Rationale Many studies demonstrate down-regulation of the norepinephrine transporter (NET) by desipramine (DMI) in vitro and in stress-naive rats. Little is known regarding regulation of the NET in stressed animals. Objective The present study was designed to investigate effects of DMI on the expression of NET and protein kinases in the stress rat. Methods Adult Fischer 344 rats were subjected to chronic social defeat (CSD) for 4 weeks. DMI (10 mg/kg, intraperitoneal (i.p.)) was administered concurrently with CSD or 1 or 2 weeks after cessation of CSD. Sucrose consumption, NET expression, and protein kinases were measured. Results CSD significantly increased messenger RNA (mRNA) and protein levels of NET in the locus coeruleus, as well as NET protein levels in the hippocampus, frontal cortex, and amygdala. These effects were nearly abolished when DMI was administered concurrently with CSD. CSD-induced upregulation of NET expression in the locus coeruleus, hippocampus, and amygdala lasted at least 2 weeks after cessation of CSD, an effect that was significantly attenuated by 1 or 2 weeks of DMI treatment starting from cessation of the CSD. Concurrent administration of DMI with CSD did not markedly interfere with CSD-induced decreases in protein levels of protein kinases A and C in these brain regions, but it did reverse the CSD-induced reduction in phosphorylated cAMP response element-binding (pCREB) protein levels in most brain regions. Conclusion These findings suggest that NET regulation by DMI occurs in both stressed and behaviorally naive rats, and DMI-induced changes in pCREB may be involved.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [ H]NE uptake capacity, an effect characterized by a robust decrease in the V of the transport of [ H]NE. As expected, reserpine did not displace the binding of [ H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [ H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [ H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca /Ca -calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [ H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [ H]NE uptake and eliminated the inhibitory effects of reserpine on [ H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca -independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.

The heterodimeric ATP-binding cassette (ABC) transporter, ABCG5/G8, is responsible for direct secretion of cholesterol and dietary sterols into the gut lumen and the bile. Inactivating mutations of ABCG5/G8 cause sitosterolemia, a rare autosomal recessive disease characterized by the accumulation of plant sterols in plasma, hypercholesterolemia and development of premature coronary heart disease. Functional and structural characterization of ABCG5/G8 is necessary to understand its mechanism and how the genetic defects impact its function. In this thesis, I expressed seventeen constructs of various disease-causing or catalytically deficient missense mutations in Pichia pastoris yeast. This establishes reagents for in vitro functional and structural studies. Secondly, I focused on two disease mutants (ABCG5-E146Q and ABCG8-R543S) and a sterol binding mutation (ABCG5-A540F) and established large-scale purification of these mutants. Using a cholesterol hemisuccinate (CHS)-dependent ATPase assay, I determined ATP hydrolysis by these three mutants and analyze their kinetic parameters. All missense mutants showed a significantly impaired ATPase activity, but the ability of ATP binding appeared unchanged between the WT and the mutants. This work demonstrates an intimate structure-function relationship in ABCG5/G8 and sheds some light on the mechanistic details of this important cholesterol-regulating ABC transporter.

Background The Breast Cancer Resistance Protein (BCRP) drug transporter is important for drug disposition and plays a critical role in regulating drug entry into the brain. Its substrate spectrum overlaps with substrates of Multi Drug Resistance Protein 1 (MDR1, P-gp), which influences and complicates the interpretation of data on drug distribution into tissues (e.g. brain). Distinguishing BCRP mediated transport from the transport by the MDR1 is often problematic. However, with new in vitro tools, this is now possible. In this project, two drug compounds, i.e. Dantrolene and Ritonavir, were investigated using these new in vitro models. The results from the experimental in vitro assay were matched with molecular dynamics (MD) simulations. Using coarse-grained (CG) simulations, a model of the BCRP transporter in a lipid bilayer was built, this model is based on the human BCRP structure revealed by Taylor et al (2017). Simulations were run for Dantrolene (a known substrate of BCRP) independently three times, and another with Ritonavir (a non-substrate) three times. Aim To determine substrate specificity for the BCRP transporter for two compounds, and to construct a CG model of BCRP transporter to see whether in silico methods can be used as an alternative for assessing substrate specificity.  Methods Madin-Darby canine kidney (MDCK) II cell line with no endogenous canine MDR1 (cMDR1) expression (MDCKcMDR1-KO), overexpressing human MDR1 (hMDR1) (MDCK-hMDR1cMDR1-KO) and stable expression of human BCRP (hBCRP) (MDCK-hBCRPcMDR1-KO) cells were cultured and used in Transwell experiments. Samples were analyzed using LC-MS/MS to determine the substrate concentrations. Apparent permeability and efflux ratio was calculated and evaluated.  MD simulations used the Martini 3 CG force field, and were run with Gromacs (version 2020.4). Tools including MODELLER, INSANE and others were used to construct the initial model (Webster, 2000; Wassenaar et al., 2015), for parameterization of substrate and non-substrate molecules. And visual inspection was done with the visual molecular dynamics (VMD) program and PyMOL. Results In vitro transport experiment confirmed that Dantrolene is a BCRP specific substrate, and Ritonavir is MDR1 specific substrate. Following simulations of these two compounds, Dantrolene is observed to stay in the transmembrane domains (TMD) for a certain period (on average several hundreds of nanoseconds), while Ritonavir is not found to bind in the TMD, which provides a proof of concept for future studies.

he company in the study seek to work more sustainable by minimizing transportations. They aim to reduce their CO2 emissions by 50%, independently on sold volume, by 2030, with 2019 as the year of reference. This report is about Epiroc Rock Drill AB’s sustainability goals inspired by Agenda 2030 and how their process deviations result in unnecessary transportations that negatively affect the environment with CO2 emissions. Epiroc Rock Drills AB aspires to gain an understanding of which process deviations lead to unnecessary transports and how much CO2 emissions they generate. In order to fulfil the aspiration, a description of the current situation was formed in which it is possible to distinguish in which parts of Underground's processes need of transportations appear and why. An analysis of the current situation was then needed to be able to answer the question of what process deviations affect Underground’s transportation needs and how much CO2 emissions they generate. To determine how much CO2 emissions the process deviations generated, a quantification of the CO2 emissions that emerged from process deviations in 2019 was included in the current situation analysis. The conclusions from the thesis were that the areas of lack of quality, delayed material, warehousing, quality assurance and washing, scrapping and 90-kits cause process deviations and affect Underground's transport needs and CO2 emissions. Summarized, the areas generated 3332 tonnes of CO2 emissions. The areas of lack of quality, delayed material and 90-kits caused the greatest amount of CO2 emissions which amounted to 3169 tonnes. The work carried out has created a foundation that Underground can continue to work with in the area of transport to achieve the goal of halving their CO2 emissions. The next step is to work with the process deviations that has been presented to minimize or eliminate them. / Företaget i studien strävar efter att arbeta mer hållbart genom att bland annat minimera transporter. De har som mål att år 2030, oberoende av såld volym, halvera sina CO2-utsläpp i linje med Agenda 2030 med 2019 som referensår. Denna rapport handlar om Epiroc Rock Drills AB:s hållbarhetsmål inspirerade av Agenda 2030 och hur deras processlöserier medför onödiga transporter vilket påverkar miljön negativt med CO2-utsläpp. Epiroc Rock Drills AB önskar att få förståelse för vilka processlöserier som leder till onödiga transporter och hur mycket CO2-utsläpp de genererar. För att uppfylla önskemålet framställdes en nulägesbeskrivning i vilken det går att utläsa i vilka delar av Undergrounds processer ett behov av transporter uppstår och varför. Med nulägesbeskrivningen som grund identifierades processlöserier som påverkar Undergrounds transportbehov och hur stora CO2-utsläpp de genererar. För att veta hur mycket CO2-utsläpp processlöserierna genererade inkluderades en kvantifiering av CO2-utsläppen som uppstod av processlöserier 2019 i nulägesanalysen.   Slutsatserna från examensarbetet var att områdena kvalitetsbrist, försenat material, lagerhållning, kvalitetssäkring och tvättning, skrotning och 90-kit orsakar processlöserier och påverkar Undergrounds transportbehov samt CO2-utsläpp. Tillsammans genererade områdena 3332 ton CO2-utsläpp. Områdena kvalitetsbrist, försenat material och 90-kit orsakade störst mängd CO2-utsläpp och uppgick till 3169 ton. Arbetet som genomförts har skapat en grund som Underground kan fortsätta arbeta med inom området transport för att nå målet att halvera sina CO2-utsläpp till 2030. Nästa steg är att arbeta med de processlöserier som lyfts för att minska eller eliminera dem.

Purpose: The purpose of this study was to investigate what measures the Nordic building company JM AB are using or plan to use to get points in the criterion, of the nordic eco-labeling company Svanen, that they call “Gröna Grepp”. The criterion “Gröna Grepp” aims to implement measures that benefit ecosystems, social sustainability, and energy efficiency. Methods: Three methods were used to complete the study. A literature study was used for searching facts about the subject and support why these measures are important. An interview study was made with the project managers at JM to investigate which points in the criterion they had taken or was planning to take. A survey was also performed with the general public to investigate their views of the measures in the criterion. Results: The study resulted in that 41 % of the investigated projects at JM are using their basic license which consists of solutions such as that 10% of the parking spaces must be adapted to be able to charge an electric car and at least 50% of the bicycle spaces are placed under a roof. The survey indicated that people in society were most interested in being able to charge electric cars and in being able to measure their energy consumption. But other aspects were also discovered from the survey and presented in the results. Conclusions: The conclusion is that the criterion Gröna grepp contributes in a positive way to several aspects. And that one should therefore invest more in Gröna grepp in the future. Taken together, one should invest more in implementing ecosystems and greenery in cities through well-planned gardens or growing boxes.

Transporter har en avgörande roll för den globala ekonomin och samhällets viktiga funktioner. Dessvärre har transportsektorn en stark koppling till de globala klimatförändringarna. I Sverige står inrikes transporter för ungefär en tredjedel av landets totala växthusgasutsläpp, för att minska utsläppen och uppnå nationella klimatmål krävs en omställning av transportsystemet som idag till stor del är beroende av fossila bränslen. För att uppnå en förändring av transportsystemet krävs det att flera olika aktörer på olika nivåer i samhället samverkar och arbetar mot samma mål. I Gävleborgs län är transportsektorn den största utsläppskällan av växthusgaser, länet har ambitionen att minska utsläppen från transporter och arbetar bland annat för att skapa ett fossilfritt transportsystem innan år 2030. I detta arbete har målet varit att bedöma vilka möjligheter små- och medelstora företag (SME-företag) har för att bidra till omställningen och till att nå klimatmålen för transportsektorn. Vidare har syftet varit att sammanställa och granska vilka förutsättningar som finns för SME-företagen i Gävleborgs län att välja förnybara drivmedel (inklusive el) och alternativa fordon vid inköp och leasing. För att uppnå syftet har kvalitativa intervjuer utförts med 6 SME-företag i länet. SME-företagens möjligheter att bidra till omställningen beror på flera olika faktorer såsom den nationella och regionala marknaden för alternativa fordon och drivmedel samt olika former av styrmedel. Resultatet visar att flera av företagen som inkluderats i detta arbete någon gång har diskuterat kring möjligheten att investera i alternativa fordon och drivmedel och anser dessutom att sådana investeringar kan skapa konkurrensfördelar för företagen, både idag och i framtiden. Däremot finns det hinder som hämmar omställningen. Ekonomiska aspekter utgör i dagsläget ett stort hinder. Ekonomiska stöd kan därför utgöra viktiga förutsättningar men resultatet visar att det råder brist på kännedom gällande sådana stöd och därmed krävs åtgärder för att på ett effektivare sätt kommunicera dessa stöd till företagen i länet. Vidare är företagen främst intresserade av elfordon men uppger samtidigt att det finns hinder i form av ett begränsat utbud av fordon, begränsad räckvidd samt bristande laddningsmöjligheter. Företagen tror att det kommer krävas både teknisk utveckling och högre krav från intressenter för att fler SME-företag ska satsa på alternativa fordon och drivmedel i framtiden, vidare menar företagen att sådana investeringar även måste vara ekonomiskt fördelaktiga.  Nyckelord: Alternativa fordon och drivmedel, Gävleborgs län, SME-företag, Transporter. / Transport plays a vital role in the global economy and the important functions of society. Unfortunately, the transport sector is strongly associated with climate change and is often pointed out as an obstacle to individual countries' ability to achieve international climate agreements and other climate goals. In Sweden, domestic transport accounts for about a third of the country's total greenhouse gas emissions, reducing emissions and achieving national climate goals requires a transition of the transport system which today is largely dependent on fossil fuels. In order to achieve a change in the transport system, it is required that several different actors at different levels in society collaborate and work towards the same goal. In Gävleborg County, the transport sector is the largest source of greenhouse gas emissions, the county has the ambition to reduce emissions from transport and create a fossil-free transport system by 2030. The aim of this work has been to assess small and medium-sized enterprises (SMEs) opportunities to contribute to the transition and to achieving the climate goals for the transport sector. Furthermore, the purpose of this work has been to compile and examine the prerequisites for SMEs in Gävleborg County to choose renewable fuels and alternatively vehicles when purchasing and leasing vehicles. To achieve this, qualitative interviews were conducted with 6 SMEs in the county. The possibilities for SMEs to contribute in the transition depend on several different factors such as the national and regional market for alternative vehicles and fuels and furthermore different types of policy regulations. The results show that several of the companies involved in this work have discussed the possibility of investing in alternative vehicles and fuels and believe that such investments can create competitive advantages, both today and in the future. However, there are obstacles that delay the transition of which economic barriers/financial aspects are a major obstacle. Financial support can therefore be an important prerequisite, however, there is a lack of knowledge regarding such support and thus measures are required to communicate these supports to the SMEs in a more efficient way. Companies are mainly interested in electric vehicles but state that there are obstacles in the form of limited supply of vehicles, limited reach and lack of charging options. The companies believe that both technical development and higher demands from stakeholders will be required in order for more SMEs to invest in alternative vehicles and fuels in the future, furthermore, the SMEs believe that such investments must also be economically advantageous. Keywords: Alternative vehicles and fuels, Gävleborg County, Small and medium-sized enterprises, Transport sector.

No description available.

Objective: Degeneration of dopaminergic neurons in the substantia nigra projecting to the striatum is responsible for the motor symptoms in Parkinson’s disease (PD). Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a well-established procedure to alleviate these symptoms in advanced PD. Yet the mechanism of action, especially the effects of STN-DBS on the availability of striatal dopamine transporter (DAT) as a marker of nigrostriatal nerve cell function, remains largely unknown. The aim of our study was therefore to evaluate whether 1) DAT availability changes within one year of STN-DBS and whether 2) the clinical outcome is predictable by DAT availability before surgical procedure (pre-op). Methods: Twenty-seven PD patients (age: 62.7 ± 8.9 years (y); duration of illness: 13.0 ± 4.9y; PD subtypes: akinetic-rigid n=11, equivalence n=13, tremor-dominant n=3) underwent [123I]FP-CIT single-photon emission computed tomography (SPECT) pre-op and one year after STN-DBS (post-op). DAT availability (specific-to-unspecific binding ratio, SBR) was assessed by volume of interest (VOI) analysis of the caudate nucleus and the putamen ipsilateral and contralateral to the clinically more affected side. Results: 1) Unified Parkinson’s Disease Rating Scale (UPDRS) III (pre-op on: on medication; pre-op off: off medication; post-op on/on: on medication/on stimulation; post-op on/off: on medication/off stimulation) improved significantly (pre-op on: 25.6 ± 12.3, pre-op off: 42.3 ± 15.2, post-op on/off: 41.4 ± 13.2; post-op on/on: 16.1 ± 9.4; pre-op on vs. post-op on/on: p = 0.006) while L-dopa equivalent daily dose (LEDD) was reduced (pre-op 957 ± 440 mg, post-op 313 ± 189 mg; p < 0.001). SBR did not differ significantly before and one year after DBS, regardless of PD subtypes. 2) Pre-op DAT availability was not related to the change in UPDRS III but the change in DAT availability was significantly correlated with the change in UPDRS III (contralateral head of the caudate VOI: p=0.014, contralateral putamen VOI: p=0.018). Conclusion Overall, DAT availability did not change significantly after one-year of STN-DBS. However, on an individual base, the improvement in UPDRS III was associated with an increase of DAT availability while DAT availability before STN-DBS surgery did not predict the clinical outcome. Whether a subtype-specific pattern of pre-op DAT availability can become a reliable predictor for successful STN-DBS has to be evaluated in larger study cohorts.:Introduction 2 1.1 Parkinson’s Disease Pathophysiology 2 1.2 Parkinson’s Disease Clinical Manifestation 4 1.2.1 Parkinson’s Disease Diagnosis 5 1.2.1.1 Unified Parkinson’s Disease Rating Scale 5 1.2.1.2 Imaging 6 1.2.2 Parkinson’s Disease Subtypes 6 1.3 Parkinson’s Disease Therapy 7 1.3.1 Pharmacologic Therapy 7 1.3.2 Surgical Therapy – Deep Brain Stimulation 9 1.3.2.1 Patient Selection 9 1.3.2.2 Operative Technique 9 1.3.2.3 Efficacy 10 1.3.2.4 Complications 11 1.3.2.5 Mechanism of action 11 2 Publication 15 3 Summary of Work 23 3.1 Background 23 3.2 DAT availability changes after STN-DBS 24 3.3 Pre-op DAT availability predicts the clinical outcome 25 3.4 DBS has a neuroprotective effect 25 3.5 Limitations and future direction 26 3.6 Conclusion 26 4 References 27 5 Attachments 35 5.1 Index of Abbreviations 35 5.2 List of figures 36 5.3 Academic Contribution 37 5.4 Declaration of the independent writing of this thesis 39 5.5 Declaration of Submission 40 5.6 Curriculum Vitae 41 5.7 Acknowledgements 43