalities in neurodegenerative disorders. Magnetoencephalography (MEG) was used to measure oscillatory brain signals in patients with Alzheimer s disease (AD), a neurodegenerative disease causing progressive cognitive decline, and mild cognitive impairment (MCI), a disorder characterized by mild but clinically significant complaints of memory loss without apparent impairment in other cognitive domains. Furthermore, to help interpret our AD/MCI results and to develop more powerful oscillatory MEG paradigms for clinical memory studies, oscillatory neuronal activity underlying declarative memory, the function which is afflicted first in both AD and MCI, was investigated in a group of healthy subjects. An increased temporal-lobe contribution coinciding with parieto-occipital deficits in oscillatory activity was observed in AD patients: sources in the 6 12.5 Hz range were significantly stronger in the parieto-occipital and significantly weaker in the right temporal region in AD patients, as compared to MCI patients and healthy elderly subjects. Further, the auditory steady-state response, thought to represent both evoked and induced activity, was enhanced in AD patients, as compared to controls, possibly reflecting decreased inhibition in auditory processing and deficits in adaptation to repetitive stimulation with low relevance. Finally, the methodological study revealed that successful declarative encoding and retrieval is associated with increases in occipital gamma and right hemisphere theta power in healthy unmedicated subjects. This result suggests that investigation of neuronal oscillations during cognitive performance could potentially be used to investigate declarative memory deficits in AD patients. Taken together, the present results provide an insight on the role of brain oscillatory activity in memory function and memory disorders.

Post-industrial Intervention : An Activity-Theoretical Expedition Tracing the Proximal Development of Forms of Conducting Interventions

Ei saatavilla

Boundaries in Action : An Activity-theoretical Study of Development, Learning, and Change in Health Care Organization for Patients with Multiple and Chronic Illnesses

This study examines boundaries in health care organizations. Boundaries are sometimes considered things to be avoided in everyday living. This study suggests that boundaries can be important temporally and spatially emerging locations of development, learning, and change in inter-organizational activity. Boundaries can act as mediators of cultural and social formations and practices. The data of the study was gathered in an intervention project during the years 2000-2002 in Helsinki in which the care of 26 patients with multiple and chronic illnesses was improved. The project used the Change Laboratory method that represents a research assisted method for developing work. The research questions of the study are: (1) What are the boundary dynamics of development, learning, and change in health care for patients with multiple and chronic illnesses? (2) How do individual patients experience boundaries in their health care? (3) How are the boundaries of health care constructed and reconstructed in social interaction? (4) What are the dynamics of boundary crossing in the experimentation with the new tools and new practice? The methodology of the study, the ethnography of the multi-organizational field of activity, draws on cultural-historical activity theory and anthropological methods. The ethnographic fieldwork involves multiple research techniques and a collaborative strategy for raising research data. The data of this study consists of observations, interviews, transcribed intervention sessions, and patients' health documents. According to the findings, the care of patients with multiple and chronic illnesses emerges as fragmented by divisions of a patient and professionals, specialties of medicine and levels of health care organization. These boundaries have a historical origin in the Finnish health care system. As an implication of these boundaries, patients frequently experience uncertainty and neglect in their care. However, the boundaries of a single patient were transformed in the Change Laboratory discussions among patients, professionals and researchers. In these discussions, the questioning of the prevailing boundaries was triggered by the observation of gaps in inter-organizational care. Transformation of the prevailing boundaries was achieved in implementation of the collaborative care agreement tool and the practice of negotiated care. However, the new tool and practice did not expand into general use during the project. The study identifies two complementary models for the development of health care organization in Finland. The 'care package model', which is based on productivity and process models adopted from engineering and the 'model of negotiated care', which is based on co-configuration and the public good.

Evaluation of driving ability of the disabled persons in the context of the psychological activity theory

In the future the number of the disabled drivers requiring a special evaluation of their driving ability will increase due to the ageing population, as well as the progress of adaptive technology. This places pressure on the development of the driving evaluation system. Despite quite intensive research there is still no consensus concerning what is the factual situation in a driver evaluation (methodology), which measures should be included in an evaluation (methods), and how an evaluation has to be carried out (practise). In order to find answers to these questions we carried out empirical studies, and simultaneously elaborated upon a conceptual model for driving and a driving evaluation. The findings of empirical studies can be condensed into the following points: 1) A driving ability defined by the on-road driving test is associated with different laboratory measures depending on the study groups. Faults in the laboratory tests predicted faults in the on-road driving test in the novice group, whereas slowness in the laboratory predicted driving faults in the experienced drivers group. 2) The Parkinson study clearly showed that even an experienced clinician cannot reliably accomplish an evaluation of a disabled person’s driving ability without collaboration with other specialists. 3) The main finding of the stroke study was that the use of a multidisciplinary team as a source of information harmonises the specialists’ evaluations. 4) The patient studies demonstrated that the disabled persons themselves, as well as their spouses, are as a rule not reliable evaluators. 5) From the safety point of view, perceptible operations with the control devices are not crucial, but correct mental actions which the driver carries out with the help of the control devices are of greatest importance. 6) Personality factors including higher-order needs and motives, attitudes and a degree of self-awareness, particularly a sense of illness, are decisive when evaluating a disabled person’s driving ability. Personality is also the main source of resources concerning compensations for lower-order physical deficiencies and restrictions. From work with the conceptual model we drew the following methodological conclusions: First, the driver has to be considered as a holistic subject of the activity, as a multilevel hierarchically organised system of an organism, a temperament, an individuality, and a personality where the personality is the leading subsystem from the standpoint of safety. Second, driving as a human form of a sociopractical activity, is also a hierarchically organised dynamic system. Third, in an evaluation of driving ability it is a question of matching these two hierarchically organised structures: a subject of an activity and a proper activity. Fourth, an evaluation has to be person centred but not disease-, function- or method centred. On the basis of our study a multidisciplinary team (practitioner, driving school teacher, psychologist, occupational therapist) is recommended for use in demanding driver evaluations. Primary in a driver’s evaluations is a coherent conceptual model while concrete methods of evaluations may vary. However, the on-road test must always be performed if possible.

Expression and function of angiotensins in the regulation of intraocular pressure - an experimental study

Glaucoma is a multifactorial long-term ocular neuropathy associated with progressive loss of the visual field, retinal nerve fiber structural abnormalities and optic disc changes. Like arterial hypertension it is usually a symptomless disease, but if left untreated leads to visual disability and eventual blindness. All therapies currently used aim to lower intraocular pressure (IOP) in order to minimize cell death. Drugs with new mechanisms of action could protect glaucomatous eyes against blindness. Renin-angiotensin system (RAS) is known to regulate systemic blood pressure and compounds acting on it are in wide clinical use in the treatment of hypertension and heart failure but not yet in ophthalmological use. There are only few previous studies concerning intraocular RAS, though evidence is accumulating that drugs antagonizing RAS can also lower IOP, the only treatable risk factor in glaucoma. The main aim of this experimental study was to clarify the expression of the renin-angiotensin system in the eye tissues and to test its potential oculohypotensive effects and mechanisms. In addition, the possible relationship between the development of hypertension and IOP was evaluated in animal models. In conclusion, a novel angiotensin receptor type (Mas), as well as ACE2 enzyme- producing agonists for Mas, were described for the first time in the eye structures participating in the regulation of IOP. In addition, a Mas receptor agonist significantly reduced even normal IOP. The effect was abolished by a specific receptor antagonist. Intraocular, local RAS would thus to be involved in the regulation of IOP, probably even more in pathological conditions such as glaucoma though there was no unambiguous relationship between arterial and ocular hypertension. The findings suggest the potential as antiglaucomatous drugs of agents which increase ACE2 activity and the formation of angiotensin (1-7), or activate Mas receptors.