BACKGROUND: The neurotransmitter noradrenaline (NA) mediates arousal, attention and mood and exerts anti-inflammatory and neuroprotective effects. Its projections reach hypothalamic nuclei which regulate the neuroendocrine stress response. Changes in noradrenergic signalling were reported in multiple sclerosis (MS) and psychiatric illness and may account for the high prevalence of comorbid depression and fatigue in MS patients. Associated studies of our study group—investigating stress response in obese and non-obese subjects—have shown increased activity of the stress axes including an association between hypothalamic-pituitary-adrenal (HPA) axis responsiveness and central noradrenaline transporter. OBJECTIVES: (i) To evaluate central NA transporter (NAT) availability in vivo in immunotherapy-naïve relapsing-remitting multiple sclerosis (RRMS) patients compared to healthy controls (HC), (ii) to measure hypothalamic-pituitary-adrenal (HPA) axis responsiveness and the arginine-vasopressin surrogate (AVP) copeptin in patients with RRMS and clinically isolated syndrome (CIS) compared to HC, (iii) to test whether HPA axis responsiveness is differentially associated to NAT availability in RRMS patients and HC. METHODS: 22 patients (11 RRMS, 11 CIS) were enrolled and compared to 22 sex- and age-matched HC. (i) Positron emission tomography (PET) was performed in 11 RRMS and 12 HC applying the NAT-selective radiotracer S,S-[11C]O-methylreboxetine ([11C]MRB) for intergroup comparison. (ii) All patients underwent the combined dexamethasone/corticotropin releasing hormone (dex/CRH) test. Plasma ACTH and cortisol curve parameters, and copeptin after dexamethasone intake were derived. (iii) MRB-PET imaging data were correlated to curve indicators and copeptin obtained from the dex/CRH test in RRMS patients. RESULTS: (i) RRMS patients show increased NAT availability in almost all subcortical regions, reaching statistical significance in the thalamus, amygdala, putamen and pons/midbrain. No association with clinical or psychometric variables was found. (ii) Immunotherapy-naïve RRMS patients show no significant changes in cortisol, ACTH or copeptin indices. (iii) There is no correlation between HPA axis indicators and NAT availability in RRMS patients. In HC, NAT availability correlated positively with cortisol curve indicators. CONCLUSION: This study supports the evidence for increased NAT availability in immunotherapy-naïve RRMS patients compared to HC. The increased NAT availability was shown in the subcortical brain regions (relevant to attention and emotional regulation) of the RRMS patients. In this cohort, no correlation with physical or psychometric scores was found. It will be further of interest, if these NAT changes longitudinally predispose to the psychiatric comorbidities which are frequently seen in MS patients or if they do in larger, more heterogenous sample sizes. Our cohort of early RRMS and CIS did not display a statistically significant alteration in the HPA axis responsiveness compared to HC. No association between NAT availability and HPA axis responsiveness could be detected in RRMS patients.:TABLE OF CONTENTS LIST OF ABBREVIATIONS…………………….………………………………………......4 LIST OF FIGURES…..….………………………….….……..…………………………..…5 I BIBLIOGRAPHIC DESCRIPTION…………………………..……………………………6 II INTRODUCTION…..…..………………………………………………………….............7 2.1 Multiple sclerosis — Background and scope……………....…………………..........7 2.1.1 Diagnostic criteria, subtypes and clinical features……….............….…………....7 2.1.2 Multiple sclerosis and its impact on daily life: fatigue.…...…...….............………9 2.2. Noradrenaline — neurotransmitter and immunomodulator…...…………….........10 2.2.1 Noradrenaline in the context of multiple sclerosis…………………….................11 2.2.2 Noradrenaline in the context of neuroinflammation and neurogenesis.............12 2.3 Noradrenaline transporter as regulator of noradrenergic transmission….…........13 2.3.1 Noradrenaline transporter imaging……………………………............................14 2.4 Neuroendocrine stress response……...…..……………………..……..……….......14 2.4.1 Noradrenaline in the context of stress response regulation…...........................15 2.4.2 Stress axis regulation in multiple sclerosis……….............……….....................16 III METHODS……………......……………………………………………………..……....19 3.1 Objectives and hypotheses.....……..…………………………………………….......19 3.2 Study design………………..….....………………………………….…..…………….20 3.3 Hypothalamic-pituitary-adrenal axis assessment using the combined dexamethasone/CRH test…….……...........……….....................................................21 3.4 Questionnaires……...…………………….....…………………………………………22 3.4.1 Beck-Depression-Inventory……………….............……….………………………22 3.4.2 Würzburger Erschöpfungsinventar bei MS….…………..….............……………22 3.5 PET imaging, imaging data processing and analysis………………….....………..23 3.6 Statistical analysis………………….………………………………………….....……23 IV RESULTS………….......……………………………………………….........................24 4.1 Changes of central noradrenaline transporter availability in immunotherapy-naïve multiple sclerosis patients – Publication….....……...…………24 4.2 HPA axis responsiveness does not differ between HC and RRMS or CIS patients…...………………………………...……………………………………….25 4.3 In RRMS patients, noradrenaline transporter availability of selected brain regions does not correlate with neuroendocrine indicators of stress responsiveness, but do positively correlate in healthy controls………………..….…..25 V SUMMARY………………….…………………………………………………………….31 5.1 Significantly changed noradrenaline transporter availability in RRMS patients in brain regions relevant to attention, vigilance and mood………….............31 5.2 Noradrenaline transporter availability is not significantly associated with psychometric and physical scores……..…………………...........................................32 5.3 HPA responsiveness does not significantly differ between early-stage RRMS patients, CIS patients and healthy controls………..…………………..............32 5.4 NAT DVR of selected brain regions do not reveal a significant association to HPA response in RRMS patients, but in healthy controls………...……..................33 5.5 Limitations..………………………………………………………………………….....35 5.6 Future directions…………………………………………………………………….....35 VI PUBLICATION BIBLIOGRAPHY…….……………………....…………….................36 VII ANHANG…….………..…...…..………..………………………………………………49 7.1 Publikationen…………..……….....…..……………………………………................49 7.1.1 Publikationen als Ko-Autorin…...…………..………..............…………………….50 7.1.1.1 Central noradrenaline transporter availability is linked with HPA axis responsiveness and copeptin in human obesity and non-obese controls……..50 7.1.1.2 Post-dexamethasone serum copeptin corresponds to HPA axis responsiveness in human obesity...............................................................................51 7.2 Erklärung zum wissenschaftlichen Beitrag der Promovendin zur Publikationspromotion…………...........………………………………………………52 7.3 Erklärung über die eigenständige Abfassung der Arbeit...…....…………...…......53

Corticotropin releasing factor (CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter (NET) in vitro. SK-N-BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [3H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status.

Glucocorticoids affect cellular and molecular events in brains by modulating the expression of many genes during stress. In the present study, we examined the regulatory effect of corticosterone on the expression and function of the norepinephrine transporter (NET) in vitro. The results show that exposure of SK-N-BE(2)C cells to corticosterone for 14 days significantly increased mRNA (up to 43%) and protein (up to 71%) levels of NET in the concentration- dependent manner. Longer exposure (21 days) resulted in greater increases in the levels of mRNAs (up to about 160%) and proteins (up to about 250%) of the NET. The up-regulatory effect of corticosterone on NET expression lasted a persistent period after cessation of exposure. Associated with the corticosterone-induced enhancement in NET expression, there was a parallel increase in the uptake of [3H]norepinephrine by SK-N-BE(2)C cells. Increased NET expression and function were abolished after exposure of cells to corticosterone in combination with mifepristone or spironolactone, two specific antagonists of corticosteroid receptors. This is consistent with the hypothesis that corticosterone-induced NET up-regulation is mediated by corticosteroid receptors. Nevertheless, there was no synergistic effect for a combination of both corticosteroid receptor antagonists. A similar up-regulation of NET protein levels was also observed after exposing PC12 cells to corticosterone. The present findings demonstrate that corticosterone up-regulates the expression and function of NET in vitro, indicating the action of corticosterone on the noradrenergic phenotype may play an important role in the correlation between stress and the development of depression.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [ 3H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.

Protein-mediated sugar transport is nominally absent in normoxic (adequately oxygenated) pigeon erythrocytes. Following exposure to metabolic inhibitors (cyanide or carbonylcyanide-p-trifluoromethoxyphenylhydrazone), pigeon red cells transport sugars by a saturable, stereoselective pathway that is inhibited by cytochalasin B or forskolin. The sugar transport capacity of fully poisoned cells is consistent with a transporter density of approximately 30 carriers per erythrocyte. Immunoblot analyses and competition ELISA indicate that pigeon red cells contain approximately 200 copies of an integral plasma membrane protein immunologically related to the glucose transporter isoform GLUT1. GLUT1 is quantitatively restricted to the plasma membrane at all times. Pigeon red cells and brain lack proteins immunologically related to the sugar transporter isoforms GLUT3 and GLUT4. Specific immunodepletion of red cell GLUT1 content results in the subsequent loss of reconstitutable protein-mediated sugar transport. These findings demonstrate that avian erythrocyte sugar transport is mediated by a GLUT1-like sugar transport protein and that sugar transport stimulation by metabolic inhibitors results from derepression of cell surface sugar transport proteins. Lysis-resealing experiments suggest that derepression is a glutathione (OSH) dependent phenomenon. This mechanism of transport regulation contrasts with insulin stimulation of sugar transport in muscle and adipose tissue which is believed to result from recruitment of intracellular sugar transporters to the plasma membrane.

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive, encapsulated bacterium capable of causing significant morbidity and mortality throughout the world. A hallmark of S. pneumoniae infection is infiltration of neutrophils (PMNs) that assist in controlling the spread infection but may also contribute to pathology. Paradoxically, studies have shown that limiting PMN infiltration into the lumen of the lung during infection actually betters clinical outcome in experimental S. pneumoniae infection. The final step in PMN luminal trafficking is a Hepoxilin A3 (HXA3)-dependent migration across the pulmonary epithelium. HXA3 is a PMN chemoattractant that forms gradients along the polarized epithelial face, drawing PMNs from the basolateral to the apical surface during proinflammatory responses. HXA3 requires assistance of an integral- membrane protein transporter to escape the cell and form the gradient. The pulmonary HXA3 transporter is currently unidentified. In this work, we identify the pulmonary HXA3 transporter as the ATP-Binding Cassette Transporter (ABC transporter) Multi-drug Resistance Associated Protein 2 (ABCC2, MRP2). We demonstrate that MRP1 and MRP2 are divergent ABC- transporters that control transepithelial PMN migration through efflux of a distinct anti-inflammatory substance and the pro-inflammatory HXA3 in the context of Streptococcus pneumoniae infection. Enrichment of MRP2 on the plasma membrane requires detection of the bacterial virulence factors pneumolysin (PLY) and hydrogen peroxide. PLY and hydrogen peroxide not only coordinate MRP2 apical membrane enrichment but also influence HXA3-dependent PMN transepithelial migration. They influence migration through stimulation of epithelial intracellular calcium increases that are crucial for HXA3 production as well as MRP2 translocation to the plasma membrane. PLY and hydrogen peroxide are not sufficient in their signaling alone, however, and require at least one additional bacterial signal to induce HXA3/MRP2 proinflammatory activities.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18326号 / 農博第2051号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4833(農学部図書室) / 31184 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

In an age where sustainability and reduced carbon dioxide emissions are highly prioritized, the transport sector faces one of its biggest challenges. Namely, reducing the environmental impact of heavy transport. In the search for solutions to make heavy transport more sustainable, electric roads and stationary charged trucks have emerged as promising alternatives. By replacing diesel trucks with electric alternatives, the solutions offer environmental benefits and contribute to Sweden's longer-term ability to achieve its climate goals. Increasing the speed of change is critical to accelerating the implementation of electrification and maximizing its positive effects on the environment and society. Therefore, it is of great interest to investigate the factors that influence the conversion process, as well as identify strategies and measures to promote a faster transition to electrified heavy transports.With this background, this study aims to investigate conditions and challenges for increasing the conversion rate of heavy transports. The study aims to gain a better understanding of the haulage companies' needs and preferences regarding the transition. Through semi-structured interviews with respondents from ten companies with varying needs, the results could be obtained, and several insights into the research question could be obtained. The analysis of the interviews, using Rogers' theory regarding the diffusion of innovation, gave rise to several barriers and driving forces for the change. The conditions that need to be created to increase the speed of the transition have then been based on the barriers and the respondents' answers. The results indicate that the speed of conversion to the electrification solutions in Sweden is controlled by the speed of adoption of the newly introduced technologies. Adoption, in turn, is affected by a series of driving forces and barriers. It is the barriers that prevent haulers from adopting the solutions. For electric roads, it has been established that the solution may be less interesting depending on the type of traffic that a company conducts. There is also uncertainty about whether electric roads will be built. Increased communication efforts by the authorities are required to spread knowledge about electric roads and address the uncertainty of potential users. When it comes to stationary charging, an electrified truck needs to achieve cost neutrality compared to a diesel truck. This can be achieved through financial support for the expansion of the charging infrastructure, subsidies, and financing services such as leasing. An additional prerequisite for increasing the adoption rate is forecasts and guarantees regarding electricity supply and electricity prices in the future. The technology for stationary charging needs to be developed further to meet the varying needs of companies in the market. The needs regarding the technology may differ from one company to another depending on the type of traffic and the goods being delivered. In order to speed up the conversion of the heaviest transports over 60 tons, the vehicles need to have reliable battery capacity. It is also necessary to offer a well-developed charging infrastructure to facilitate the electrification of all three types of transports, namely local, regional, and long-distance transport. Furthermore, knowledge-raising efforts by the authorities are required to counteract uncertainties regarding policies and future solutions. This study suggests initiatives such as the “effektkommissionen” of Region Skåne as a suitable communication channel between the actors involved. The initiative has the potential to address various uncertainties and make the solution more visible to potential users. By creating the proposed conditions, an increased rate of adoption can be achieved, which in turn, leads to Sweden fulfilling its climate goals. / I en tid där hållbarheten och minskade koldioxidutsläpp är högt prioriterade står transportsektorn inför en av sina största utmaningar, nämligen att minska miljöpåverkan från tunga transporter. I jakten på lösningar för att göra tunga transporter mer hållbara, har elvägar och stationärt laddade lastbilar framträtt som ett lovande alternativ. Genom att ersätta diesellastbilar med eldrivna alternativ erbjuder elektrifieringslösningarna betydande miljöfördelar, samt bidrar till att Sverige på längre sikt kan uppnå sina klimatmål. Att öka omställningshastigheten är avgörande för att påskynda implementeringen av elektrifieringen, samt maximera dess positiva effekter på miljön och samhället. Därför är det av stort intresse att undersöka faktorerna som påverkar omställningsprocessen, samt identifiera strategier och åtgärder för att främja en snabbare övergång till elektrifierade tunga transporter. Mot bakgrund av detta syftar denna studie till att undersöka möjliga förutsättningar och utmaningar för att öka omställningshastigheten för tunga transporter. Denna studie ämnar till att få bättre förståelse för åkeriföretagens behov och preferenser kring omställningen. Genom semistrukturerade intervjuer med respondenter från tio företag av varierande behov kunde resultaten erhållas och flera insikter på forskningsfrågan kunde fås. Analysen av intervjuerna med hjälp av Rogers teori kring diffusion av innovation gav upphov till flera barriärer och drivkrafter för omställningen. Förutsättningarna som behöver skapas för att öka omställningshastigheten har sedan baserats på barriärerna och respondenternas svar. Resultaten tyder på att omställningshastigheten till elektrifieringslösningarna i Sverige styrs utav adoptionshastigheten av dem nyintroducerade teknologierna. Adoptionen i sin tur påverkas av en rad drivkrafter och barriär. Det är framför allt barriärerna som utgör hindren för åkeriernas adoption av lösningarna. För elvägar har det konstaterats att lösningen kan vara en mindre intressant lösning beroende på typen av trafik som ett företag utför. Det råder dessutom en osäkerhet kring huruvida elvägar kommer att byggas. Det krävs ökade kommunikations insatser av myndigheterna för att sprida kunskaperna om elvägar och adressera osäkerheten hos potentiella användare. När det gäller stationär laddning behöver en elektrifierade lastbil uppnå kostnadsneutralitet jämför med en diesellastbil. Detta kan uppnås genom ekonomiskt stöd till utbyggnaden av laddinfrastrukturen, subventioner samt finansieringstjänster som leasing. En ytterligare förutsättning för att öka adoptionen är prognoser och garantier gällande elförsörjningen samt elpriserna i framtiden. Teknologin för stationär laddning behöver utvecklas vidare för att möta företagens varierande behov i marknaden. Behoven när det gäller teknologin kan skilja sig från ett företag till ett annat beroende på typen av trafik samt godsen som levereras. För att påskynda omställningen av de tyngsta transporterna över 60 ton behöver fordonen ha en tillförlitlig batterikapacitet. Det är dessutom nödvändigt att erbjuda en väl utbyggd laddinfrastruktur för att möjliggöra elektrifieringen av samtliga tre typer av transporter, nämligen lokal, regional, och fjärrtransporter. Vidare krävs kunskapshöjande insatser av myndigheterna för att motverka osäkerheter gällande styrmedel och framtida lösningar. Denna studie föreslår initiativ såsom effektkommissionen av Skåne Region som en lämplig kommunikationskanal mellan de involverade aktörerna. Initiativet har potentialen att adressera olika osäkerheter och göra lösningen mer synlig. Genom att skapa de föreslagna förutsättningarna kan en ökad omställningshastighet uppnås vilket i sin tur leder till att Sverige uppfyller sina klimatmål och ambitionen om att vara ett föregångsland.