Endoplasmic reticulum stress and cell death mechanisms in the cell model of Huntington’s disease

This thesis clarifies important molecular pathways that are activated during the cell death observed in Huntington’s disease. Huntington’s disease is one of the most common inherited neurodegenerative diseases, which is primarily inherited in an autosomal dominant manner. HD is caused by an expansion of CAG repeats in the first exon of the IT15 gene. IT15 encodes the production of a Huntington’s disease protein huntingtin. Mutation of the IT15 gene results in a long stretch of polyQ residues close to the amino-terminal region of huntingtin. Huntington’s disease is a fatal autosomal neurodegenerative disorder. Despite the current knowledge of HD, the precise mechanism behind the selective neuronal death, and how the disease propagates, still remains an enigma. The studies mainly focused on the control of endoplasmic reticulum (ER) stress triggered by the mutant huntingtin proteins. The ER is a delicate organelle having essential roles in protein folding and calcium regulation. Even the slightest perturbations on ER homeostasis are effective enough to trigger ER stress and its adaptation pathways, called unfolded protein response (UPR). UPR is essential for cellular homeostasis and it adapts ER to the changing environment and decreases ER stress. If adaptation processes fail and stress is excessive and prolonged; irreversible cell death pathways are engaged. The results showed that inhibition of ER stress with chemical agents are able to decrease cell death and formation of toxic cell aggregates caused by mutant huntingtin proteins. The study concentrated also to the NF-κB (nuclear factor-kappaB) pathway, which is activated during ER stress. NF-κB pathway is capable to regulate the levels of important cellular antioxidants. Cellular antioxidants provide a first line of defence against excess reactive oxygen species. Excess accumulation of reactive oxygen species and subsequent activation of oxidative stress damages motley of vital cellular processes and induce cell degeneration. Data showed that mutant huntingtin proteins downregulate the expression levels of NF-κB and vital antioxidants, which was followed by increased oxidative stress and cell death. Treatment with antioxidants and inhibition of oxidative stress were able to counteract these adverse effects. In addition, thesis connects ER stress caused by mutant huntingtin to the cytoprotective autophagy. Autophagy sustains cellular balance by degrading potentially toxic cell proteins and components observed in Huntington’s disease. The results revealed that cytoprotective autophagy is active at the early points (24h) of ER stress after expression of mutant huntingtin proteins. GADD34 (growth arrest and DNA damage-inducible gene 34), which is previously connected to the regulation of translation during cell stress, was shown to control the stimulation of autophagy. However, GADD34 and autophagy were downregulated at later time points (48h) during mutant huntingtin proteins induced ER stress, and subsequently cell survival decreased. Overexpression GADD34 enhanced autophagy and decreased cell death, indicating that GADD34 plays a critical role in cell protection. The thesis reveales new interesting data about the neuronal cell death pathways seen in Huntington’s disease, and how cell degeneration is partly counteracted by various therapeutic agents. Expression of mutant huntingtin proteins is shown to alter signaling events that control ER stress, oxidative stress and autophagy. Despite that Huntington’s disease is mainly an untreatable disorder; these findings offer potential targets and neuroprotective strategies in designing novel therapies for Huntington’s disease.

Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

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.

Bone Stress Injuries of the Foot and Ankle

Bone stress injuries of the foot have been known for more than 150 years. For a century, their primary diagnostic imaging tool has been radiography. However, currently the golden standard for establishing the diagnosis of stress injuries is magnetic resonance imaging (MRI). Although the injury type has been fairly well documented in the earlier literature, little information is available on the healing of stress injuries located in e.g. the talus and calcaneus. The current study retrospectively evaluated the stress injuries of the foot and ankle treated at the Central Military Hospital over a period of eight years in patients who underwent MRI for stress injury of the foot. The imaging studies of the patients were reevaluated to determine the exact nature of the stress injury. Moreover, the hospital records of the patients were reviewed to determine the healing of stress injuries of the talus and calcaneus. Patients with a stress fracture in the talus were recalled for a follow-up examination and MRI scan one to six years after the initial injury to determine if the fracture had completely healed, clinically and radiologically. The bone stress injuries of the foot were found to affect more than one bone in a majority of the cases. The talus and the calcaneus were the bones most commonly affected. In the talus, the most common site for the injuries was the head of the bone, and in the calcaneus, the posterior part of the bone. The injuries in these bones were associated with injuries in the surrounding bones. Stress injuries in the calcaneus seemed to heal well. No complications were seen in the primary healing process. The patients were, however, sometimes compelled to refrain from physical training for up to months. In the talus, minor degenerative findings of the articular surface were seen in half of the patients who participated in a follow-up MRI scan and radiographs taken one to six years after the initial injury. Half of the patients also reported minor exercise related symptoms in the follow-up. The symptoms were, however, not noticeable in everyday life.

Glycemic Control in Diabetic Pregnancies: Effects on Fetal and Maternal Outcome

Background: Both maternal and fetal complications are increased in diabetic pregnancies. Although hypertensive complications are increased in pregnant women with pregestational diabetes, reports on hypertensive complications in women with gestational diabetes mellitus (GDM) have been contradictory. Congenital malformations and macrosomia are the main fetal complications in Type 1 diabetic pregnancies, whereas fetal macrosomia and birth trauma but not congenital malformations are increased in GDM pregnancies. Aims: To study the frequency of hypertensive disorders in gestational diabetes mellitus. To evaluate the risk of macrosomia and brachial plexus injury (Erb’s palsy) and the ability of the 2-hour glucose tolerance test (OGTT) combined with the 24-hour glucose profile to distinguish between low and high risks of fetal macrosomia among women with GDM. To evaluate the relationship between glycemic control and the risk of fetal malformations in pregnancies complicated by Type 1 diabetes mellitus. To assess the effect of glycemic control on the occurrence of preeclampsia and pregnancy-induced hypertension in Type 1 diabetic pregnancies. Subjects: A total of 986 women with GDM and 203 women with borderline glucose intolerance (one abnormal value in the OGTT) with a singleton pregancy, 488 pregnant women with Type 1 diabetes (691 pregnancies and 709 offspring), and 1154 pregnant non-diabetic women (1181 pregnancies and 1187 offspring) were investigated. Results: In a prospective study on 81 GDM patients the combined frequency of preeclampsia and PIH was higher than in 327 non-diabetic controls (19.8% vs 6.1%, p<0.001). On the other hand, in 203 women with only one abnormal value in the OGTT, the rate of hypertensive complications did not differ from that of the controls. Both GDM women and those with only one abnormal value in the OGTT had higher pre-pregnancy weights and BMIs than the controls. In a retrospective study involving 385 insulin-treated and 520 diet-treated GDM patients, and 805 non-diabetic control pregnant women, fetal macrosomia occurred more often in the insulin-treated GDM pregnancies (18.2%, p<0.001) than in the diet-treated GDM pregnancies (4.4%), or the control pregnancies (2.2%). The rate of Erb’s palsy in vaginally delivered infants was 2.7% in the insulin-treated group of women and 2.4% in the diet-treated women compared with 0.3% in the controls (p<0.001). The cesarean section rate was more than twice as high (42.3% vs 18.6%) in the insulin-treated GDM patients as in the controls. A major fetal malformation was observed in 30 (4.2%) of the 709 newborn infants in Type 1 diabetic pregnancies and in 10 (1.4%) of the 735 controls (RR 3.1, 95% CI 1.6–6.2). Even women whose levels of HbA1c (normal values less than 5.6%) were only slightly increased in early pregnancy (between 5.6 and 6.8%) had a relative risk of fetal malformation of 3.0 (95% CI 1.2–7.5). Only diabetic patients with a normal HbA1c level (<5.6%) in early pregnancy had the same low risk of fetal malformations as the controls. Preeclampsia was diagnosed in 12.8% and PIH in 11.4% of the 616 Type 1 diabetic women without diabetic nephropathy. The corresponding frequencies among the 854 control women were 2.7% (OR 5.2; 95% CI 3.3–8.4) for preeclampsia and 5.6% (OR 2.2, 95% CI 1.5–3.1) for PIH. Multiple logistic regression analysis indicated that glycemic control, nulliparity, diabetic retinopathy and duration of diabetes were statistically significant independent predictors of preeclampsia. The adjusted odds ratios for preeclampsia were 1.6 (95% CI 1.3–2.0) for each 1%-unit increment in the HbA1c value during the first trimester and 0.6 (95% CI 0.5–0.8) for each 1%-unit decrement during the first half of pregnancy. In contrast, changes in glycemic control during the second half of pregnancy did not alter the risk of preeclampsia. Conclusions: In type 1 diabetic pregnancies it is extremely important to achieve optimal glycemic control before pregnancy and maintain it throughout pregnancy in order to decrease the complication rates both in the mother and in her offspring. The rate of fetal macrosomia and birth trauma in GDM pregnancies, especially in the group of insulin-treated women, is still relatively high. New strategies for screening, diagnosing, and treatment of GDM must be developed in order to decrease fetal and neonatal complications.

Work Stress in the Nursing Profession : An Evaluation of Organizational Causal Attribution

The goal of this study was to examine the role of organizational causal attribution in understanding the relation of work stressors (work-role overload, excessive role responsibility, and unpleasant physical environment) and personal resources (social support and cognitive coping) to such organizational-attitudinal outcomes as work engagement, turnover intention, and organizational identification. In some analyses, cognitive coping was also treated as an organizational outcome. Causal attribution was conceptualized in terms of four dimensions: internality-externality, attributing the cause of one’s successes and failures to oneself, as opposed to external factors, stability (thinking that the cause of one’s successes and failures is stable over time), globality (perceiving the cause to be operative on many areas of one’s life), and controllability (believing that one can control the causes of one’s successes and failures). Several hypotheses were derived from Karasek’s (1989) Job Demands–Control (JD-C) model and from the Job Demands–Resources (JD-R) model (Demerouti, Bakker, Nachreiner & Schaufeli, 2001). Based on the JD-C model, a number of moderation effects were predicted, stating that the strength of the association of work stressors with the outcome variables (e.g. turnover intentions) varies as a function of the causal attribution; for example, unpleasant work environment is more strongly associated with turnover intention among those with an external locus of causality than among those with an internal locuse of causality. From the JD-R model, a number of hypotheses on the mediation model were derived. They were based on two processes posited by the model: an energy-draining process in which work stressors along with a mediating effect of causal attribution for failures deplete the nurses’ energy, leading to turnover intention, and a motivational process in which personal resources along with a mediating effect of causal attribution for successes foster the nurses’ engagement in their work, leading to higher organizational identification and to decreased intention to leave the nursing job. For instance, it was expected that the relationship between work stressors and turnover intention could be explained (mediated) by a tendency to attribute one’s work failures to stable causes. The data were collected from among Finnish hospital nurses using e-questionnaires. Overall 934 nurses responded the questionnaires. Work stressors and personal resources were measured by five scales derived from the Occupational Stress Inventory-Revised (Osipow, 1998). Causal attribution was measured using the Occupational Attributional Style Questionnaire (Furnham, 2004). Work engagement was assessed through the Utrecht Work Engagement Scale (Schaufeli & al., 2002), turnover intention by the Van Veldhoven & Meijman (1994) scale, and organizational identification by the Mael & Ashforth (1992) measure. The results provided support for the function of causal attribution in the overall work stress process. Findings related to the moderation model can be divided into three main findings. First, external locus of causality along with job level moderated the relationship between work overload and cognitive coping. Hence, this interaction was evidenced only among nurses in non-supervisory positions. Second, external locus of causality and job level together moderated the relationship between physical environment and turnover intention. An opposite pattern of interaction was found for this interaction: among nurses, externality exacerbated the effect of perceived unpleasantness of the physical environment on turnover intention, whereas among supervisors internality produced the same effect. Third, job level also disclosed a moderation effect for controllability attribution over the relationship between physical environment and cognitive coping. Findings related to the mediation model for the energetic process indicated that the partial model in which work stressors have also a direct effect on turnover intention fitted the data better. In the mediation model for the motivational process, an intermediate mediation effect in which the effects of personal resources on turnover intention went through two mediators (e.g., causal dimensions and organizational identification) fitted the data better. All dimensions of causal attribution appeared to follow a somewhat unique pattern of mediation effect not only for energetic but also for motivational processes. Overall findings on mediation models partly supported the two simultaneous underlying processes proposed by the JD-R model. While in the energetic process the dimension of externality mediated the relationship between stressors and turnover partially, all the dimensions of causal attribution appeared to entail significant mediator effects in the motivational process. The general findings supported the moderation effect and the mediation effect of causal attribution in the work stress process. The study contributes to several research traditions, including the interaction approach, the JD-C, and the JD-R models. However, many potential functions of organizational causal attribution are yet to be evaluated by relevant academic and organizational research. Keywords: organizational causal attribution, optimistic / pessimistic attributional style, work stressors, organisational stress process, stressors in nursing profession, hospital nursing, JD-R model, personal resources, turnover intention, work engagement, organizational identification.

Developmental origins of physiological stress reactivity

The model of developmental origins of health and disease proposes that organisms during fetal period utilize cues that enable their adaptation in the postnatal environment they are likely to live, having short-term advantages when trying to survive in environment but simultaneously in the long run have costs for health. A large body of epidemiological research has found that low birth weight, a marker of intrauterine conditions, is associated with cardiovascular (CV) disease. Since the reported associations of birth weight with normal variation in the resting blood pressure (BP), a major predictor of CV disease risk, have been modest, a key candidate mediating the link has been CV and hypothalamus-pituitary-adrenal axes (HPAA) reactivity to stress. In addition, not only weight at birth but also gestational age and early postnatal growth may have independent associations to stress reactivity. The aim of this thesis was to investigate whether pre- and postnatal growth and gestational age are associated with CV and HPAA activity before, during and after stress in childhood and in late adulthood. Altogether 287 men and women aged 60-70 and 299 boys and girls aged 7-9 underwent Trier Social Stress Test. Several indices of HPAA and CV were measured and birth size and gestational age were obtained from birth records. Results showed that low birth weight was associated with low HPAA activity during psychosocial stress, and rapid gain in BMI during years 7-11 was related to heightened stress reactivity to psychosocial stress. Size at birth in children and gestational age and early postnatal (0-2 years) gain in height in adults were associated with CV stress responses; however, in a sex-specific manner. Given that CV stress responses and HPAA activity are markers of CV disease vulnerability, our results may partly explain the associations between early environment and later CV disease.