Protein folding and stability are essential for protein function. Changes in these characteristics can lead to altered physiological states and to the development of certain pathologies. While extensive research has focused on the stability of soluble proteins, membrane protein stability has received much less attention. Understanding the stability of membrane proteins can provide insight into folding mechanisms and the etiology of various pathologies. The purpose of this project is to prepare molecular tools to perform comparative studies of homologous membrane proteins that are found in various environments. To this end, thermophilic (Pyrococcus abyssi), mesophilic (Escherichia coli), and psychrophilic (Exiguobacterium 255-15) transmembrane Zn2+ transporting ATPases were cloned, expressed, and functionally characterized to correlate thermostability with optimal functional temperatures. In addition, the lipid environments and composition (rigid or fluid lipids) may also be involved in determining the stability of membrane proteins. Toward exploring the role of extremophilic lipids, Archaeoglobus fulgidus and Thermotoga maritima were grown and lipids were extracted. Availability of these molecular tools will enable physical-chemical studies toward understanding the structural factors that determine functional stability.

Intrahospitala transporter (IHT) kan rädda liv men är en logistisk utmaning och ett riskfyllt moment. IHT av intensivvårdspatienten innebär en ökad risk för patienten att råka ut för en transportrelaterad komplikation. Omvårdnad utanför intensivvårdsavdelningen och under transporter ingår i specialistsjuksköterskans arbetsområde. Sjuksköterskans omvårdnadsansvar vid transport av patienten inkluderar alla aspekter av omvårdnad, medicinsk behandling, övervakning, underhåll av apparatur samt dokumentation. Höga krav ställs på specialistsjuksköterskans kompetens och ansvar vilket påverkar både arbetsmiljö och patientsäkerhet. För att förebygga och förhindra komplikationer i samband med IHT behöver specialistsjuksköterskan ökade kunskaper. Syftet med litteraturstudien var att beskriva specialistsjuksköterskans arbetsmiljö och patientsäkerhetsarbete i samband med intrahospitala transporter. En allmän litteraturstudie genomfördes och resultatet baserades på nio vetenskapliga artiklar. Kompetensnivån inom teamet varierade och flera faktorer påverkade säkerheten i utförandet. Användandet av checklistor och riktlinjer ökar patientsäkerheten samt riskmedvetenheten hos specialistsjuksköterskan. För att upprätthålla god arbetsmiljö och patientsäkerhet i samband med IHT krävs kontinuerlig utbildning samt att checklistor och riktlinjer implementeras och efterföljs. Inom området rekommenderas mer forskning som beskriver specialistsjuksköterskans upplevelser gällande arbetsmiljö och patientsäkerhetsarbete med syfte att öka riskmedvetenheten, vårdkvalitén samt patientsäkerhetsarbetet i samband med IHT. / Intrahospital transports (IHT) can save lives but is a logistical challenge and a risky moment. The IHT of intensive care patient involves an increased risk for the patient with transport related adverse events. Responsibility of caring outside the intensive care unit and during transport is part of the specialist nurse's work. The nurse responsibility for transporting the patient includes all aspects of nursing, medical treatment, monitoring and maintenance of equipment as well as documentation. High demands are placed on the specialist nurse's competence and responsibility, which affects both the working environment and patient safety. To prevent and avoid complications associated with IHT, the specialist nurse needs increased knowledge. The purpose of the literature study was to describe the specialist nurse's work environment and patient safety work in connection with intrahospital transport. A general literature study was conducted. The result was based on nine scientific articles. Level of competence within the team was varied and several factors affected the safety and performance. Using checklists and guidelines increases patient safety and increase the risk awareness of the nurse. In order to maintain a good working environment and increase patient safety awareness in connection with IHT, continuous training is required and that implementation of checklists and guidelines to be followed is required. Within this field, more research that describes the specialist nurse's experiences current working environment and patient safety is recommended. The purpose for further research would be to increase risk awareness, health quality and patient safety work in connection with IHT.

Methylphenidate (trade name: Ritalin) resulted in a conditioned place preference, but in contrast to work in juveniles, there were no sex differences. In addition, methylphenidate produced a significant decrease in the dopamine transporter compared to controls that may have implications towards development and plasticity of the dopamine system.

Glukosetransporter spielen eine wichtige Rolle in der Versorgung des Gehirns mit Nährstoffen und somit für den Erhalt der physiologischen Zellintegrität. Glukose wird über die Blut-Hirn-Schranke (BHS) mittels spezifischen transmembranen Transportproteinen der SLC-Genfamilie (GLUT, SGLT) befördert. Dabei scheint während physiologischen Bedingungen hauptsächlich der Glukosetransporter GLUT1 (SLC2A1) für die Energieversorgung des Gehirns zuständig zu sein. Die Erforschung der SGLT-Expression ist in den letzten Jahren ein wichtiger Ansatzpunkt für neue Behandlungsstrategien vieler Erkrankungen, wie Diabetes Mellitus, maligne Neoplasien oder eines Herzinfarkts, geworden. Jedoch ist über deren Expression und Funktion im menschlichen Gehirn nur wenig bekannt. Besonders die Lokalisation entlang der BHS bleibt fraglich. Ein Großteil bisheriger Forschungsarbeiten beschäftigt sich hauptsächlich mit der Expressionsanalyse des Transporters SGLT1 im tierischen Gehirn in vivo (Poppe et al. 1997; Balen et al. 2008; Yu et al. 2013). Es konnte aufgezeigt werden, dass SGLT1 mRNA exklusiv in Neuronen und nicht an der BHS exprimiert wird. Dies wird durch in vitro Analysen einer humanen Hirnendothelzelllinie bestätigt. Demnach kann kein SGLT1 unter physiologischen Bedingungen nachgewiesen werden (Sajja et al. 2014). Im menschlichen Hirngewebe besitzen SGLTs somit keine zentrale Funktion für den Glukosetransport an der BHS. Im Gegensatz dazu konnte eine Expression von SGLT sowohl in vivo als auch in vitro während hypoglykämischen Bedingungen belegt werden (Vemula et al. 2009; Sajja et al. 2014). Die Expression der SGLT-Transporter während einer ischämischen Hypoglykämie führt zu der Annahme, dass diese Transporter für die Aufrechterhaltung der Energieversorgung des geschädigten Hirngewebes notwendig sind. Um die physiologischen Mechanismen nach einem Glukosemangel zu untersuchen, wurden SHT-Modelle etabliert (Salvador et al. 2013). In einem experimentellen Modell des Schädel-Hirn-Traumas im Rahmen eines DFG-gefördertes Projekts ist ein Expressionsverlauf von Glukosetransportern im Maushirn und in Hirnendothelzellen erarbeitet worden (Wais 2012; Salvador et al. 2015). Somit könnten SGLTs als Ansatzpunkt für den Nachweis der Überlebenszeit nach einem SHT fungieren. Die vorliegende Arbeit fokussiert sich auf die Expression der Natrium-abhängigen Glukosetransporter SGLT1 und SGLT2 im menschlichen Gehirn. Hierbei liegt das Hauptaugenmerk auf der Lokalisation dieser Transporter an der menschlichen BHS von post mortalem Hirngewebe. Weiterhin wird untersucht ob die Expressionsstärke von SGLT1 und SGLT2 eine Aussage über die Überlebenszeit von Verstorbenen nach einer traumatisch bedingten Hirnveränderung zulässt. Die Lokalisation von SGLT1 und SGLT2 an der menschlichen BHS konnte durch die Etablierung eines Protokolls zur Isolation von Hirnkapillaren erfolgen. Vorab wurden alle verwendeten Antikörper auf ihre Spezifität mittels siRNA Transfektion und Blockierung der Immunfluoreszenzsignale mittels immunisierten Peptids getestet. Somit ist die Spezifität der detektierten SGLT1- und SGLT2-Expression in menschlichen Hirnkapillaren gewährleistet. Anschließend wird untersucht, in welchen zeitlichem Verlauf nach einer traumatisch bedingten Hirnveränderung die verschiedenen Formen der Glukosetransporter exprimiert werden und ob ggf. der Umfang und die Verteilung von SGLT1, SGLT2 und GLUT1 sowie das Verhältnis zueinander Auskünfte über eine vitale bzw. postmortale Entstehung eines Traumas bzw. dessen Überlebenszeit zulässt. Hierfür wird ein Expressionsschema der Glukosetransporter generiert, abhängig von Todeszeitpunkt und Todesursache. Es konnte festgestellt werden, dass GLUT1 nicht als Target für die Ermittlung der Überlebenszeit nach einem Trauma geeignet ist. Dahingegen zeigen SGLT1 und SGLT2 eine signifikante Änderung der Expressionsstärke im contusionalen Gewebe in Abhängigkeit von der Überlebenszeit. Obwohl diese vorläufigen Daten einen neuen Ansatzpunkt für die forensische Fragestellung aufzeigen, müssen weitere Experimente mit einem erhöhten Umfang der Probenanzahl und kürzere Zeitspannen der Überlebenszeiträume durchgeführt werden. / The transport of glucose across the endothelial cells of the human blood-brain barrier (hBBB) plays a major role for energy supply of the brain and therefor for neuronal integrity. Glucose enters the brain cells through specific transmembrane transporter proteins of the SLC-gene family (GLUT, SGLT). Under physiological conditions glucose uptake across the BBB seems to be mediated primarily by facilitated diffusion through glucose transporter 1 (GLUT1). Although SGLTs are a known drug target for diabetes and furthermore play a role in other disease like cancer and cardiac ischemia, active glucose transport by SGLTs is hardly observed and very little is known about their expression or activity in human brain. Especially the function along the BBB remains uncertain. Up to now, expression analysis focused on SGLT1 and has been confirmed in vivo by analyzing brain tissue of animals (Poppe et al. 1997; Balen et al. 2008; Yu et al. 2013). Here detection mainly occurs in neurons, no SGLT1 mRNA in capillaries of the BBB could be found. Similarly in vitro experiments with a human brain microvascular endothelial cell line reveals no expression of SGLT1 under physiological conditions (Sajja et al. 2014). In human brain, SGLT1 is hardly expressed and so far could not be found along the BBB. In contrast to these findings, expression of SGLT1 could be detected in vivo as well as in vitro under hypoglycemic conditions (Vemula et al. 2009; Sajja et al. 2014). The occurrence of these transporters during ischemic hypoglycemia could lead to the conclusion that the secondary active glucose transport by SGLTs is necessary for additional glucose supply in injured brain. To investigate if SGLTs are required for the reconstruction of energy supply after glucose deficiency, traumatic brain injury (TBI) models were established to study secondary physiological mechanisms along the BBB (Salvador et al. 2013). In an experimental CCI (controlled cortical impact) mouse model within a DFG-funded project, an expression pattern of glucose transporters in the mouse brain and in brain endothelial cells has been developed (Wais 2012; Salvador et al. 2015). Thus it could lead as a Target for evidence of the time of survival after TBI. This study focuses on the sodium-dependent glucose transporters SGLT1 and SGLT2 expression in human brain. The main topic is to localize the sodium-dependent glucose transporters along the human BBB of post mortem brain tissue and to examine whether SGLT expression allow a conclusion to be drawn about the survival time of a patient after TBI. First of all the localization of SGLT1 and SGLT2 at the human BBB could be shown by establishment a capillary isolation protocol of human post mortem brain tissue. Therefore the antibody specificity was tested by a siRNA transfection protocol and blocking the immunofluorescence signal with an immunized peptide. Thus, specific SGLT1 and SGLT2 expression at the endothelial lining of the capillary lumen could be demonstrated. After attaching the value of SGLTs at the human BBB, the relationship of the glucose transporter expression in TBI tissue according to the survival time of the patient is presented. Hereby it should be clarified whether the expression and distribution of the transporters GLUT1, SGLT1 and SGLT2 as well as the relation to each other provide information on a vital or post mortal development of a trauma or its survival time. It could determine that GLUT1 is not suitable as a target for the representation of survival time after TBI. However, SGLT1 and SGLT2 show a significant change in the expression profile of traumatic brain regions. Here an increase according to the survival time after trauma can be shown. Although these preliminary data suggest a novel target for forensic questions, more experiments with an increased scope of survival time frames should be carried out.

ABC (ATP-binding-cassette)-Transporter katalysieren den ATP-abhängigen Transport diverser niedermolekularer Substanzen durch die biologische Zellmembran. Ihr Vorkommen erstreckt sich auf alle drei Domänen des Lebens. Der Maltose Transporter von E.coli gehört zu dieser Superfamilie der ABC-Transporter. Die Kristallstrukturen des Transporters MalFGK2 wurden kürzlich gelöst für dessen inaktiven Zustand als auch für dessen katalytischen Zwischenzustand. Um den Transportmechanismus besser verstehen zu können, müssen die Kristallstrukturen des Transporters und seiner Komponenten unter physiologischen Bedingungen genau geprüft werden, um den daraus katalytischen Mechanismus zu bewerten. Im rahmen der Dissertation konnte mittels Lösungs-NMR kann gezeigt werden, dass die periplasmatische Schleife P2 von MalF eine unabhängige Faltung aufweist und eine wohl definierte Tertiärstruktur einnimmt, die vergleichbar ist mit der im Kristall vorliegenden Konformation. MalF-P2 interagiert unabhängig von der Transmembranregion von MalF und MalG mit dem Maltose-Bindeprotein in An- und Abwesenheit des Substrats mit einem KD im mikromolaren Bereich. NMR Untersuchungen zu den an der Interaktion beteiligten Aminosäuren stehen in Einklang mit den Kristallstrukturdaten. Die Analyse residualer dipolarer Kopplungen (RDC) zeigt, dass die Konformation der zwei individuellen Domänen von MalF-P2 in Abwesenheit von MalE erhalten bleibt und der im Kristall ähnelt. Die Zugabe von MalE induziert eine Änderung der relativen Orientierung der zwei Domänen von MalF-P2 um so dem räumlichen Anspruch des Liganden gerecht zu werden. Besonders betroffen hiervon ist die Domäne 2 von MalF-P2, deren Konformation abweicht von der in der Kristallstruktur. Die Struktur der Domäne 1 dagegen bleibt konserviert, während sich lediglich ihre relative Orientierung zu Domäne 2 ändert. MD Simulationen des MalF-P2-MalE-Komplexes deuten auf eine stark dynamische Interaktion von MalF-P2 mit der MalE Bindungsregion hin. NMR CPMG Kinetikstudien weisen auf die Bildung eines ungewöhnlichen Knicks in alhpa-Helix alpha2 während der Assoziation hin. Diese konformelle Änderung der alpha-Helix findet auf einer Zeitskala von Millisekunden statt, was im Einklang mit der Austauschrate der Komplexbildung ist. / ABC (ATP-binding-cassette)-transporters catalyze the ATP-dependent transport of diverse solutes across the cellular membrane. They are present in all three kingdoms of life. The E.coli maltose transporter belongs to the ATP binding cassette (ABC) transporter superfamily. Recently, the crystal structures of the full transporter MalFGK2 in its resting and a catalytic intermediate state was solved. At the present state of research, it is of particular interest to scrutinize the X-ray structures of the transporter and its components under physiological conditions as well as to evaluate their implications for the catalytic mechanism. In the context of the PhD thesis, it could be shown using solution-state NMR that the periplasmic loop P2 of MalF folds independently in solution and adopts a well-defined tertiary structure, which is similar to the one found in the crystal structure. MalF-P2 interacts with the maltose binding protein, independent of the transmembrane region of MalF and MalG, with a KD in the µM range, in the presence and absence of substrate. NMR studies showed good agreement of the residues interacting in solution to those identified in the X-ray structure. Analysis of residual dipolar coupling (RDC) experiments shows that the conformation of the two individual domains of MalF-P2 is preserved in the absence of MalE, and resembles the conformation in the X-ray structure. Upon titration of MalE to MalF-P2, the two domains of MalF-P2 change their relative orientation in order to accommodate the ligand. In particular, a conformational change of domain 2 of MalF-P2 is induced, which is distinct to the conformation found in the X-ray structure. Domain 1 retains its structure but changes its relative orientation to domain 2. MD simulations of the MalF-P2 – MalE complex show a highly dynamic interaction of MalF-P2 to the MalE interface. From NMR CPMG kinetic studies, a peculiar kink of alpha-helix alpha2 can be seen introduced upon association. The transition time of this conformational change of the alpha-helix is on the ms timescale, which is matching the exchange rate of the complex formation.

The release of the neurotoxic alkaloid saxitoxin by cyanobacterial cells was previously thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export by membrane transporters. Transporter proteins associated with STX biosynthesis in Cylindrospermopsis raciborskii T3 (sxtF and sxtM) and Anabaena circinalis 131C (naDt) were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation. An sxtM homologue was discovered in A. circinalis 131C, as part of an sxt cluster, and found to be uniquely associated with STX-producing strains. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub-family and membrane topology analysis predicted 12 membrane-spanning regions. To characterise the functional substrates of the putative STX-transporters, they were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics, ciprofloxacin and ofloxacin. Disruption of gene homologues of naDt and the sxt MATE genes in Synechocystis sp. PCC6803 yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcription of the putative STX transporters, and the putative STX biosynthesis gene sxtA, was studied in C. raciborskii T3 and A. circinalis 131C under alkali and Na+ stress. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C and was correlated with a down-regulation of the putative transport and biosynthetic genes. In C. raciborskii T3, alkali stress promoted higher extracellular but lower intracellular STX levels, which also correlated with large increases in transcription of the putative STX transport genes.

<p>Road transports stand for a great part of Sweden’s total CO2-emissions. Transports are expected to increase over the coming years, and with those the emissions. A greater environmental awareness leads to increased demands for environmentally friendly transport. The aim of this thesis is to study the consequences of changing transportation modes from road to railway. The thesis contains three major parts; a literature study, a case study and analysis with the national freight system Samgods.</p><p>The literature study intends to give the reader an insight in the potentials as well the obstacles of the railway. Environmental aspects are discussed as well as political views, both positive and negative. Socioeconomic and more company specific incentives that would lead to more transfers from road to railway are presented and discussed.</p><p>A case study including two companies, Swep and KappAhl, both with relations which transfer potentials they wish to be examined. Differences in costs for today’s truck transportation are calculated and compared to train transportations. The Swedish national freight system, Samgods, is used to examine the distance relationships between closest terminals and final destinations and how intermodal solutions would be designed and the economic consequences thereof. The environmental impacts are calculated for each relation for both companies to present the saving that can be made if the transportation mode is changed, and also to show the companies their environmental impact today at a specific relation.</p><p>The result of this thesis is that KappAhl would make a great saving when it comes to the CO₂-emissions. If they are willing to increase transportation costs with 4.5 % they could reduce their CO₂-emissions with 95 % by using the railway instead of only road transportations. For Swep, the comparison between today’s transportation costs and a future cost for railway transport could not be implemented due to non suitable transportation solutions in Samgods. The environmental impact, however, was calculated for today’s transportations and a future railway solution. The result was a great saving for each of the calculated emission types. The analysis made with Samgods showed that freight transported with container is less sensitive to the distance to nearest terminal than freight transported without container. Furthermore, according to Samgods it is cost-effective to transport freight with combined transport if the distance to the final destination is more than 4.6 times the distance to the nearest terminal. The corresponding value for transports without container is 6.9 times. The relationship between nearest terminal and final destination is applicable for distances from 60 km to 300 km from the final destination.</p>

No description available.

No description available.

<p>Årsskiftet 2003/2004 trädde Lagen (2003:778) om skydd mot olyckor i kraft. Därmed upphävdes Räddningstjänstlagen. Lagtexten kräver att kommuner skall upprätta en riskhanteringsplan för tänkbara olyckstillfällen. Vänersborgs kommun har beslutat göra så och en del i detta arbete är att sammanställa en riskanalys. Denna rapport innehåller en riskanalys för farligt gods inom Vänersborgs tätort samt riksväg 45. Exempel på farligt gods är brandfarliga vätskor, frätande ämnen och gaser. Vid en riskanalys identifieras alla de risker som kan tänkas vara aktuella. Sannolikheten och konsekvenserna av dessa risker vägs samman till en risknivå, vilken kan åskådliggöras i t.ex. en riskmatris. Med risknivåerna som grund görs en riskvärdering, där storheter omvandlas till dimensionslösa värden (2 döda per 100 år blir t.ex. "låg risk"). Utifrån de värderade riskerna kan beslut tas om eventuella riskreducerande åtgärder. Inom ramen för denna rapport innebär riskerna avåkning eller kollision med läckage av gas, brandfarliga vätskor eller frätande ämnen som följd. Konsekvenserna av detta kan bli toxicitet, frätskador, brand, gasexplosion eller i extrema fall "Boiling Liquid Expanding Vapour Explosion" (BLEVE).</p>