This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.

Introduktion. Autoimmun tyreoidit (AITD) är en kronisk autoimmun sjukdom där immunförsvarets antikroppar (ab) attackerar tyreoideaproteinerna tyreoideaperoxidas (TPO) och/eller tyreoglobulin (TG). Studier visar att selentillskott hos patienter med AITD kan minska tyreoideaantikroppar, storleken och antalet noduler hos en förstorad tyreoidea. Syftet med detta arbete var att undersöka hur selentillskott påverkar serumnivåer av TPOab, samt tyreoideahormonnivåer vid AITD.   Metod. Arbetet är en litteraturstudie och därför har metoden varit att samla relevant litteratur genom PubMed med sökningar som ”selenium autoimmune thyroiditis”, ”selenium thyroid” och ”autoimmune thyroiditis”. Bland sökresultaten valdes nio artiklar ut baserat på studiekvalitet, publikationsår och relevans. Bland artiklarna granskades och sammanställdes uppmätta nivåer av TPOab samt tyreoideahormonnivåer, vilka valdes som indikation på effekt utav selentillskott. Resultat. Resultaten var inkonsekventa. Majoriteten av studierna (7 av 9) tydde på att oral administrering av selentillskott effektivt minskade serumkoncentrationerna av TPOab hos patienter med AITD i alla åldersgrupper. De studier som resulterade i störst minskning av TPOab pågick i 3-12 månader. Utav de 9 studerade artiklarna var det endast en studie som inte rapporterade någon som helst positiv klinisk effekt hos patienterna. Två av studierna visade att selen förhindrar vidare försämring av tyreoideans ekogenitet, vilket tyder på att selen kan hejda inflammationsprocessen men ej reversera tyreoideaskadorna den orsakat. Majoriteten av studierna (7 av 9) visade att selentillskott ej ger några signifikanta förändringar i tyreoideahormonerna: TSH, fT4 och fT3. Diskussion. Varför AITD-patienter svarar olika på selenadministrering är ännu okänt, men kan misstänkas bero på selenbehandlingens varaktighet, patienternas intratyroidnivåer av selen vid studiens början, förekomst av jodbrist, samt patienternas ålder och sjukdomsprogression. Slutsats. Att ha adekvata fysiologiska nivåer av selen är av stor vikt för att bevara tyreoideans hälsa och förebygga tyreoidearelaterade sjukdomar. Majoriteten utav de granskade studierna visar att tillskott av selen kan minska antalet TPOab. Selentillskott kan även ha immunrelaterade fördelar men verkar inte påverka nivån tyreoideahormonnivåer. Inga negativa effekter påvisades vid intag av selentillskott vilket gör dess administrering säker. Fler studier behöver dock göras för att fastställa effektiviteten av selentillskott vid AITD. / Introduction. Autoimmune thyroiditis (AITD) is a chronic autoimmune disease in which the immune system's antibodies (ab) attack the thyroid proteins thyroid peroxidase (TPO) and/or thyroglobulin (TG). Studies show that selenium supplementation in patients with AITD can reduce thyroid antibodies and the size and number of nodules in an enlarged thyroid. The purpose of this study was to investigate how selenium supplementation affects the serum levels of thyroid peroxidase antibodies (TPOab) and thyroid hormone levels in autoimmune thyroiditis. Method. This is a literature study and therefore the method has been to gather relevant literature through searches on PubMed such as "selenium autoimmune thyroiditis", "selenium thyroid" and "autoimmune thyroiditis". Among the search results, nine articles were selected based on quality, publication year and relevance. Among the articles, measured levels of TPOab and thyroid hormone levels were examined and compiled, and were chosen as an indication of the effect of selenium supplementation. Results. The results were inconsistent. The majority of the studies (7 of 9) suggest that oral administration of selenium supplements effectively reduced serum concentrations of TPOab in patients with AITD in all age groups. The studies that resulted in the largest decrease in TPOab lasted for 3-12 months. Out of the 9 examined studies, only one study did not report any positive clinical effect in patients. Two of the studies showed that the selenium prevents further impairment of thyroid echogenicity, suggesting that selenium can inhibit the inflammatory process but not reverse the pre-existing thyroid damage it’s caused. The majority of studies (7 out of 9) show that selenium supplementation does not produce significant changes in the thyroid hormones: TSH, fT4 and fT3. Discussion. Why AITD-patients respond differently to selenium administration is still unknown, but it may be due to the duration of selenium treatment, the patients' intrathyroid levels of selenium at the onset of the study, the presence of iodine deficiency, as well as the age and disease progression of the patients. Conclusion. Having adequate physiological levels of selenium is of great importance in preserving thyroid health and preventing thyroid-related diseases. The majority of the studies show that selenium supplementation can reduce the number of TPOab. Selenium supplementation may also have immune related benefits but does not appear to affect the thyroid hormone levels. No adverse effects were observed during selenium supplementation, which makes its administration safe. However, more studies are needed to determine the effectiveness of selenium supplementation for AITD.

Disturbances of host integrity have the potential to evoke activation of innate immunologic and hemostatic protection mechanisms in blood. Irrespective of whether the activating stimulus is typically immunogenic or thrombotic, it will generally affect both the complement system and platelets to a certain degree. The theme of this thesis is complement and platelet activity, which is intersected in all five included papers. The initial aim was to study the responses and mechanisms of the complement cascade in relation to platelet activation. The secondary aim was to use an applied approach to regulate platelets and complement on model biomaterial and cell surfaces.    Complement activation was found in the fluid phase in response to platelet activation in whole blood. The mechanism was traced to platelet release of stored chondroitin sulfate-A (CS-A) and classical pathway activation via C1q. C3 was detected at the platelet surface, though its binding was independent of complement activation. The inhibitors factor H and C4-binding protein (C4BP) were detected on activated platelets, and their binding was partly dependent on surface-exposed CS-A. Collectively, these results showed that platelet activation induces inflammatory complement activation in the fluid phase. CS-A was shown to be a central molecule in the complement-modulatory functions of platelets by its interaction with C1q, C4BP, and factor H. Platelet activation and surface adherence were successfully attenuated by conjugating an ADP-degrading apyrase on a model biomaterial. Only minor complement regulation was seen, and was therefore targeted specifically on surfaces and cells by co-immobilizing a factor H-binding peptide together with the apyrase. This combined approach led to a synchronized inhibition of both platelet and complement activation at the interface of biomaterials/xenogeneic cells and blood. In conclusion, here presents a novel crosstalk-mechanism for activation of complement when triggering platelets, which highlights the importance of regulating both complement and platelets to lower inflammatory events. In addition, a strategy to enhance the biocompatibility of biomaterials and cells by simultaneously targeting ADP-dependent platelet activation and the alternative complement C3-convertase is proposed.

Serous epithelial ovarian cancer (EOC) is a potent suppressor of the immune defense. Here, we studied interactions between EOC and the immune system that lead to escape from tumor immune surveillance. We explored: 1) tumor escape from cytotoxicity by exosome-mediated modulation of the NK-cell receptors NKG2D and DNAM-1; 2) cytokine mRNA profiles in the EOC microenvironment and peripheral blood and their role in the suppression of the anti-tumor immune responses; 3) expression of long non-coding (lnc) RNAs in EOC tumors and exosomes. We found that EOC-secreted exosomes carried MICA/B and ULBP1-3, ligands of NKG2D, and could downregulate the NKG2D receptor and impair NKG2D-mediated cytotoxicity. In contrast, the DNAM-1 receptor ligands PVR and nectin-2 were seldom found in exosomes and were not associated with the exosomal membrane leaving the DNAM-1 receptor-mediated cytotoxicity intact. We compared cytokine mRNA expression in the tumor microenvironment and in immune cells of peripheral blood in EOC patients and patients with benign ovarian conditions. EOC patients were unable to mount an IFN-gamma mRNA response needed for tumor cell elimination. Instead, there was a significant up-regulation of inflammation and immune suppression i.e. responses promoting tumorigenesis and T-regulatory cell priming that suppress anti-tumor immunity. In addition, we studied lncRNAs in tissues and sera exosomes from EOC and benign ovarian conditions aiming to assess the lncRNA(s) expression profile and look for lncRNA(s) as possible marker(s) for early diagnosis. We found a deregulated lncRNAs expression in EOC tissues that correlated well with the lncRNAs expression in exosomes. Candidate lncRNAs with the highest expression and abundance were suggested for evaluation as EOC diagnostic markers in a future large cohort study. Our studies of EOC tissue and EOC exosomes highlight the immunosuppressive tumor microenvironment and the complex tumor exosome-mediated network of immunosuppressive mechanisms, and provide a mechanistic explanation of the observation that NKG2D-mediated cytotoxicity does not function in EOC patients and is partially replaced by the accessory DNAM-1 dependent cytotoxic pathway. The deregulated lncRNAs expression in EOC tissues and exosomes might serve for diagnostic purposes but could also be a potential risk of spreading tumor-derived lncRNAs in EOC exosomes to recipient cells throughout the body.

Background & Aims: Celiac disease (CD) is a chronic inflammatory small-bowel enteropathy caused by permanent intolerance to gliadin in wheat gluten, and related proteins in ray and barley. It is disputed whether CD patients tolerate oats. The only treatment of CD is lifelong gluten-free diet (GFD). Only individuals that carry the HLA-DQ2 and/or DQ8 alleles, and eat gluten can develop CD. Dysbiosis in the gut microbiota is a suggested risk factor for CD. T cells in small intestinal mucosa, including intraepithelial lymphocytes (IELs), are known to be important in the pathogenesis of CD. In contrast, the role of intestinal epithelial cells (IECs) is poorly understood. In this thesis we investigated the role of IECs in the immune pathology of CD from duodenal mucosa of children with CD, clinical controls and treated CD. We also investigated the role of CD associated bacteria and oats supplemented GFD on the mucosal immune system. Results: A new CD-associated bacterium, Prevotella jejuni, was isolated and characterized. It is a saccharolytic and proteolytic anaerobe. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were upregulated in IECs in active CD. In two in vitro models for intestinal epithelium, small intestine enteroids and T84 polarized tight monolayers, we showed that 70% of these genes were upregulated by interferon (IFN)-γ via the IRF1 pathway. IRF1 was also upregulated by the CD-associated bacteria P. jejuni and Actinomyces gravenitzii. IECs expressed the NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin (IL)-18, which induces IFN-γ in IELs. P. jejuni bound the intestinal epithelial cell lines T84, Caco2, HT29, and INT407, while Lachnoanaerobaculum umeaense preferentially bound Caco2. P. jejuni caused decreased transepithelial resistance over tight monolayers, while L. umeaense caused an increase. P. jejuni upregulated mRNAs for the detoxification molecules CYP1A1, CYP1A2, CYP1B1, and TIPARP, the chemokines CX3CL1, CXCL1, and CXCL10, the sialyltranserase ST3GAL4, and the inflammation promoting protein S100A3 in tight monolayers. L. umeaense upregulated the chemokines CCL20 and CXCL10, and down-regulated TLR2. In a randomized, double-blinded intervention trial comparing two study-groups, standard GFD and oat-containing GFD, we found that mRNAs for several immune effector molecules and tight junction proteins were only reduced in patients receiving GFD, but not in a substantial fraction of patients on GFD with oats. The down-regulatory cytokines IL-10 and TGF-β1, the cytotoxicity-activating NK-receptors NKG2C and NKG2E, and the tight junction protein claudin-4 remained elevated in the study group on GFD with oats. Conclusions: IECs are far from inactive in CD. A key factor in the epithelial reaction in CD appears to be over-expression of IRF1 in IECs. Dual activation of IRF1 and IRF1-regulated genes, both directly by P. jejuni and indirectly by IFN-γ via the IL-18-inflammasome, would drastically enhance the inflammatory response and lead to the pathological situation seen in active CD. P. jejuni harms the intestinal epithelium, i.e., it is a likely risk factor for CD, while L. umeaense strengthen barrier function and local immunity, possibly acting as a protective. A fraction of CD patients should avoid oats in the diet. / Doctoral thesis

Bakgrund: Diabetes Mellitus typ I är ett globalt hälsoproblem som skördar många liv varje år och påverkar livskvaliteten för de drabbade. Diabetes typ I är en autoimmun sjukdom som leder till destruktion av insulinproducerande betaceller i pankreas och således rubbad glukosreglering. Huvudsakliga patogena immunceller inkluderar autoantikroppar, exempelvis riktade mot glutaminsyra dekarboxylase 65, och autoreaktiva T-celler. Diagnos sker generellt baserat på förhöjda halter plasmaglukos och eventuellt stimulering av C-peptid för att utreda status för den endogena betacellsfunktionen. Diabeteskomplikationer är en vanlig dödsorsak hos diabetespatienter. År 2019 orsakades 4,2 miljoner dödsfall av diabetes eller diabeteskomplikationer. I dagsläget är administrering av exogent insulin enda behandlingsmöjligheten för typ I diabetespatienter. Det har dock länge forskats på alternativ i form av preventiv behandling men i dagsläget finns inga preventiva behandlingar på marknaden. Syfte: Litteraturstudiens syfte var att undersöka prospektiva möjligheter till diabetespreventiv behandling inom områdena glutaminsyra dekarboxylase 65 vaccin och coxsackievirus B vaccin med avseende på effekt samt jämföra dessa två prospektiva behandlingsmöjligheter. Metod: Arbetet har utförts genom granskning av artiklar från databasen PubMed. För litteraturgranskning av studier om GAD-behandling valdes tre kliniska studier utifrån sökning med ”type 1 diabetes”, ”diabetes mellitus”, ”type 1”, ”GAD” och ”vaccine” som sökord. För artiklar om CVB och CVB-vaccin användes ”type 1 diabetes”, ”vaccine” och ”coxsackievirus” som sökord. Resultat: Kliniska studier på GAD-behandling visar ingen signifikant skillnad mellan GAD-alum och placebo i helgruppsanalyser. Vid vissa stratifierade analyser för exempelvis kön, ålder, eller antal riskfaktorer detekteras signifikanta skillnader genom ökad mängd stimulerad C-peptid eller progression till klinisk diabetes. Den prospektiva kohortstudien över diabetesincidens påvisar att CVB är en riskfaktor för diabetes hos människa. Vidare visar de prekliniska studierna på signifikant minskad diabetesincidens i CVB-vaccinerade studiepopulationer jämfört med placebo. Slutsats: Varken behandling med GAD-alum eller CVB-vaccin är möjligt att använda som preventiv behandling i nuläget. Dock visar studierna på lovande framtidsmöjligheter för CVB-vaccin som primärprevention och GAD-alum som sekundär- eller tertiärprevention. / Background: Diabetes Mellitus type I is a global health issue, causing numerous deaths each year and also influencing the quality of life of those affected. Type I diabetes is an autoimmune disease where the individuals own immune system causes destruction of insulin producing beta cells in the endocrine islets of pancreas. Main immunological features include, autoantibodies directed towards glutamic acid decarboxylase 65, and autoreactive T-cells. Diagnosis is generally based on elevated levels of plasma glucose and stimulated C-peptide, together disclosing the status of the beta cell function. The lack of endogen insulin causes disturbances in the glucose metabolism which leads to prevailing tissue damage in cells and organs of the diabetic individual’s body. Furthermore, insufficient control of plasma glucose is related to development of diabetes complications. Diabetic complications are known to be a major cause of death in diabetic patients. Diabetes and diabetic complications caused 4,2 million deaths in 2019. Insufficient adherence to treatment regimen during a long period of time is known to increase the risk for some common diabetes complications. Administration of exogenous insulin is the only current treatment available for type I diabetes, albeit recurrent attempts to find a cure or successful preventive treatment for diabetes mellitus type I. Recent promising research on diabetes preventive treatment includes the autoantigen glutamic acid decarboxylase-65 and vaccine against coxsackievirus B. Aim: The purpose of this literature study was to examine prospective possibilities for diabetes preventive treatments. Further, the purpose was to compare the promising preventive treatments of GAD65-vaccine and CVB-vaccine concerning effect and prospective treatment regimens. Methods: The thesis is a literature study based on articles found by searching the database PubMed. Clinical studies examining the effect of GAD-treatment was found by using key words such as ”type 1 diabetes”, ”diabetes mellitus”, ”type 1”, ”GAD” and ”vaccine”. Studies examining the effect of CVB and CVB-vaccines was primarily preclinical and prospective cohort studies, found by searching for the key words ”type 1 diabetes”, ”vaccine” and ”coxsackievirus”. Results: Clinical studies of GAD-treatment does not demonstrate a statistically significant difference between treatment with GAD-alum compared to placebo in full group analysis. Stratified groups occasionally prove significant differences in quantity of stimulated C-peptide or progression to clinical diabetes by age, gender or amount of risk factors for example. The prospective cohort study examining the incidence of diabetes, demonstrates that CVB is a risk factor for type I diabetes in humans. Furthermore, the preclinical studies detect a significant decrease in diabetes incidence in CVB-vaccinated mice compared with placebo. Conclusion: Neither treatment with GAD-alum nor CVB-vaccine is currently ready for use. However, the studies show a promising prospective possibility for CVB-vaccine as a primary prevention and GAD-alum as a secondary or tertiary prevention of type I diabetes.

Vid blodgivning donerar blodgivare blod frivilligt. Blodet kan sedan användas inom sjukvården för exempelvis blodtransfusion, vilket kräver blodprodukter kompatibla med patienten. Förekomst av leukocyter i blodprodukter medför en ökad risk för febrila transfusionsreaktioner hos transfunderade patienter. Därför krävs det att vid framställning leukocytreducera blodprodukter och utföra kvalitetskontroll. Med analysen B-leukocytpartikelkoncentration (LPK) kan totalantalet leukocyter i helblod beräknas. Flödescytometri är en metod som kan analysera optiska och fluorescerande egenskaper hos exempelvis celler i en suspension, vilket kan användas för att kvantifiera cellantal. BD Leucocount™-Kit (BD Biosciences) är avsett för flödescytometrisk analys av antalet kvarvarande leukocyter i leukocytreducerade blodprodukter. Vid framställning av blodprodukter ska helblodspåsen vila vid rumstemperatur i minst 3 timmar efter blodgivning. I Falun används antingen ett dagsprogram där produktion sker samma dag som blodgivningen, eller ett övernattningsprogram där produktion sker dagen därpå. Prover från 505 kontrollerade erytrocytenheter, samlade i Falun, har påvisat en skillnad i leukocytkoncentration beroende på vilket program som använts. Anledningen till att erytrocytenheternas leukocytinnehåll skiljer sig är inte känt. Syftet med denna studie är därav att undersöka om vilotiden har någon effekt på leukocytkoncentrationen i helblodspåsar. LPK varierade mellan helblodspåsarna. Ett ökande leukocytantal observerades över tid i majoriteten av helblodspåsar, inklusive medelvärde. Däremot kunde inte hypotesprövning påvisa statistisk signifikans. Hypotesen om att leukocytantalet ökar över tid går emot grundläggande hematologi. Utifrån resultaten i denna studie kan inte hypotesen bevisas. Vidare studier bör genomföras. / During blood donation, blood donors donate blood voluntarily. The blood can then be used in healthcare for, for example, blood transfusions, which requires blood products compatible with the patient. The presence of leukocytes in blood products increases the risk of febrile transfusion reactions in transfused patients. Therefore, leukocyte-reduction in blood products is necessary during production. Each blood center must perform quality control on produced blood products. With the analysis B-leukocyte particle concentration (LPK), the total number of leukocytes in whole blood can be calculated. Flow cytometry is a method that can analyze the optical and fluorescent properties of, for example, cells in a suspension, which can be used to quantify cell numbers. The BD Leucocount™-Kit (BD Biosciences) is intended for flow cytometric analysis of the number of leukocytes remaining in leukocyte-reduced blood products. When producing blood products, the whole blood bag should rest at room temperature for at least three hours after the donation. In Falun, either a day program is used where production takes place on the same day as the blood was donated, or an overnight program where production takes place the next day. Samples from 505 controlled erythrocyte units, collected in Falun, have shown a difference in leukocyte concentration depending on the program used. The reason why the leukocyte content of erythrocyte units differs is not known. The purpose of this study is therefore to investigate whether the resting period has any effect on the leukocyte concentration in whole blood bags. The LPK varied between the whole blood bags. An increasing leukocyte count was observed over time in most of the whole blood bags. However, hypothesis testing did not show statistical significance. The hypothesis that leukocyte counts increase goes against basic hematology. Based on the results of this study, the hypothesis cannot be proven. Further studies should be conducted. / <p>Vårdförbundet tilldelade Leo Svahn stipendium 2021 för <em>bästa kandidatuppsats inom biomedicinsk laboratorievetenskap</em>.</p>

The mastoid, located in the temporal bone, houses an air cell system whose cells have a variation in size that can go far below current conventional clinical CT scanner resolution. Therefore, the mastoid air cell system is only partially represented in a CT scan. Where the conventional clinical CT scanner lacks level of minute details, micro-CT scanning provides an overwhelming amount of ne details. The temporal bone being one of the most complex in the human body, visualization of micro-CT scanning of this boneawakens the curiosity of the experimenter, especially with the correct visualization settings. This thesis first presents a statistical analysis determining the surface area to volume ratio of the mastoid air cell system of human temporal bone, from micro-CT scanning using methods previously applied for conventional clinical CT scans. The study compared current results with previous studies, with successive downsampling the data down to a resolution found in conventional clinical CT scanning. The results from the statistical analysis showed that all the small mastoid air cells, that cannot be detected in conventional clinical CT scans, do heavily contribute to the estimation of the surface area, and in consequence to the estimation of the surface area to volume ratio by a factor of about 2.6. Such a result further strengthens the idea of the mastoid to play an active role in pressure regulation and gas exchange. Discovery of micro-channels through specific use of a non-traditional transfer function was then reported, where a qualitative and a quantitative pre-analysis were performed and reported. To gain more knowledge about these micro-channels, a local structure tensor analysis was applied where structures are described in terms of planar, tubular, or isotropic structures. The results from this structural tensor analysis suggest these microchannels to potentially be part of a more complex framework, which hypothetically would provide a separate blood supply for the mucosa lining the mastoid air cell system. The knowledge gained from analysing the micro-channels as locally providing blood to the mucosa, led to the consideration of how inflammation of the mucosa could impact the pneumatization of the mastoid air cell system. Though very primitive, a 3D shape analysis of the mastoid air cell system was carried out. The mastoid air cell system was first represented in a compact form through a medial axis, from which medial balls could be used. The medial balls, representative of how large the mastoid air cells can be locally, were used in two complementary clustering methods, one based on the size diameter of the medial balls and one based on their location within the mastoid air cell system. From both quantitative and qualitative statistics, it was possible to map the clusters based on pre-defined regions already described in the literature, which opened the door for new hypotheses concerning the effect of mucosal inflammation on the mastoid pneumatization. Last but not least, discovery of other structures, previously unreported in the literature, were also visually observed and briefly discussed in this thesis. Further analysis of these unknown structures is needed.

<p>Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated.</p><p><i>Nostoc punctiforme</i> PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by <i>hupSL</i>. The transcription of <i>hupSL</i> was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in <i>N. punctiforme</i>, the <i>hypFCDEAB</i> operon. These genes were found to be situated upstream of <i>hupSL</i> in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon.</p><p>The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of <i>N. punctiforme</i> was constructed, where <i>hupL</i> was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the <i>hupL</i>^- mutant and in wild type <i>N. punctiforme</i> was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup^- mutant of the related strain <i>Nostoc</i> PCC 7120 were also studied. </p><p>This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.</p>

Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated. Nostoc punctiforme PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by hupSL. The transcription of hupSL was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in N. punctiforme, the hypFCDEAB operon. These genes were found to be situated upstream of hupSL in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon. The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of N. punctiforme was constructed, where hupL was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the hupL- mutant and in wild type N. punctiforme was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup- mutant of the related strain Nostoc PCC 7120 were also studied. This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.