Background: A small cross sectional area (CSA) of the paraspinal muscles may be related to low back pain among military aviators but previous studies have mainly concentrated on spinal disc degeneration. Therefore, the primary aim of the study was to investigate the changes in muscle CSA and composition of the psoas and paraspinal muscles during a 5-year follow up among Finnish Air Force (FINAF) fighter pilots. Methods: Study population consisted of 26 volunteered FINAF male fighter pilots (age: 20.6 (±0.6) at the baseline). The magnetic resonance imaging (MRI) examinations were collected at baseline and after 5 years of follow-up. CSA and composition of the paraspinal and psoas muscles were obtained at the levels of 3-4 and 4-5 lumbar spine. Maximal isometric strength tests were only performed on one occasion at baseline. Results: The follow-up comparisons indicated that the mean CSA of the paraspinal muscles increased (p <0.01) by 8% at L3-4 level and 7% at L4-5 level during the 5-year period. There was no change in muscle composition during the follow-up period. The paraspinal and psoas muscles' CSA was positively related to overall maximal isometric strength at the baseline. However, there was no association between LBP and muscle composition or CSA. Conclusions: The paraspinal muscles' CSA increased among FINAF fighter pilots during the first 5 years of service. This might be explained by physically demanding work and regular physical activity. However, no associations between muscle composition or CSA and low back pain (LBP) experienced were observed after the five-year follow-up. © 2019 The Author(s).

Thermal tolerance has a major effect on individual fitness and species distributions and can be determined by genetic variation and phenotypic plasticity. We investigate the effects of developmental and adult thermal conditions on cold tolerance, measured as chill coma recovery (CCR) time, during the early and late adult stage in the Glanville fritillary butterfly. We also investigate the genetic basis of cold tolerance by associating CCR variation with polymorphisms in candidate genes that have a known role in insect physiology. Our results demonstrate that a cooler developmental temperature leads to reduced cold tolerance in the early adult stage, whereas cooler conditions during the adult stage lead to increased cold tolerance. This suggests that adult acclimation, but not developmental plasticity, of adult cold tolerance is adaptive. This could be explained by the ecological conditions the Glanville fritillary experiences in the field, where temperature during early summer, but not spring, is predictive of thermal conditions during the butterfly's flight season. In addition, an amino acid polymorphism (Ala-Glu) in the gene flightin, which has a known function in insect flight and locomotion, was associated with CCR. These amino acids have distinct biochemical properties and may thus affect protein function and/or structure. To our knowledge, our study is the first to link genetic variation in flightin to cold tolerance, or thermal adaptation in general.

Mitochondria are vital cellular organelles that produce the majority of the energy required by cells and are therefore represented as "the power house of the cell". Cellular energy, in the form of adenosine triphosphate (ATP), is generated through oxidative phosphorylation (OXPHOS), which is energized by the redox reactions of the electron transport chain (ETC) in mitochondria. Mitochondria are essential for maintaining metabolic homeostasis, fatty-acid metabolism, nucleotide synthesis and cellular redox balance, in addition to producing ATP. Given the multitude of mitochondrial functions, failure of any of them can lead to mitochondrial dysfunction that can have drastic consequences. It can lead to cell loss, organ failure and even death. Mitochondrial disorders are commonly associated with debilitating childhood onset diseases with no known cure. Current therapeutic approaches are largey symptomatic, such as dietary modification for diabetes, anti-convulsant drugs to control seizures, physical exercise for hypotonia, pacemaker implants for cardiac rhythm abnormalities and cochlear implants for sensorineural deafness. However, these are not a permanent solution or cure for severe disorders caused by genetic mutations or irreversible physical damage. Cardiomyopathy, failure of the heart muscle, is one of the most common symptoms of mitochondrial disorders, while cardiomyopathies almost always are associated with mitochondrial dysfunction. In severe forms of cardiomyopathy, the ultimate treatment option of heart transplantation is invasive and carries high risk, and the waiting list for a functional healthy heart is increasingly outstripping availability. I have investigated the use of a mitochondrial enzyme, the alternative oxidase (AOX), which is found in plants and lower eukaryotes, as well as in primitive metazoans but not in mammals as a new approach to better understand the underlying molecular mechanisms of diseases involving mitochondrial dysfunction. On a broader perspective, this would also aid in the development of a possible treatment for diseases associated with mitochondrial dysfunction. If catalytically engaged, AOX branches the mitochondrial ETC by oxidizing ubiquinol, which accumulates in the reduced form if the cytochrome pathway is impaired. By doing so it reduces oxygen directly to water, which is typically carried out by cytochrome oxidase, and thus maintains redox balance by recycling NADH and FADH2, two by-products of the Krebs cycle and upstream metabolism. Focusing on different types of cardiomyopathy, my strategy has been to introduce the AOX gene from the tunicate Ciona intestinalis, a close relative of vertebrates, into mouse models of disease, using genomic manipulation. I have studied and characterized two transgenic mouse models expressing C. intestinalis AOX. The first was designed to express AOX constitutively and ubiquitously. I verified this at the RNA and protein level revealing high-level expression in all tissues tested, including the heart, with the exception of the adult brain. The mice did not show any observable phenotype, making them nearly indistinguishable from their wild-type littermates under non-stressed conditions. Heart function, as measured by ejection fraction and left ventricular mass, treadmill performance and grip strength were all normal in one-year-old AOX-expressing mice. Weight was indistinguishable from that of wild-type littermates on chow or high-fat diet; although on ketogenic diet AOX-expressing mice showed a slightly mitigated weight gain, at least when caged in same-sex groups. A second AOX transgenic line was characterized, in which AOX expression was designed to be activatable by Cre-loxP-mediated removal of a STOP cassette (SNAP coding sequence, pA signal) located downstream of a CAG promoter and upstream of the AOX coding sequence. After verification of the transgenic insertion by PCR and Southern blotting, test activation by breeding to mice ubiquitously expressing Cre recombinase under the control of a β-actin promoter resulted in successful activation, based on ubiquitous expression of AOX and concomitant loss of SNAP expression. This allowed me to test the tissue-specificity of AOX-mediated modification of pathological phenotypes in mouse models of cardiomyopathy. Two different mouse models of cardiomyopathy were investigated. In the first, inflammatory cardiomyopathy was induced by overexpressing monocyte chemo-attractant protein 1 (MCP1) in the cardiomyocytes, which leads to loss of cardiac function commencing during early adulthood (12 weeks of age). Previously published data showed mitochondrial structural damage, decrease in ATP levels; and suggested induced oxidative stress as a pathological mechanism. Although the majority of oxidative stress in the cardiomyocytes of Mcp1 mice is induced by the infiltrating monocytes, via NADPH oxidase pathway, the contribution of mitochondria to the production of reactive oxygen species (ROS) is not well understood. I hypothesized that mitochondrial ROS production, especially via a defective or overloaded cytochrome pathway in the mitochondrial respiratory chain, might play a key role in the underlying molecular mechanisms. Therefore we wanted to test whether AOX could mitigate it, as it has been shown to alleviate oxidative stress under conditions where the cytochrome pathway is dysfunctional. I found that expressing AOX in this model preserved cardiac ejection fraction at 12 weeks of age but not at later time points. At the molecular level, I determined that mitochondrial complex I-linked respiration was severely affected in Mcp1-overexpressing mice irrespective of AOX expression. However, AOX preserved complex II mediated respiration. In theory, this should maintain mitochondrial redox homeostasis and limit oxidative stress and damage within mitochondria, but at the expense of ATP production and mitochondrial-ROS signaling. Ultrastructural analysis revealed mitochondrial damage in the cardiac tissue of 12 week-old Mcp1-overexpressing mice, which was alleviated by AOX expression. Despite this, AOX had no effect on the survival of Mcp1-overexpressing mice, whilst cardiomyocyte-specific AOX expression actually resulted in earlier death than Mcp1 alone. Autophagic markers were mildly elevated in all cases, and metabolic changes consistent with OXPHOS dysfunction were essentially unaffected by AOX expression in the Mcp1 mouse model. Overall, I conclude that AOX is insufficient to block lethal damage to mitochondria and/or other cellular components in this inflammatory cardiomyopathy model, despite transient beneficial effects. Previously, West et al (2011) and Mills et al (2016) reported on the significance of mitochondrial ROS in inducing macrophage activity in a LPS-induced inflammatory mouse model. Additionally, Mills et al (2016) also showed AOX expression prevented LPS-induced lethality in mice, where succinate-dependent ROS production in the ETC was reported to be the underlying cause for the observed severe phenotype. However, since AOX was unable to prevent lethality in Mcp1 mice, it is indicative that the ROS production in this inflammatory model might not be dependent on the ETC. The second model I studied was of Cox-deficient mitochondrial cardiomyopathy, induced by cardiomyocyte-specific knockout of Cox10, an essential enzyme of heme a biosynthesis. Cox10 knockout in the heart was lethal within the first days or weeks of life, and concomitant AOX expression had hardly any effect on this. Mice with heterozygous knockout of Cox10 survived for months but eventually succumbed to heart failure. However, this phenotype was produced by the cardiomyocyte-specific Cre expression alone, and was again exacerbated by AOX activation, despite transient improvements in cardiac performance in young mice. In conclusion, AOX can serve as a valuable tool to study disease mechanisms where mitochondrial dysfunction is proposed to play a key role in the pathophysiology. Although AOX did not rescue the disease models I studied, it did shed light on underlying molecular mechanisms. Considering the fact that AOX showed a partial, if transient, functional rescue in both of the heart failure models investigated, it might yet be relevant as a potential therapeutic option. Further research is necessary to fully understand the therapeutic potential of AOX, either by itself or in combination with other treatments that address different pathological processes, as well as the apparently negative effects of AOX itself during disease progression.

The Rosaceae family accounts for more than 90% of the total fresh fruit production in Finland and for more than 25% worldwide in 2013. Thus, improving the yield potential of Rosaceae species works in favor of economic growth and food security. To achieve that, expanding the knowledge on Rosaceae species is required. Fragaria vesca (F. vesca), particularly the everbearing F. vesca semperflorens accession ‘Hawaii-4’ (H4), provides several features that makes it formidable as a model organism for the study of physiological processes in Rosaceae species, where the CENTRORADIALIS/TERMINALFLOWER 1/SELF-PRUNING (CETS) genes play a remarkable role. In this Thesis, I have studied the expression patterns and functions of the CETS genes by performing gene expression analysis, generation of H4 transgenic lines and observation on H4 plants (both transgenic and wild type) grown under different sets of conditions. My results confirmed the expression patterns and functions previously reported for F.vesca TERMINAL FLOWER1 and F.vesca FLOWERING LOCUS T1. I showed that F.vesca CENTRORADIALIS-LIKE2 is a floral repressor and demonstrated that F.vesca FLOWERING LOCUS T3 is a flowering promoter. Additionally, our data suggest that F.vesca MOTHER OF FT is directly related with stolon formation and that F.vesca CENTRORADIALIS-LIKE1 is most likely a floral repressor. This new information could be used in the future to improve the efficiency of Rosaceae crops farming by adapting flowering and vegetative responses and also for breeding more productive Rosaceae crops.

Vertebrate brain is one of the most complex and mysterious objects for biological research. Embryonic brain development involves stereotypic brain structure formation, and a vast number of precise intercellular connections are established for the generation of the highly complex circuitry of the brain. This work aims at explaining HMGB1 and AMIGO1 function in modulating vertebrate brain development. Hmgb1 knockdown zebrafish morphants produced by injection of morpholino oligonucleotides display severe defects in the forebrain and gross deteriorated catecholaminergic system. The morphant is also deficient in survival and proliferation of neural progenitors. Similar central nervous system (CNS) developmental defects have been observed in HMGB1 knockout mouse embryo. The HMGB1 null mouse embryonic brain cells showed much lower proliferating and differentiating activities compared to wild type animals. HMGB1 knockdown and knockout model respectively from zebrafish and mouse have confirmed that AMIGO1 expression is directly regulated by HMGB1. AMIGO1 regulates expression of Kv2.1 potassium channel during development, but the colocalization of AMIGO1 and Kv2.1 has only been observed in mouse and zebrafish adult brain. Furthermore, knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. The same defect can be also induced by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. The impaired formation of neural circuitry is reflected in enhanced locomotor activity and attenuated escape responses. Our data demonstrate that HMGB1 is a critical factor for embryonic CNS development involved in many important developmental events. HMGB1 is essential for the neurogenesis and differentiation occurring at the developmental stage when forebrain structures are forming. Amigo1 is required for the development of neural circuits under the regulation of HMGB1. The mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. HMGB1 and AMIGO1 are both crucial for embryonic brain development and neural circuit formation.

Birth asphyxia is a major cause of infant and childhood death, disability and neurodevelopmental delay worldwide. During birth, impairment of respiration is reflected in elevated levels of CO2 and diminished levels of O2 in the neonate. The fundamental presentation and diagnostic criterion of birth asphyxia is severe acidosis, most commonly measured in umbilical blood. Resuscitation is associated with normalization of blood pH values and arterial blood gases. Within hours of a moderate or severe asphyxic insult during birth, severe seizures are triggered. In the present study, asphyxic conditions during birth are modeled as an induced hypoxia and hypercapnia in postnatal day 6 rat pups. Respiratory conditions are altered so that pups breathe 20 % CO2 with either 9 % O2 or 4 % O2 (N2 balanced) for 60 or 45 minutes, respectively. Brain extracellular and intraneuronal pH became rapidly acidotic during asphyxic conditions. After experimental asphyxia, immediate restoration of normoxia and normocapnia was associated with a large seizure burden. Seizures in the postasphyxia period were tightly correlated with a recovery and alkaline overshoot in brain pH. Enhanced acid extrusion from the brain was attributed to increased Na/H exchange across the blood-brain barrier. Pharmacologic blockade of Na/H exchange in the blood-brain barrier with amiloride or its analog abolished brain alkalosis and seizures. These findings suggest that a brain-confined alkalosis is generated by Na/H exchangers in the blood-brain barrier when normocapnic conditions are immediately restored after experimental birth asphyxia. A putative therapeutic strategy was tested, where the CO2 level of inhaled air in the postasphyxic period was reduced in steps, so that normocapnic conditions are gradually restored. This graded restoration of normocapnia was achieved by exposing the pup to 10 % CO2 in air for 30 minutes, followed by 5 % CO2 in air for a further 30 minutes, and finally with room air. A dramatic attenuation of brain alkalosis and seizures was induced by graded restoration of normocapnia. Immediate restoration of normocapnia after asphyxia was associated with adverse outcome in juvenile and adult rats, manifest as compromised sensorimotor coordination, altered emotional reactivity to acute stress, diminished inhibition of fear-motivated behavior, impaired memory and learning, abnormal social interaction, and increased seizure susceptibility. Graded restoration of normocapnia after asphyxia was associated with significant and favorable improvement of outcome, such that behavioral deficits were rescued, and seizure threshold was not significantly different from control animals. The findings of the this study suggest a central role for Na/H exchange in the blood-brain barrier in mediating the postasphyxia brain alkalosis as measured in the present study as well as in human babies. Importantly, the findings also suggest a putative therapeutic strategy in which recovery from acidosis during neonatal resuscitation is controlled through a graded restoration of normocapnia.

Parkinson s disease (PD) is a neurodegenerative disorder affecting seven to ten million people worldwide. The average age of diagnosis is 60, but some forms can affect even young adults. In the US alone the direct and indirect expenses for PD exceed $25 billion each year. PD is best characterized by the death of dopaminergic neurons of the substantia nigra pars compacta, which causes symptoms ranging from rigidity to postural instability. As the disease progresses, other areas of the brain become affected, generating psychiatric and cognitive dysfunctions. Current therapies effectively reduce motor symptoms of PD, but do not stop its progression. Neurotrophic factors regulate neuronal growth, differentiation, and survival, and several of them have been shown to protect and regenerate dopaminergic neurons in animal models of PD. The glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) have reached clinical trials, but they did not replicate the promising results of the preclinical studies. Several reasons, including stability of recombinant proteins and their diffusion in the brain tissue, could explain the results of the clinical trials. Stability might have been a problem especially with GDNF, which has been delivered mostly as bacterially-produced recombinant protein. In this work we analysed GDNF produced in mammalian cells and compared it to bacterially-produced GDNF. E. coli produced-GDNF is less stable than mammalian GDNF. This difference is probably due to the purification/renaturation method used with the E. coli-produced factor. Processing and stability of GDNF are affected also by cell line and medium used for its production. In mammalian cells glycosylation of GDNF is fundamental for its processing into the mature molecule. The diffusion problem affects both GDNF and NRTN, which do not diffuse far enough from the infusion site because of their heparin-binding properties. Heparin and the closely related heparan sulphates are abundant in the extracellular matrix and on the cell surface, and hinder the diffusion of GDNF and NRTN. The diffusion issue might not be a significant problem in the animal experiments, but might limit the results achieved with humans, who have significantly bigger brain size compared to rats and monkeys. In this work we have developed NRTN mutant variants with lower affinity for heparin and characterized their activity in vitro and in a unilateral 6-OHDA rat model of PD. All NRTN variants were biologically active. Especially the variant N4 showed better diffusion and rescued a higher number of dopaminergic fibres than E. coli-produced GDNF. Toxin-treated rats administered with N4 also showed functional recovery in behavioural assays. However, as a caveat the mutations introduced could have drawbacks influencing NRTN recycling/degradation and signalling. In this respect lack of heparin-binding could affect NRTN accumulation on the cell surface and inside the cells, therefore causing a slower initiation of the signal. Taken together our results help understanding basic features of GDNF and NRTN, such as the roles of glycosylation and of heparin binding. They also point out several important features that have to be taken into account when producing and/or modifying growth factors for clinical use, and underlines that mammalian molecules with reduced heparin binding could be beneficial for treating PD patients.

The time of arrival of interneurons and oligodendrocytes to the neocortex is critical for proper functional brain development. Aberrances in this sequence can be detrimental, and involved in different developmental diseases. Thus, understanding the mechanisms for temporal control of the genesis and migration of neural cells is crucial. The aim of this study was to focus on the ventral telencephalon, a major source of interneurons and oligodendrocytes, in more detail. A more sensitive method was developed for detecting and quantifying oligodendrocyte precursor cells, e.g. Olig2. The device decloaking chamber was compared to the microwave oven-based heat-induced epitope retrieval (HIER) method by studying the labeling of Olig2 marker in paraffin-embedded sections from embryonic mouse brain. The results demonstrated that the decloaking chamber-based HIER method is the most suitable technique for the detection of single Olig2-labeled cells in the ventral telencephalon. This qualitative result was reflected in the quantitative analyses: more Olig2-labeled cells were quantifiable with the decloaking chamber- than with the microwave oven-approach. Thus, the decloaking chamber-based HIER method constitutes a sensitive technique for the detection of oligodendrocyte precursor cells, and therefore for its quantification in the developing ventral telencephalon. The development of telencephalon depends on fundamental processes, which include proliferation and migration of neural cells. The Na-K-Cl cotransporter isoform 1 (NKCC1) is an important protein for the process of volume regulation, and has been implicated in cell division. Within the developing brain, the ventral telencephalon showed the highest expression of NKCC1. This expression corresponded to neural progenitor cells in the lateral ganglionic eminence (LGE). Using NKCC1 knockout mice, it was demonstrated that NKCC1 influenced cell cycle reentry. Consequently, mice lacking NKCC1 have impaired Sp8-expressing interneurons and Olig2-labeled cells. Thus, NKCC1 is crucial in vivo for cell cycle decision, thereby altering the production of oligodendrocyte and interneuron progenitor cells in the LGE. Once interneurons are born, they migrate to the neocortex. The implication of syndecan-3 was assessed in their tangential migration. The results showed that the Glial cell line-derived neurotrophic factor GDNF interacts with syndecan-3 to promote the tangential migration of calbindin-expressing interneurons within the telencephalon. Consistently, mice lacking syndecan-3 have an accumulation of migrating interneurons in the LGE. In summary, two important mechanisms were found for temporal and spatial control of cortical oligodendrocytes and interneurons.

In the present series of studies, contributions of descending pain facilitatory and inhibitory pathways to behavioral pain responses were assessed under physiological conditions in humans and rats, under a simulated weightlessness condition in rats, and in an experimental model of Parkinson’s disease (PD) in rats. A long-term activation of descending pain modulatory pathways was induced by noxious conditioning stimulation to allow determining whether the studied experimental procedures cause decreases or increases in the activity of descending pathways. Acute muscle nociception was induced by intramuscular injection of 5.8% hypertonic saline. Secondary mechanical hyperalgesia was used as an index of the magnitude of descending facilitation, and secondary heat hypoalgesia as an index of the magnitude of descending inhibition. To explore whether heating-needle stimulation (H.N.S.) can promote analgesia due to modulation of descending pain controls, the effect of H.N.S. on descending facilitation and inhibition was also determined in different experimental conditions of the present study. To study whether thalamic dopamine is involved in descending modulation of pain, the effects induced by microinjections of dopamine or dopamine D2 receptor antagonist into the thalamic mediodorsal (MD) / ventromedial (VM) nuclei on descending pain modulation were evaluated. Furthermore, expression of Fos, a marker of neuronal activation, was determined in the spinal cord dorsal horn to reveal a spinal neuronal correlate for descending pain modulation. In the current series of studies, simulated weightlessness and a partial depletion of dopamine in the striatum enhanced endogenous descending facilitation and depressed descending inhibition. In the spinal dorsal horn, changes in Fos expression in the superficial layers were associated with changes in descending facilitation and those in the middle/deep layers with changes in descending inhibition. Intramuscular (i.m.) H.N.S. at an innocuous temperature of 43 C triggered descending inhibition without concomitant activation of descending facilitation both in physiological conditions and in experimental model of PD. In the PD model, dopamine D2 receptors in the thalamic VM nucleus were involved in mediating the enhancement of descending pain inhibition induced by H.N.S. applied at the temperature of 43 C. In humans, i.m. H.N.S. applied at the non-painful temperature of 43 C enhanced selectively descending inhibition, and it may be explained by a difference in the density of capsaicin- and heat-sensitive fibers innervating these heating-needle stimulation targets. Injection of capsaicin that selectively activates C-fibers produced stronger pain in acupoints than in non-acupoints.