Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes. In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting. In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway. Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent. The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion. In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients.   In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.

Due to global warming, our planet will experience more extreme weather conditions. Plants can protect themselves against these abiotic stress conditions with their stress response, which includes alternative splicing of certain genes. Alternative splicing is a post-transcriptional process where a single gene gives rise to different mRNAs, which in turn produces different proteins. In plants, this is usually done by intron retention. One type of protein that may be involved in this stress response are the non-specific lipid transfer proteins (LTPs). Indeed, evidence of intron retention has been found in the LTP genes in the liverwort Marchantia polymorpha, called MpLTPd. To investigate whether this alternative splicing is caused by abiotic stress or not, I subjected the moss to two different types of stress trials, drought and cold, and compared the general expression of the intron in MpLTPd2 and MpLTPd3 from the stressed samples to samples from a moss grown under normal conditions. I found that the expression of the intron did change in the stressed moss, but none of the differences were significant. This suggests that alterative splicing in MpLTPd2 and MpLTPd3 is not caused by cold and drought and that the intron-containing protein plays no role in the protection of M. polymorpha against abiotic stress.

Investigation of tumor growth has traditionally been studied focusing only on the cancer cells. However, tumors consist of a complex tissue organization where heterotypic signaling occurs between different cell types. The cross-talk between tumor cells and other surrounding cell types may ultimately prove to be as important for the tumor cell behavior as the internal signaling cascades in the tumor cell itself.Myeloid cells, such as granulocytes and monocytes, and thrombocytes play an important role in the tumor tissue, as a tumor can be compared to a wound healing process without the normal regulation mechanisms. Platelets are thought to facilitate tumor cell extravasation by binding to the tumor cell and recruiting myeloid cells that secrete factors aiding tumor migration through the endothelial cells. Studying the content of granules and vesicles of the platelets and myeloid cells can provide important knowledge about how the tumor interactions are mediated and which key proteins that controls these processes.Serglycin is an intracellular proteoglycan that attaches chains of negatively charged glycosaminoglycans. It is thought to have a function in retaining and storing proteins in hematipoietic cells. In this project the impact of the loss of serglycin on platelets and myeloid cells was investigated, using a spontaneous insulinoma serglycin knockout mouse model. The results suggests that serglycin does not affect the amount of neutrophil granulocytes and monocytes in peripheral blood, nor does it seem to affect the amount of platelets sequestered to the tumor tissue. A co-staining for platelets and MMP9 positive granulocytes was also performed in order to assess if granulocyte-platelet interactions in the tumor were affected by loss of serglycin. Interactions between these cells were observed in both genotypes. Von Willebrand factor levels in the tumor tissue also remained unchanged upon loss of serglycin. However, preliminary experiments indicated that serglycin seems to play a role in the intracellular amounts of vimentin and VEGFB in undifferentiated primary bone marrow derived monocytes.

Trypanosoma brucei is a causative agent of African sleeping sickness. It is an extracellular parasite which circulates in the blood, lymph and eventually invades the central nervous system. There is a great need for new medicines against the disease and specific properties of nucleoside kinases in the pathogen can be exploited as targets for chemotherapy.  T. brucei contains a gene where two thymidine kinase sequences are fused into a single open reading frame. These types of tandem thymidine kinases were found only in different types of parasites, which made us to believe that it might be beneficial for them. Each thymidine kinase sequence in these tandem enzymes are here referred to as a domain. By cloning and expressing each domain from T. brucei separately, we found that domain 1 was inactive and domain 2 was as active as the full-length enzyme. T. brucei thymidine kinase phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine and to some extent purine nucleosides like deoxyinosine and deoxyguanosine. Human thymidine kinase increases the affinity to its substrates when it forms oligomers. Similarly, the T. brucei two thymidine kinase sequences, which can be viewed as a pseudodimer, had a higher affinity to its substrates than domain 2 alone.  T. brucei lacks de novo purine biosynthesis and it is therefore dependent on salvaging the required purine nucleotides for RNA and DNA synthesis from the host. Purine salvage is considered as a target for drug development. It has been shown that in the presence of deoxyadenosine in the growth medium, the parasites accumulate high levels of dATP and the extensive phosphorylation of deoxyadenosine leads to depleted ATP pools. Initially, we wondered if deoxyadenosine could be used as a drug against T. brucei. However, we found that T. brucei is partially protected against deoxyadenosine because it was cleaved by the enzyme methylthioadenosine phosphorylase (MTAP) to adenine and ribose-1-phosphate. At higher concentration of deoxyadenosine, 3 the formed adenine was not efficiently salvaged into ATP and started to inhibit MTAP instead. The deoxyadenosine was then instead phosphorylated by adenosine kinase leading to accumulation of dATP. The MTAP reaction makes deoxyadenosine itself useless as a drug and instead we focused on finding analogues of deoxyadenosine or adenosine that were cleavage-resistant and at the same time good substrates of T. brucei adenosine kinase. Our best hit was then 9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine (FANA-A). An additional advantage of FANA-A as a drug was that it was taken up by the P1 nucleoside transporter family, which makes it useful also against multidrug resistant parasites that often have lost the P2 transporter function and take up their purines solely by the P1 transporter. In parallel with our study of nucleoside metabolism in T. brucei, we also have a collaboration project where we screen essential oils from plants which are used in traditional medicine. If the essential oils are active against the trypanosomes, we further analyze the different components in the oils to identify new drugs against African sleeping sickness. One such compound identified from the plant Smyrnium olusatrum is isofuranodiene, which inhibited T. brucei proliferation with an IC50 value of 3 μM.

Tumour immunity is a balance between immune mediators that promote tumor progression versus mediators that promote tumor rejection. Infiltrating lymphocytes in human colorectal cancer tissues are independent prognostic factors for a better survival and a high number of cytotoxic CD8+ T-cells have been associated with a better prognosis in terms of a longer and disease free survival for the patient. In our syngeneic rat model we induce colon carcinoma subperitoneally by injecting a colon cancer cell line BN7005, a cell line expressing the epitope (Lewis Y) for the BR96 antibody. Tumours are dissected out and treated with different fixatives and then either frozen, snap-frozen or embedded in paraffin followed by sectioning. Immunohistochemistry using monoclonal antibodies against the tumour infiltrating leukocytes was performed on the tissue. The results were seen as an infiltration of different leukocytes in the tumours.

Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers. Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells. Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer. Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors. The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection. At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project. / Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer

Solute carriers (SLCs) are membrane-bound transporter proteins, important for nutrient, ion, drug and metabolite transport across membranes. A quarter of the human genome codes for membrane-bound proteins, and SLCs make up the largest group of transporter proteins. Due to their ability to transport a large repertoire of substances across, not just the plasma membrane, but also the membrane of internal organelles, they hold a key position in maintaining homeostasis affecting metabolic pathways. Unfortunately, some of the more than 400 identified SLCs are still not fully characterized, even though a quarter of these are associated with human disease. In addition, there are about 30 membrane-bound proteins with strong resemblance to SLCs, of which very little is known. The aim of this thesis is to characterize some of these putative SLCs, focusing on their localization and function in the central nervous system. Since many of the known SLCs play a vital part in metabolism and related pathways, the response to different nutritional conditions has been used as a key method. MFSD14A and MFSD14B, characterized in Paper I, are putative SLCs belonging to the Major Facilitator Superfamily (MFS) and found to be neuronal, differentially expressed in the mouse central nervous system and transiently upregulated in mouse embryonic cortex cultures due to amino acid deprivation. They were also altered in areas of the mouse brain after starvation as well as after high fat diet. In Paper II, the effect on gene regulation due to complete amino acid starvation was monitored in a mouse hypothalamic cell line and 47 different genes belonging to SLCs, or putative SLCs, were found to be affected. Of these, 15 genes belonged to already known amino acid transporters, whereas 32 were putative SLCs with no known function or SLCs not known to react to amino acids. The three SV2 proteins, SV2A, SV2B and SV2C, were studied in Paper III using human neuroblastoma cell lines. The high metabolic state of cancers often result in an upregulation and alteration of transporter proteins, and alterations of the SV2 proteins were found following different treatments performed in this study. Paper IV focused on putative SLCs of MFS type and their role in glucose metabolism. Mouse embryonic cortex cultures were subjected to glucose starvation and the gene expression of 19 putative transporters were analyzed. All but four of the putative transporters were affected either at 3h or 12h of glucose deprivation. In conclusion, several SLCs and putative SLCs studied in this thesis are strongly affected by alteration in metabolism, either due to amino acids or glucose or both. This makes the putative SLCs dynamic membrane-bound proteins, possibly transporters, highly affected by nutritional status and most likely regulated to maintain homeostasis.

The redox state in the cell plays a major role in determining vital functions and its major imbalance can lead to severe cell injury or death. Redox active proteins and cytokines involved in this process includes thioredoxin (Trx), protein disulfide isomerase (PDI), and tumor necrosis factor (TNF) superfamilies. Trx is a multipotent protein and key regulator of cellular redox balance operating in synergy with Trx reductase and NADPH (the Trx system). Trx has gene regulatory activity of several transcription factors. It also controls in a fascinating way redox-sensitive “on-off” decisions for apoptotic or hypertrophic pathways. Trx protects against H2O2 and TNFmediated cytotoxicity, a pathway in which TNF receptor-binding generates ROS. TNF is an autocrine growth factor and survival factor in vitro and in vivo for B-type of chronic lymphocytic leukemia (B-CLL) cells. The overall aim of this study was to investigate the importance of redox active proteins and cytokines in inflammation and cancer. We focused on: i) the role of Trx, TrxR, and selenium in carcinogenesis and in resistant cancer cells. ii) the importance of Trx in cancer cells and the redox regulation of TNF and its receptors TNFR1 and TNFR2. iii) the potential role of Trx as a key regulator in cellular redox balance, in the pathogenesis of cardiac dysfunction; its relationship to stress response parameters. iv) whether unmutated CLL (UCLL) responses to PKC and ROS pathways were different from mutated CLL (M-CLL) responses. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy resistant cells and suggest that redox regulation through the Trx system is an important target for cancer therapy. Trx was strikingly elevated in heart failure cases compared with controls signifying an adaptive stress response that is higher the more severe the disease. TNF autocrine release was redox modulated and the TNF receptors interacted at the cell surface membrane with the redox-active PDI, which excerted a stringent redox-control of the TNFR signaling. The proliferative response as well as increase of autocrine TNF and Trx were higher in U-CLL than in M-CLL. The overall conclusion of the four papers included in this thesis is that redox-active proteins and cytokines plays an important role in control and regulation of cancer and inflammation. Furthermore, redox regulation via thioredoxin by selenium, may offer novel treatment possibilities for resistant tumors disease.

Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes ultimately determining cellular function. Therefore, to develop a complete understanding of cellular processes, obtaining knowledge about protein function and interaction at a molecular level is critical. Consequently, the investigation of proteins in their native environment or in partially purified mixtures is a major endeavour in modern life sciences. Due to their high chemical similarity, the inherent problem of studying proteins in complex mixtures is to specifically differentiate one protein of interest from the bulk of other proteins. Site-specific protein functionalisation strategies have become an indispensable tool in biochemical- and cell biology studies. This thesis presents the development of a new enzymatic site-specific protein functionalisation strategy that is based on the reversible covalent phosphocholination of short amino acid sequences in intact proteins. A synthetic strategy has been established that allows access to functionalised CDP-choline derivatives carrying fluorescent reporter groups, affinity tags or bioorthogonal handles. These CDP-choline derivatives serve as co-substrates for the bacterial phosphocholinating enzyme AnkX from Legionella pneumophila, which transfers a phosphocholine moiety to the switch II region of its native target protein Rab1b during infection. We identified the octapeptide sequence TITSSYYR as the minimum recognition sequence required to direct the AnkX catalysed phosphocholination and demonstrated the functionalisation of proteins of interest carrying this recognition tag at the N- or C-terminus as well as in internal loop regions. Moreover, this covalent modification can be hydrolytically reversed by the action of the Legionella enzyme Lem3, which makes the labeling strategy the first example of a covalent and reversible approach that is fully orthogonal to current existing methodologies. Thus, the here presented protein functionalisation approach holds the potential to increase the scope of possible labeling strategies in complex biological systems. In addition to the labeling of tagged target proteins, a CDP-choline derivative equipped with a biotin affinity-tag was synthesised and used in pull-down experiments to investigate the substrate scope of AnkX and to elucidate the role of protein phosphocholination during Legionella pneumophila infection. / Proteiner utgör huvudbeståndsdelen av alla biomolekyler i en cell. Dessa är involverade i alla cellulära processer som bestämmer cellens egenskaper. För att förstå de cellulära processerna är det nödvändigt att förstå proteinernas funktion på molekylär nivå. Att studera proteiner i deras naturliga omgivning, det vill säga inuti en cell eller i ett cellextrakt, är en stor utmaning i dagens livsvetenskaper. Eftersom proteiner är kemiskt lika varandra så är det svårt att skilja ett från tusentals andra. Att specifikt märka proteiner för att skilja ut dem från bakgrunden har blivit ett viktigt arbetssätt i modern biokemi och cellbiologi. Avhandlingen beskriver utvecklandet av en ny metod för reversibel och kovalent enzymatisk märkning baserat på fosfokolinering/defosfokolinering av en kort aminosyrasekvens i intakta proteiner. En syntesmetod för att framställa onaturliga CDP-kolinderivat har etablerats vilket tillåter oss att framställa CDP-kolin som bär en funktionalitet, vilket kan vara ett färgämne eller en affinitetstagg. Dessa onaturliga CDP-kolinderivat accepteras som co-substrat av enzymet AnkX från Legionella pneumophila vilket transfererar den funktionaliserade delen av CDP-kolinderivatet till en kort aminosyrasekvens baserad på AnkX’s naturliga substrat vid infektion, det lilla GTPaset Rab1. Under avhandlingsarbetets gång identifierades den kortaste aminosyrasekvensen som känns igen av AnkX, endast de åtta aminosyrorna TITSSYYR är nödvändiga för igenkänning av AnkX. Dessa åtta aminosyror kan genetiskt infogas i början, slutet eller mitt i ett protein för igenkänning och funktionalisering via AnkX och våra syntetiska CDP-kolinderivat. Vid Legionellainfektion i eukaryota celler klyvs fosfokolineringen efter en viss tid, eftersom Legionella pneumophila producerar ett fosfodiesteras, Lem3, som tar bort de fosfokolineringar som AnkX har installerat när de inte längre behövs. Vi har använt Lem3 för att ta bort märkning i sekvensen TITSS(PC)YYR, vilket gör vår strategi helt reversibel. Vi har kunnat demonstrera att AnkX-Lem3 systemet accepterar ett brett spektrum av CDP-kolinderivat, vilket gör metoden till den första av sitt slag, eftersom den är fullt reversibel. Vi har vidare undersökt vilka proteiner AnkX reagerar med inuti celler, vi använde oss av ett CDP-kolinderivat funktionaliserat med biotin, vilket har tillåtit oss att fiska ut alla de proteiner som fosfokolineras av AnkX. Förutom de små GTPaserna i Rab-familjen så identifierade vi även IMPDH2, ett enzym som reglerar det hastighetsbestämmande steget i syntesen av guanosin-nukleotider. Detta är mycket intressant, eftersom det leder till frågan ifall Legionella pneumophila manipulerar sin värdcell genom att förändra mängden GTP i förhållande till ATP.

The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo. Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase. This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>