The infection of the human gastric mucosa by the bacterium Helicobacter pylori can lead to the development of gastritis, gastroduodenal ulcers, and cancer. The factors that determine disease development in a small percentage of infected individuals are still not fully understood. In this thesis, we aimed to identify and functionally characterize novel virulence factors of H. pylori and to understand their effect on host cell responses. In Paper I, we found that JHP0290, an uncharacterized secreted protein of H. pylori, induced macrophage apoptosis concomitant to the release of pro-inflammatory cytokine TNF via the regulation of the Src family of kinases and ERK MAPK pathways. In paper II, we demonstrated that JHP0290 exhibits both proliferative and anti-apoptotic activity, together with a faster progression of the cell cycle in gastric epithelial cells. During these responses, ERK MAPK and NF-κB pathways were activated. Paper III revealed a pro-apoptotic effect of another H. pylori-secreted protein HP1286 in macrophages via the TNF-independent and ERK-dependent pathways. No apoptosis was observed in HP1286-treated T cells or HL60 neutrophil-like cells, suggesting cell-type specific effect of HP1286. In Paper IV, we observed the pro-inflammatory activity of H. pylori secreted protein HP1173 in macrophages. The protein was found to induce TNF, IL-1β, and IL-8 in macrophages through MAPKs, NF-κB, and AP-1 signaling pathways. Furthermore, differential expression and release of JHP0290, HP1286, and HP1173 homologues was observed among H. pylori strains (papers II, III, IV).  Due to their ability to regulate multiple host cell responses, proteins JHP0290, HP1286, and HP1173 could play an important role in bacterial pathogenesis.

Along with proteins, a myriad of different molecular biomarkers, such as post-translational modifications and autoantibodies, could be used in an attempt to improve disease detection and progression. In this thesis, I build on several iterations of the proximity ligation assay to develop and apply new adaptable methods to facilitate detection of proteins, autoantibodies and post-translational modifications. In paper I, we present an adaptation of the solid-phase proximity ligation assay (SP-PLA) for the detection of post-translational modification of proteins (PTMs). The assay was adapted for the detection of two of the most commons PTMs present in proteins, glycosylation and phosphorylation, offering the encouraging prospect of using detection of PTMs in a diagnostic or prognostic capacity.  In paper II, we developed a variant of the proximity ligation assay using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer, termed PLARCA. With a detection limit considerably lower than ELISA, PLARCA detected femtomolar levels of these proteins in patient samples. In paper III, we aim to compare detection values of samples collected from earlobe capillary, venous plasma, as well as capillary plasma stored in dried plasma spots (DPS) assessed with a 92-plex inflammation panel using multiplex proximity extension assay (PEA). Despite the high variability in protein measurements between the three sample sources, we were able to conclude that earlobe capillary sampling is a suitable less invasive alternative, to venipuncture. In paper IV, we describe the application of PLARCA and proximity extension assay (PEA) for the detection of GAD65 autoantibodies (GADA). Thus, offering highly sensitive and specific autoimmunity detection.

I hjärnan finns neuron och gliaceller. Förut trodde man att neuroner var dem enda som hade en viktig funktion i hjärnan men på senare tid har upptäckt att gliaceller har en större betydande roll än man tidigare trott. Gliaceller är ett samlingsnamn som innefattar bland annat microglia celler, oligodendrocyter och astrocyter. Östrogen är ett steroidhormon som har många viktiga funktioner i kroppen som bland annat reproduktionen, immunförsvaret, skelettet och endokrina system. Östrogen binder till östrogenreceptorer och de finns 3 stycken olika som kallas för östrogenreceptor alfa (α), östrogenreceptor beta (β) och G-proteinkopplade östrogenreceptor (GRP30). Alla dessa östrogenreceptorer har man funnit i hjärnan. Syftet med detta projektarbete är att ge en djupare förståelse om östrogens signalering i hjärnans gliaceller och om östrogens signalering kan ge någon relevant funktion till framtida farmakologiska behandlingar.  Systematisk litteraturstudie gjordes och sökningar på databasen PubMed. Begränsade antalet träffar med sökord, inklusionskriterier och exklusionskriterier. Artiklar granskades sedan via ett urvalssystem och relevanta artiklar användes för att besvara syfte och frågeställningar.  Östrogensignaleringen på gliaceller har många olika effekter. En signalering på östrogenreceptor β på oligodendrocyter leder till mognad, differentiering, bättre överlevd och att remyeliniseringen aktiverades. Medan en östrogens signalering på microglia cellens östrogenreceptorer α, β och GRP30 leder dämpning av inflammation och förbättrad kognitiv funktion. Östrogensignaleringen på astrocyter ger flera olika effekter såsom metabolismen av glukos, progesteron syntesen, glutamattransportören GLT-1, tillväxtfaktorn TGF-α, upptaget av glutamat samt ökad proteinproduktion av AMPA-receptor. Den nya kunskapen om östrogens signalering på hjärnans gliaceller kan leda till framtida farmakologiska behandlingar vid hjärnskada och ischemisk stroke. Östrogenet har visat på neuronskyddande effekter via signalering på gliaceller. Svagheten är att de endast är djurstudier som ligger till grund för kunskapen om östrogens signalering på gliaceller. I framtiden skulle det behövas styrkas med studier gjorda på människa. En styrka är att djurstudierna ger en fingervisning om östrogen signaleringen eftersom hjärnans uppbyggnad är likvärdig.

Det finns idag flera tusen genetiska sjukdomar som inte kan botas med hjälp av dagens läkemedelsbehandlingar. Detta är något forskarna försöker finna en lösning på. Två nya potenta genredigeringsverktyg har utvecklats och tros kunna bota och behandla många av de idag kända genetiska sjukdomarna. Detta är clustered regularly interspaced short palindromic repeats med CRISPR-associerade proteiner, CRISPR/Cas9 och prime editing. Tekniker som utvecklats från det adaptiva immunförsvaret hos prokaryoter. Både CRISPR/Cas9 och prime editing är RNA-guidade system med DNA som mål, de är även möjliga att programmera. Syftet med denna litteratursökning var att: 1) Jämföra teknikerna CRISPR/Cas9 och prime editing, 2) Undersöka vilka idag pågående kliniska prövningar som finns där någon av teknikerna används vid behandling av sjukdom. 3) Undersöka vilka sjukdomstillstånd som tros kunna botas och/eller behandlas med hjälp av någon av teknikerna samt 4) undersöka hur forskare ser på de etiska aspekterna av dessa tekniker. Information har hämtats under arbetets gång, främst från PubMed, Google och clinicaltrials.gov. Det finns idag 16 pågående studier där CRISPR/Cas9 används som behandlingsmetod. För prime editing finns det inga pågående studier. Sjukdomarna som forskarna hoppas kunna behandla med hjälp av metoderna är många, men de har kommit längst i utvecklingen av läkemedel för cancer, blodsjukdomar och ögonsjukdomar. De etiska diskussionerna har varit många och den stora frågan som diskuteras är hur tekniken skall regleras för att inte utnyttjas till sådant som potentiellt kan vara skadligt. Detta är två tekniker med hopp om nya behandlingsmetoder för genetiska sjukdomar, dock är de endast i början av sin utveckling och mer forskning och förfining av metoderna krävs innan de kan tillämpas kliniskt. / Today, there are thousands of genetic diseases that cannot be cured with the help of today's drug treatments. This is something the researchers are trying to find a solution to. Two new potent gene editing tools have been developed and are believed to be able to treat or cure many of today's genetic diseases. These are Clustered regularly interspaced short palindromic repeats with CRISPR-associated proteins, CRISPR/Cas9 and prime editing. Techniques developed from the adaptive immune system of prokaryotes. Both CRISPR/Cas9 and prime editing are RNA-guided DNA-targeted systems that are programmable. The purpose of this literature search was to: 1) compare the CRISPR/Cas9 and prime editing techniques, 2) investigate the current clinical trials in which any of the techniques are used to treat disease. 3) investigate which diseases that are believed to be cured and/or treated by using one of the techniques, and 4) investigare how researchers view the ethical aspects of these techniques. Information was gathered during a period between January to May 2020, mainly from PubMed, Google and clinicaltrials.gov. There are currently 16 ongoing studies using CRISPR/Cas9 as a treatment method. For prime editing there are no ongoing studies. The diseases that the researchers hope to be able to treat using the methods are many, but they have come the farthest in the development of a drug for cancer, blood diseases and eye diseases. There have been many discussions about the ethical side, but the big question being discussed is how the technology should be regulated so that it may not be used to harm instead of treat. These two techniques give hope of new treatment methods of genetic diseases, however, they are in the early stages of their development and more research and refinement of the methods is required before they can be applied clinically.

En kartläggning och en kritik av det sätt på vilket den svenska rättsordningen för närvarande hanterar avtal i strid med lag eller goda seder.

Purpose This project aims to explore the classification method of kinase inhibitors with five-channel cell painting image data based on the deep learning model. Methods A ResNet50 transfer learning model was used as the starting point to build the deep neural network (DNN) model, where different DNN parameters were selected to make the deep learning model more suitable for the cell painting data. Two different adaptive layers (adaptive average pooling 3D and convolution 2D) were added separately before the ResNet50 transfer learning model to adapt the five-layer cell painting image to the neural network. In addition, the skimage.transform.resize function was used to compress the five-layer cell painting image. Results The proposed deep learning model demonstrates the effectiveness in all three classification experiments. The proposed model performs particularly well in classifying among control, EGFR, PIKK and CDK kinase inhibitors families. It achieves an F1-score of 0.7764 on all four targets and has a 93\% accuracy rate in the PIKK kinase inhibitors family. The adaptive average pooling 3D layer successfully adapts the five-layer images to the model, resulting in an improved effect. The training time of the model is significantly reduced to one-fortieth by compressing the image size. Conclusion The proposed model achieved convincing effectiveness in classifying families, which showed progress in building the deep learning model to classify kinase inhibitors on five-channel cell painting data. This study also proved the feasibility of directly inputting five-channel cell painting images to DNN. In addition, the speed of the model increased sharply by compressing the image size without an obvious loss of data information.

Acute spinal cord injury is often caused by collisions with motor vehicles, falls or violence. This injury could potentially lead to paraplegia or tetraplegia, causing great economic and personal loss. The patophysiology is biphasic, with primary and secondary mechanisms. Regarding secondary spinal cord injury, glutamate and interleukin-1 beta (IL-1<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" />) activates N-methyl-d-aspartate (NMDA)-receptors leading to prolonged excitotoxity, causing neuronal death and subsequently glial scarring. Cross-linked-hyaluronic acid gel and interleukin-1 receptor antagonist (IL-1RA) are believed to have a neuroprotective effect. The major aim of this study was to evaluate neuroprotection in the central neural system. Briefly, spinal cord slice cultures from mice (p9-12) were chemically injured with NMDA and treated with two hyaluronic acid-based gels with integrated, or added, IL1RA. RNA was extracted and transcripted to cDNA. The gene expression of Neuronal nuclear protein, <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" />-aktin and IL-1<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Cbeta" data-classname="equation" data-title="" /> were studies with RT-qPCR. Results showed that gel integrated with IL1RA had significant therapeutic effect, resembling undamaged cultures. Furthermore, axonal outgrowth was investigated in dorsal root ganglion (DRG) in which two preparations of the method were evaluated. Results demonstrated that changing medium every other day was more preferred, compared to adding 20 µl medium every day.  In conclusion, gels integrated with IL1RA have neuroprotective properties and in DRG preparations, medium should be changed every other day for optimal results.

If you could trap a moment of painful sorrow or a deep joy, what form would you like to keep? This project is exploring how designing with biology can allow new sets of affordances and ways of expressing and interacting with the artifacts. Hence to this idea, the researcher is exploring the concept of crystalizing tears and turning them to gold, both metaphorically and literally. Showcasing how wearable made of living things could allow us more humane, poetic, and symbiotic relationships, compare to transactional interactions we currently have with wearables based on computation. As the outcome of the project, the researcher uses a biochemical phenomenon to commemorate chapters in life. Beyond that, the researcher is opening a field of what could be possible in the context of designing with biology and biological matters, how we are going to use them, and what kind of relationship we could build, as we move from machines to organisms, from pixels to cells. In its core, the researcher desired to show that Interaction designers must become material researchers, rather than inhabiting in the realm of familiar mediums. And this is what this thesis is all about, and hopefully, it does not stop there.

Background Broth microdilution (BMD) is a gold-standard reference method to determine minimum inhibitory concentration (MIC) of antibiotics. For this, a standardized concentration of bacterial inoculum (2e5–8e5 colony-forming units, CFU/ml) is added to progressively higher concentrations of antibiotics. Bacteria stop growing at a particular antibiotic concentration termed MIC. Like other assays, various biological and/or technical factors can affect BMD results.   Aims To investigate the effects of inoculum concentration (5e4–5e6 CFU/ml), growth-medium concentration (cation-adjusted Mueller-Hinton Broth (CAMHB)), ranging 0.5x to 2x (1x as standard)) and age (&lt;6-months or &gt;1-year old) of fastidious medium on MIC results. And to compare BMD results using 5 different brands of CAMHBs and 1 cation-non-adjusted MH-broth (non-CAMHB).   Methods 12 isolates of bacteria (gram-positive (n=3), gram-negative(n=5), fastidious isolates (n=7)) and custom-made antibiotics-containing plates for gram-positive (11 antibiotics) or gram-negative bacteria (10 antibiotics) were used. Overnight-grown colonies were used to prepare BMD solutions (MH-broth + inoculum +/- fastidious) which were plated on antibiotic-plates as well as diluted prior to plating on agar-plates. Antibiotic- and agar-plates were incubated (18–20hr, 35°C) and used to determine MICs (following European Committee on Antimicrobial Susceptibility Testing instructions) and actual number of viable bacteria in BMD solutions, respectively.   Results Increasing inoculum concentration increased MICs of all antibiotics except cefoxitin. Piperacillin–tazobactam, levofloxacin, benzylpenicillin and ampicillin were especially sensitive to increase in inoculum and showed a 4-fold increase in &gt;50% isolates. MICs for tobramycin, tigecycline and gentamicin increased by 2-fold in &gt;50% isolates every time MH-broth concentration increased. Age of fastidious medium had no decipherable pattern of effects on MIC. All MH-broths gave similar results except when testing daptomycin which gave higher MICs with non-CAMHB compared to CAMHB.    Conclusion This research reveals some technical factors affecting MIC results. These results could help define parameters for automated BMD-performing-systems. However, this research shows only trends as more replicates are needed to determine statistically significant results.

Autoimmuna sjukdomar är en samling komplexa, kroniska, inflammatoriska sjukdomstillstånd som kännetecknas av dysreglering av immunsystemet, vilket resulterar i inflammation och skada av vävnader, celler och organ. Dessa sjukdomar har en betydande inverkan på individens livskvalitet och bidrar ofta till ökad dödsrisk där komorbiditeter föreligger. Emellertid medför den varierande symptombilden för olika autoimmuna sjukdomar betydande utmaningar för att uppnå noggrann diagnos, prognos och utvärdering av behandling. Det finns därför ett påtagligt behov av att upptäcka nya biomarkörer.  I denna studie utfördes en omfattande analys av 944 plasmaprover med hjälp av OlinkR Explore-plattformen, vilket genererade data för 1463 unika proteiner. Baserat på uttrycksdata identifierades proteiner förknippade med de sex utvalda autoimmuna sjukdomarna multipel skleros, myosit, reumatoid artrit, systemisk skleros, Sjögrens sjukdom och systemisk lupus erythematosus samt några av deras definierade subgrupper. Dessa potentiella biomarkörer kommer eventuellt att underlätta tidig diagnos, sjukdomsdifferentiering och prognos. Flertalet av dessa proteiner har ännu aldrig kopplats till de här specifika sjukdomarna i litteraturen, särskilt inte från plasmaprover, vilket ger spännande nya perspektiv för biomarkörsutveckling. Det är dock av största vikt att genomföra robusta valideringsstudier i oberoende kohorter.  Sammanfattningsvis belyser våra resultat den potentiella brukbarheten hos dessa proteomiska plasmabiomarkörer för att förbättra tidig sjukdomsdetektering, karakterisering av subgrupper och sjukdomsdifferentiering att stimulera. Förhoppningsvis kan dessa resultat stimulera till vidare forskning inom området för biomarkörer och potentiella framsteg inom individbaserad medicin. / Autoimmune diseases are complex, chronic, inflammatory conditions characterized by dysregulation of the immune system, resulting in inflammation and damage to various tissues, cells and organs. These diseases significantly impact individuals’ quality of life and often contribute to increased mortality risk in the presence of comorbidities. However, due to the diverse array of symptoms associated with different autoimmune diseases, accurate diagnosis, prognosis, and treatment evaluation pose significant challenges. Thus, there is a pressing need for the discovery of novel biomarkers.  In this study, a comprehensive analysis of 944 plasma samples using the OlinkR Explore platform was conducted, generating data on 1463 unique proteins. Based on the expression data, associated proteins were identified for six selected autoimmune diseases, namely multiple sclerosis, myositis, rheumatoid arthritis, systemic sclerosis, Sjögren’s syndrome, and systemic lupus erythematosus, as well as some of their defined subgroups. These are prospective biomarkers and have the potential to aid in early diagnosis, therapeutic intervention, subgroup identification, disease differentiation, and disease prognosis. Notably, some of these proteins have not been previously associated with the specific diseases in the existing literature, especially not in plasma samples, thereby offering intriguing new perspectives for biomarker development. However, it is of great importance to conduct robust validation studies in independent cohorts to confirm the outcomes of this study.  In summary, our findings highlight the potential utility of these proteomic plasma biomarkers in improving the early detection, subgroup characterization, and disease differentiation of autoimmune diseases. The identification of these proteins will hopefully stimulate further investigation in the field of biomarker research and potential advancements in personalized medicine.