<p dir="ltr">Phosphatidylinositol phosphates (PIPs) and soluble inositol phosphates (IPs) serve as critical secondary messenger molecules that regulate cellular processes. The INPP5 family of phosphatases play an essential role in regulating levels of PIP-5’ and IP-5’ molecules. Src homology 2-containing-inositol phosphatases (SHIP), are a subgroup of the INPP5 family that consists of two members, SHIP1 and SHIP2. Both SHIP proteins have been identified to hydrolyze PI(3,4,5)P3 into PI(3,4)P2. Interestingly, the dysregulation of PI(3,4,5)P3 and SHIP proteins have been observed in multiple diseases, such as cancer, diabetes, and neurodegenerative disease. Recently, SHIP1 was identified as a potential risk factor for the development of Late-onset Alzheimer’s Disease (LOAD). Furthermore, knockdown and inhibition of SHIP1 using small-molecule inhibitors were shown to reduce phenotypes associated with LOAD. Taking these studies together suggests SHIP1 to be a potential therapeutic target for the treatment of LOAD.</p><p dir="ltr"><br></p><p dir="ltr">Despite SHIP1’s therapeutic potential, the development of specific small-molecule inhibitors that target SHIP1 has been challenging. One explanation for this challenge is that very little is known about the overall structure and function of SHIP1. In this thesis I will discuss in detail how we generated multiple SHIP1 constructs to improve our understanding of SHIP1’s overall structure and function in an <i>in vitro </i>setting.</p><p><br></p><p dir="ltr">Efficient protein production is essential for studying enzyme structure and function. The choice of expression system can impact protein yield and stability. The E. coli (BL21) and Baculovirus expression systems are two commonly used systems for protein production. While E. coli is cost-effective and can yield a large amount of protein, the Baculovirus system offers advantages in terms of protein folding and post-translational modifications. Using both systems to generate SHIP1 protein, we demonstrate that the Baculovirus system significantly enhances SHIP1 solubility for all generated constructs, making it the preferable choice for investigating the structure and function of SHIP1.</p><p><br></p><p dir="ltr">SHIP1, a 133 kDa protein, which comprises five established domains: an N-terminal Src Homolgy 2 (SH2) domain, 2.) a pleckstrin homology-related (PH) domain, 3.) an inositol phosphatase catalytic (Ptase) domain, 4.) a C2 domain, and 5.) a C-terminal domain containing proline-rich regions (PXXP) and tyrosine phosphorylated (NPXY) motifs. Despite their regulatory roles in phosphatase activity, protein-protein interactions, and membrane association, limited information is available about their structures and how they contribute SHIP1’s biochemical functions. In this study, we utilized baculovirus-expressed SHIP1 constructs to investigate the impact of each domain on macromolecular structure. Interestingly, a previously unrecognized domain within SHIP1 that directly impacts the enzyme's oligomeric state was identified. This work highlights that SHIP1's individual domains can significantly impact its overall structure and function, providing valuable insights for the development of potential therapeutics in the treatment of LOAD.</p><p><br></p><p dir="ltr">Accurate determination of phosphatase kinetics is vital for understanding the enzymatic activity and its potential involvement in disease. Using our baculovirus generated SHIP1 constructs, we employed in-vitro assays, including the malachite green (MG) and the 2-amino-6-mercapto-7-methylpurine riboside (MESG) coupled enzyme assays, to gain insight into SHIP1 kinetics. Results from the MG assay shows that SHIP1 can hydrolyze the PI(3,4,5)P3 diC8 substrate more efficiently than I(1,3,4,5)P4. Additionally, SHIP1’s PH domain was observed to increase the turnover of PI(3,4,5)P3 diC8. Furthermore, dimerization of SHIP1 was not observed to alter SHIP1 kinetics in any way. Lastly, no major differences in I(1,3,4,5)P4 kinetics were observed with the addition of SHIP1’s N-terminus. These results offer the first comprehensive biochemical characterization of SHIP1 across its substrates and N-terminal domains.</p><p><br></p><p dir="ltr">The development of potent and specific small-molecule inhibitors that target SHIP1 remains challenging. One potential cause for this challenge is that no structures of SHIP1 have been solved in complex with active compounds, making structure-based drug design impossible. In this study, we developed a covalent compound, <b>TAD-58547</b>, from a previously published fragment-based screen that was conducted on SHIP1’s Ptase and C2 domain. <b>TAD-58547 </b>was shown to effectively inhibited SHIP1's Ptase and C2 domains at modest potency. Using X-ray crystallography, this compound was observed to form a covalent interaction with a cysteine residue near the Phosphatase-C2 domain interface. Intriguingly, the inhibitor's potency was observed to be reduced in the presence of the SH2 domain. In addition to testing <b>TAD-58547</b> against our SHIP1 constructs, we investigated the effect of SHIP1’s N-terminus on the potency of a literature compound, <b>TAD-58616</b>. This compound was shown to inhibit all our tested constructs at low µM concentrations. Furthermore, using x-ray crystallography <b>TAD-58616 </b>was solved in complex with SHIP1’s Ptase and C2 domain. Intriguingly, density for <b>TAD-58616 </b>was shown to interact with a site previously identified from the fragment-based screen. While we initially determined this site to be a result of crystal packing, fragments bound to this site may have the potential to inhibit SHIP1. The work presented in this study reinforced the importance of testing inhibitors against physiological relevant forms of SHIP1, when developing potential therapeutics.</p><p><br></p><p dir="ltr">Lastly, new evidence has suggested that the binding of phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) and immunoreceptor tyrosine-based inhibitory motifs (p-ITIM) to SHIP1’s N-terminal SH2 domain is essential for its “Anchorage and Activation” at the plasma membrane (PM). With this model it is believed that SHIP1’s SH2 domain, places the phosphatase into an auto-inhibited state. Upon binding to immune receptor proteins and adaptor proteins that contain ITAM/ITIM sequences, SHIP1 becomes un-auto-inhibited, allowing it to efficiently hydrolyze PI(3,4,5)P3 embedded in the PM. While this model does support the notion that SHIP1 activity is mediated by its PM localization, our biophysical and biochemical characterization add another level of complexity to this regulatory event. Taking all these results together, we propose a novel model for SHIP1 called “Anchorage and Assist” and suggest innovative therapeutic strategies for targeting SHIP1.</p><p><br></p><p dir="ltr">In conclusion, this thesis highlights the importance of choosing suitable expression systems for efficient protein production. Additionally, it offers insight into SHIP1's regulatory mechanisms through the discovery of a novel domain impacting its oligomeric state. Furthermore, the accurate determination of SHIP1 kinetics enhances our understanding of this phosphatase and its potential implications in disease. Also, the identification and crystallization of a novel and previously determined inhibitor scaffolds in complex with SHIP1 increases our ongoing efforts to develop a small-molecule inhibitor that specifically targets SHIP1. Lastly, using recently published data, detailing SHIP1 PM localization and activation, we proposed a new model for SHIP1 activity and suggest novel therapeutic strategies for targeting SHIP1.</p>

Den pågående klimatkrisen kan otvivelaktigt förklaras av antropogena aktiviteter på jorden. Växthusgaserna som orsakar den globala uppvärmningen uppstår huvudsakligen genom förbränning av fossila ämnen och mätningar visar på exceptionella koncentrationer av växthusgaser i atmosfären – för koldioxid de högsta på 800 000 år. Planetens medeltemperatur har rubbats och lämnar idag inte någon del av planeten oberörd. Den globala uppvärmningen och klimatförändringarna bekämpas genom globala överenskommelser, så som Parisavtalet, om drastiskt minskade nettoutsläpp av växthusgaser. Sveriges krav på utsläppsminskningar genom EU ligger på 50 procent fram till 2030. Näringslivet har en väsentlig och ansvarsfull roll för en hållbar utveckling, den gröna omställningen och genomförandet av de globala klimatmålen. GHG-protokollet är en icke-vinstdrivande organisation som etablerades i slutet av 1990-talet utifrån det ökande behovet av ett globalt standardiserat ramverk för redovisning och rapportering av växthusgasutsläpp. Enligt GHG-protokollet sorteras utsläppen som direkta eller indirekta i tre scope: scope 1 (direkta), 2 (indirekta) och 3 (indirekta, som uppstår i värdekedjan utanför företagets grindar). Genom en fallstudie på Ramudden, ett företag inom bygg- och anläggningssektorn, utför den här studien klimatberäkningar inom scope 3 på ett trafikprojekt gällande en tunnelavstängning. Studiens utförande baseras på primär datainsamling från Ramudden, sekundär datainsamling från internationellt erkända databaser och med metodstöd genom GHG-protokollet. Målsättningen med studien är att utreda var de största växthusgasutsläppen uppstår i tunnelavstängningen, inom områdena material, transport och avfall, och var utsläppen kan minskas. Resultatet visar att de största utsläppen härrör från området transport, specifikt förbränning av diesel. Störst utsläppsreducering anses kunna uppnås inom området transport genom undvikande av nyinköpt material och byte från fossila bränslen till förnybara. Slutsatserna som dras är att inköp av nya produkter följer med höga växthusgasutsläpp genom transporten av dem. Vidare ger fossil diesel (miljöklass 1) sex gånger större växthusgasutsläpp än fossilfri diesel i form av HVO. / The current climate crisis can undoubtedly be explained by anthropogenic activities on Earth. The greenhouse gases enter the atmosphere through the burning of fossil fuels and cause global warming. Measurements show exceptional concentrations of greenhouse gases – for carbon dioxide the highest in 800 000 years. The planet´s average temperature has been thrown out of balance and does not leave any part of the planet unaffected.  Global warming and climate change are combated through global agreements, such as The Paris Agreement, with demands on drastically reduced net emissions of greenhouse gases. Sweden´s requirement within the EU is a 50 percent emission reduction until 2030. The business sector has an essential and responsible role for sustainable development, the green transition, and the implementation of global climate goals. The GHG protocol is a non-profit organization established in the late 1990s that arose out of the growing need for a globally standardized framework for accounting and reporting greenhouse gas emissions. The GHG protocol classifies emissions as direct or indirect emissions, into three scopes: scope 1 (direct), 2 (indirect) and 3 (indirect emissions that occur in the value chain and are not included in scope 2). Through a case study on Ramudden, a company in the building and construction sector, this study performs scope 3 climate calculations on a traffic project regarding a tunnel closure. The execution of the study is based on primary data collection from Ramudden, secondary data from international databases and a methodological guidance from the GHG protocol. The aim of this study is to examine where the largest greenhouse gas emissions occur within the project, in the areas of material, transport and waste, and identify where the emissions most effectively can be reduced. The result shows that the greatest emissions come from the transport area, specifically diesel emissions. The greatest emission reductions are achievable by avoiding purchases of new equipment and therefore avoiding its transportation emissions, and by switching from fossil fuels to renewable. The conclusions are that the purchase of new equipment generates large emissions from the equipment transportation. Furthermore, fossil diesel (environmental class 1) produces six times greater greenhouse gas emissions than fossil-free diesel (HVO).

Physical inactivity is a growing concern in the world. Exergames, which are physically demanding games, offer a solution to motivate individuals to participate in regular physical activity, and virtual reality (VR) is the latest addition to this field. Visual physical activity data through wearable trackers has also shown promising results to motivate individuals to stay active. This thesis researched the possibility to increase VR players physical activity levels, beneficial movements, and immersion, by involving their physical activity data in the form of a physical activity bar in the gameplay, with a reward being granted upon completion. This was explored through a within-subject user study with 20 participants where they first played a VR game without the physical activity bar and then again with it. The results showed an increase for all activity levels and immersion, however, the conclusion was that players are more likely to perform beneficial movements that fit the gameplay. / Fysisk inaktivitet är ett växande bekymmer i världen. Exergames, som är fysiskt krävande spel, erbjuder en lösning för att motivera individer att delta i regelbunden fysisk aktivitet, och virtuell verklighet (VR) är det senaste tillskottet på detta område. Visuell fysisk aktivitetsdata genom bärbara spårare har också visat lovande resultat för att motivera individer att hålla sig aktiva. Det här examensarbetet undersökte möjligheten att öka VR-spelares fysiska aktivitetsnivåer, speciella rörelser och immersion, genom att involvera deras fysiska aktivitetsdata i form av fysisk aktivitetsdata i spelet, med en belöning som delas ut när de är klara. Detta undersöktes genom en användarstudie inom ämnet med 20 deltagare där de först spelade ett VR-spel utan fältet för fysisk aktivitet och sedan igen med det. Resultaten visade en ökning för alla aktivitetsnivåer och immersion, men kom till slutsatsen att spelare är mer benägna att utföra speciella rörelser som passar spelet.

Network screening and countermeasure selection are two crucial steps in the highway improvement process. In network screening, potential improvement locations are ranked and prioritized based on a specific method with a set of criteria. The most common practice by transportation agencies has been to use a simple scoring method, which, in general, weighs and scores each criterion and then ranks the locations based on their relative overall scoring. The method does not deal well with criteria that are qualitative in nature, nor does it account for the impacts of correlation among the criteria. The introduction of Analytic Hierarchy Process (AHP) provides agencies with a method to include both quantitative and qualitative criteria. However, it does not address the issue on correlation. This dissertation explores the use of both Analytic Network Process (ANP) and Fuzzy Analytic Network Process (FANP) for their potential capabilities to address both issues. Using urban four-lane divided highways in Florida for bicycle safety improvements, both ANP and FANP were shown to provide more reasonable rankings than AHP, with FANP providing the best results among the methods. After the locations are ranked and prioritized for improvements, the next step is to evaluate the potential countermeasures for improvements at the selected top-ranked locations. In this step, the standard practice has been to use Crash Modification Factors (CMFs) to quantify the potential impacts from implementing specific countermeasures. In this research, CMFs for bicycle crashes on urban facilities in Florida were developed using the Generalized Linear Model approach with a Zero-Inflated Negative Binomial (ZINB) distribution. The CMFs were tested for their spatial and temporal transferability and the results show only limited transferability both spatially and temporally. The CMFs show that, in general, wider lanes, lower speed limits, and presence of vegetation in the median reduce bicycle crashes, while presence of sidewalk and sidewalk barrier increase bicycle crashes. The research further considered bicycle exposure using the bicycle activity data from the Strava smartphone application. It was found that increased bicycle activity reduces bicycle crash probabilities on segments but increases bicycle crash probabilities at signalized intersections. Also, presence of bus stops and use of permissive signal phasing at intersections were found to increase bicycle crash probabilities.