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Klimatkrisen är ett problem som behöver angripas från flera områden. Ett område med stor negativ miljöpåverkan i Sverige är transportsektorn och i synnerhet har godstransporter en stor påverkan. I takt med att konsumtionen i samhället ökar för varje år så ökar även godstransporterna kopplade till detaljhandeln i samma  takt, vilket leder till ökade utsläpp och negativ miljöpåverkan. Detaljhandeln har även krav på att erbjuda deras konsumenter en sömlös handel där de fritt ska kunna handla och returnera i olika kanaler, vilket leder till ökad komplexitet i deras logistiknätverk. För att minska utsläppen från godstransporterna måste detaljhandelsföretagen använda mer hållbara transporter genom bland annat mer miljövänliga fordon, bränslen och bättre transportplanering.   Eftersom detaljhandelsföretag i stor utsträckning tar hjälp av tredjepartslogistiker med deras allt mer komplicerade logistiklösningar så det upp till båda två parterna att tillsammans utveckla hållbara transporter. En litteraturgenomsökning visade att det fanns behov av mer forskning med fokus på samarbetet mellan detaljhandelsföretag och tredjepartslogistiker. Detta eftersom detaljhandelsföretaget är nära konsumenterna vilket visat sig leda till att deras logistik har stor negativ effekt på miljön. Denna studie har därav för avsikt att besvara följande problemformulering: Hur kan samarbetet mellan svenska detaljhandelsföretag med omnistrategi och deras tredjepartslogistiker utvecklas med fokus på hållbara transporter? Denna studie genomförde fallstudier på detaljhandelsföretag och tredjepartslogistiker för att undersöka och skapa en djupare förståelse för vilka utmaningar och möjligheter som finns hos de två parterna till att utveckla hållbara transporter tillsammans samt vilka drivkrafter de har för att etablera dem. Studien har en abduktiv ansats där den teoretiska referensramen behandlar teorier och tidigare studier inom hållbara transporter, intressenters påverkan, inköpsprocessens utvärdering och uppföljning samt hur kommunikation och motivation påverkar ett samarbete. Studien genomförde kvalitativa semistrukturerade intervjuer med några av Sveriges största detaljhandelsföretag och 3PL-företag.   Respondenternas svar tyder på att den största drivkraften till att utveckla hållbara transporter är samhällets krav på hållbarhet, men även att konkurrenter blir viktigare att ta hänsyn till, i synnerhet för tredjepartslogistiker. Det framkom att det fanns skillnad mellan större och mindre tredjepartslogistiker där detaljhandelsföretagen upplevde mindre aktörer som enklare att samarbeta med. Satsningar på  IT-system är nödvändiga för att skapa bättre möjligheter för utvärdering och uppföljning av hållbara transporter, men även för att möjliggöra ett mer standardiserat sätt att mäta och rapportera CO2-utsläpp. Samarbetet kan förbättras genom fler gemensamma projekt mellan detaljhandelsföretaget och deras tredjepartslogistiker, detta för att förbättra relationen samt öka motivationen hos den part som är mindre motiverad till att utveckla hållbara transporter. De utmaningar som kvarstår är konsumentens ovilja att betala för hållbara transporter samt begränsningar inom teknologi och infrastruktur för fossilfria fordon.

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23843号 / 薬博第850号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 木村 郁夫, 教授 中山 和久, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

<p>Pap-Pit is a low affinity phosphate transporter found in <em>S. meliloti</em> and many other microorganisms. Pit is the transporter and Pap is the Pit accessory protein. Pap has been shown to be required for the function of Pap-Pit system in <em>S. meliloti</em>. In this study, <em>pap-pit</em> or <em>pit</em> alone from three species of bacteria have been expressed <em>in trans</em> in the <em>E. coli</em> Pi uptake mutants to check their ability to complement the Pi uptake deficiency of the hosts. A visualization tag, SNAP-tag, has been fused to <em>S. meliloti</em> Pap to help determine the subcellular localization of Pap. Here we show that there is an optimal level of Pap-Pit in the cells, and Pap appears to modulate this level to optimize the function of the system. We also demonstrate that Pap is probably localized intracellularly along the cell membrane. In addition, a <em>S. meliloti pap-pit </em>deletion strain has been prepared and to be used as the background strain for site-directed mutagenesis in Pap. The highly conserved surface amino acids in Pap have been identified to be the candidates for the site-directed mutagenesis.</p> / Master of Science (MSc)

This dissertation is focused on the application of Markov State Models on protein folding and designing of small drug-like molecules, as well as application of computational tools on the study of biological processes. The central focus of protein folding is to understand how proteins obtain their unique three-dimensional structure from their aminoacid sequences. The function of protein critically depends on its three- dimensional structure; hence, any internal (such as mutations) or external (such as high temperature) perturbation that obstructs three-dimensional structure of a protein will also interfere with its function. Many diseases are associated with inability of protein to form its unique structure. For example, sickle cell anemia is caused by a single mutation that changes glutamic acid to valine. Molecular dynamics (MD) simulations could be utilized to study protein folding and effects of perturbations on protein energy landscape; however, due to its inherent atomic resolution, MD simulations usually provide enormous amount of data even for small proteins. A thorough analysis and extraction of desired information from MD provided data could be extremely challenging and is well beyond human comprehension. Markov state models (MSMs) are proved to be apt for the analysis of large scale random processes and equilibrium conditions, hence it could be applied for protein folding studies. MSMs can be used to obtain long timescale information from short timescale simulations. In other words, the combination of many short simulations and MSMs is a powerful technique to study the folding mechanism of many proteins, even the ones with folding times over millisecond. This dissertation is centered on the use of MSMs and MD simulation in understanding protein folding and biological processes and is constructed as the following. The first chapter provides a brief introduction into MD simulation and the different techniques that could be used to facilitate simulations. Protein folding and its challenges are also discussed in chapter one. Finally, chapter one ends with describing MSMs and technical aspects of building them for protein folding studies. Chapter two is focused on using MD simulations and MSMs to design small protein like molecules to prevent biofilm propagation by disrupting its lifecycle. The biofilm lifecycle and strategy for its interruption is described first. Then, the designed molecules and their conformational sampling by MD simulations are explained. Next, the application of MSMs in obtaining and comparing equilibrium population of all designs are discussed. At the end of chapter two, the molecular descriptions of best designs are explained. Chapter three is focused on the effects of mutations on the energy landscape of a sixteen residue protein from c-terminal hairpin of protein G, GB1. Three mutations, tz4, tz5, and tz6 are discussed, and their folding rates and folding mechanisms are compared with wild-type GB1 using MSMs built from a significantly large MD simulation data set (aggregating over 9 millisecond). Finally, chapter four is focused on the application of MD simulations on understanding the selectivity of Na,K-ATPase, a biologically critical protein that transports sodium ions outside and potassium ions inside against their concentration gradient in almost all eukaryotic cells. Multiple MD approaches, including metadynamics and free energy perturbation methods are used to describe the origins of selectivity for Na,K-ATPase. / Chemistry

Phosphate (PO4-3 or Pi) is an essential molecule necessary for sustaining life and it plays important roles in nucleic acid and cell membrane integrity. However, phosphate is found in growth-limiting concentrations in most environments. Bacteria have developed a diverse set of transport systems to uptake and scavenge phosphate from their environment for use in cellular processes. In the soil bacterium, Sinorhizobium meliloti, one such Pi transport system is the Pap-Pit system. Pit is a membrane transporter for Pi and is associated with a cytosolic protein of unknown function known as Pap (Pit-accessory protein). Interestingly, the stop codon of pap overlaps with the start codon of pit by a single nucleotide. In previous work, the pap gene appeared to be required immediately upstream of pit in an operon for functional Pi transport. Thus, in a pap deletion mutant, when pap is present in trans, there is no Pi transport. This suggests a possible translational coupling mechanism between Pap and Pit, in which the translation of Pap is required for the translation of Pit. Here, an alkaline phosphatase (phoA/lacZ) and a β-glucuronidase (gusA) translational reporter were fused to Pit as a measure of its translation and to understand the role of translational coupling in the Pap-Pit system. Growth complementation experiments with a conditionally Pi transport deficient S. meliloti mutant carrying various mutations in both pap and pit have also been performed in an attempt to determine the function of Pap in Pi uptake. The results presented here provide evidence that pap and pit are translationally coupled, and this is necessary for functional Pi transport via Pap-Pit. / Thesis / Master of Science (MSc) / Microbes require phosphorus in the form of inorganic phosphate (Pi) as an essential nutrient, but it is often found in growth-limiting concentrations in the environment. Bacteria have developed a diverse set of Pi transport systems to scavenge and take up phosphate from the environment. In the soil bacterium, Sinorhizobium meliloti, one such Pi transport system is the Pap-Pit system. Pit is a membrane transporter for Pi and is associated with a cytosolic protein of unknown function known as Pap. Various mutations in both pap and pit have been constructed in an attempt to determine the function of Pap in Pi uptake via Pit. The pap gene appears to be required immediately upstream of pit in an operon for functional Pi transport. The pap and pit genes overlap by a single nucleotide and this may suggest a translational coupling mechanism that is required for functional Pi transport via Pap-Pit.

The objectives of these studies were to investigate the effects of two special proteins, spray-dried plasma protein (SDPP), a high quality protein source, and Peptiva®, a mixture of peptides manufactured from marine products, on growth performance, nitrogen balance and enzyme and nutrient transporter mRNA expression in the brushborder membrane in weanling pigs. The results indicated that 6 % SDPP increased ADG and ADFI in the first 10 d after weaning (P < 0.05) without carry-over benefits in subsequent phases. There were potential additive effects of SDPP and Cu on growth promotion. Trends for interaction of diet and pen sanitation were observed for G:F with more pronounced response to SDPP (P = 0.07) and Cu (P = 0.11) supplementation in the sub-sanitary pens. In the duodenum, reduced crypt depth with Cu supplementation (P < 0.01) and a trend for greater villous length with SDPP supplementation (P = 0.09) were observed. Pigs reared in the sub-sanitary pens had lower ADG (P < 0.05) as well as shorter villous length and less crypt depth (P < 0.05) than those from sanitary pens. To investigate the potential impact of dietary proteins on gene expression in the intestine, 54 weanling pigs were fed either 6 % SDPP, 0.5 % Peptiva®, or soy control diets, and were killed 3 or 10 d after weaning. Northern blot results revealed significant diet by intestinal segment interactions (P < 0.05) for aminopeptidase A and aminopeptidase N. Aminopeptidase A was evenly distributed along the small intestine in the Peptiva® group, but decreased dramatically in the ileum in other groups. Aminopeptidase N increased from the proximal to the distal intestine in the soy protein and SDPP groups, whereas in the Peptiva® group, relative abundance was highest in the jejunum and lowest in the duodenum. Most of the enzyme and nutrient transporter mRNA abundance was observed in the distal segements of the small intestine and changed as the animals matured. Due to the low abundance of cytokine mRNA expression in the intestine, mRNA levels of cytokine were quantified by Real-Time PCR. The results indicated that the pigs fed the SDPP diet tended to have lower pro-inflammatory cytokine IL-1-β and TNF-α compared to other treatments. Tumor necrosis factor--α and IL-10 mRNA abundance increased from the proximal to the distal intestine, and was higher (P < 0.05) in the ileum than in the duodenum and jejunum. The mRNA abundance of IL-1-β, IL-10, and TNF-α also increased as the animals matured (P < 0.01). In summary, SDPP increased growth performance of weanling pigs, which were associated with changes in intestinal morphology and function. Peptiva® influenced aminopeptidases distribution along the small intestine. The mRNA abundance for digestive enzymes, nutrient transporters, and cytokines were differentially regulated along the small intestine as pigs matured. / Ph. D.

Mosquitoes affect human health worldwide as a result of their ability to vector multiple diseases. Mosquitocide resistance is a serious public health challenge that warrants the development of improved chemical control strategies for mosquitoes. Previous studies demonstrate the mosquito blood-brain barrier (BBB) to interfere with the target-site delivery and action of anticholinesterase chemistries. The ATP-binding cassette (ABC) transporters are efflux proteins that assist in maintaining the BBB interface and serve as a first line of defense to mosquitocide exposures. To date, there are three subfamilies (ABC -B, -C, -G) of ABC transporters; however, knowledge of these chemistries interacting with mosquito ABC transporter(s) is limited. Here, I report that tacrine and bis(7)-tacrine are relative non-toxic anticholinesterases at solubility limits; however, the addition of verapamil enhances toxicity of both tacrine and bis(7)-tacrine to mosquitoes. Verapamil significantly increases the mortality of mosquitoes exposed to tacrine and bis(7)-tacrine compared to the tacrine- and bis(7)- tacrine-only treatments. Tacrine and bis(7)-tacrine reduce acetylcholinesterase activity in mosquito head preparations compared to the untreated mosquitoes; however, the addition of verapamil significantly increases the anticholinesterase activity of tacrine and bis(7)-tacrine compared to the tacrine-and bis(7)-tacrine-only treatments. Tacrine and bis(7)-tacrine increase ATPase activity in Aedes aegypti at lower concentrations compared to that of verapamil (Fig. 3). The differential increase in ATPase activity suggests that tacrine and bis(7)-tacrine are more suitable substrates for ABC transporter(s) compared to verapamil and, thus, provides putative evidence that ABC transporter(s) is a pharmacological obstacle to the delivery of these anticholinesterases to their intended target site. / Master of Science in Life Sciences