The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host. We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae. Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells.

Unilateral cortisol producing adenoma (CPA) is the most common cause of ACTH-independent Cushing’s syndrome and is surgically curable. On the other hand, adrenocortical carcinomas (ACCs) are rare and aggressive tumours. Although the overall survival of the patients with ACC is very poor, the outcome can be heterogeneous and vary significantly between the patients. This thesis comprises studies showing genetic and genomic events occurring in CPAs and ACCs, their functional impact and clinical correlations. The Wnt/β-catenin and cAMP/PKA signalling pathways are crucial in adrenal homeostasis and frequent mutations in members of these pathways (CTNNB1, GNAS, and PRKACA) are found in CPAs. Mutational analysis revealed that ~60% of the CPAs harboured mutations in either of these genes. Transcriptome signature exhibited increased expression of genes involved in steroidogenesis in PRKACA/GNAS mutated (Cluster1) tumours in comparison to CTNNB1 mutated /wildtype (Cluster2) tumours. In addition we have also observed that gain of chromosome arm 9q was the most frequent arm level copy number variation (CNV) occurring in CPAs and were exclusively present in Cluster2 tumours. We also discovered novel PRKACA mutations occurring in ACCs, causing activation of cAMP/signalling pathway.    Comprehensive analysis of Wnt/β-catenin signalling pathway in ACCs revealed novel interstitial deletions occurring in CTNNB1 leading to deletion of the N-terminus of β-catenin. This is a novel and yet another frequent event leading to activated Wnt/β-catenin signalling and downstream targets in ACCs. Both, mutations occurring in CTNNB1 and nuclear expression of its protein were associated with poor overall survival. Through multiregional sampling approach we discovered intra-tumour heterogeneity in ACC tumours. Although all the multiregions within a tumour showed presence of shared basal CNVs, they encompassed private CNVs, different ploidy levels and private mutations in known driver genes. We found intra-tumour heterogeneity in CTNNB1, PRKACA, TERT promoter and TP53 mutations as well as ZNRF3 and CDKN2A/2B homozygous deletions.

Glioblastoma multiforme is the most malignant primary brain tumor that affects adults, recognized by the World Health Organization as an aggressive grade IV astrocytoma. Patients diagnosed with this type of tumor are left with a poor prognosis even with the most advanced treatment available. The cancer is quite heterogeneous and is typically categorized into four different subtypes depending on genetic aberrations and patient characteristics. Furthermore, researchers have discovered a subpopulation of glioblastoma cells, known as cancer stem cells, which are thought to be resistant to current therapies and responsible for tumor reoccurrence and relapse. Previous studies, in addition to this one, have found that the differentiation of glioblastoma cells downregulate nestin protein expression, the selected stem cell marker, and upregulate glial fibrillary acid protein expression, the selected differentiation marker, using immunofluorescence. Thus, one alternative treatment option is to understand the mechanism underlying the differentiation of cancer stem cells. Four cell cultures representative of each glioblastoma subtype will be endogenously tagged using the genome editing system, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9). The representative stem cell marker, nestin, will be tagged with a green fluorescent protein, while the chosen differentiation marker, glial fibrillary acid protein, will be tagged with a red fluorescent protein. Several drugs were screened to analyze whether the drugs had a differentiation effect on the glioblastoma cells. As a result, strong evidence indicated that bone morphogenetic protein four upregulated glial fibrillary acid protein expression levels to the same extent as the differentiation control media using 5% fetal bovine serum. The goal of this study is to establish a method to directly monitor the differentiation process of glioblastoma cells as a novel molecular screening method. In this case, all glioblastoma cells, even the ones resistant to treatment, can be eliminated through an initial “pre-treatment” by forcing differentiation of cancer stem cells, making the cells more susceptible to the chemotherapy drugs. In the long run, glioblastoma patients would have a chance at a more positive prognosis; a longer life that is free of glioblastoma. / Master Thesis in Applied Biotechnology

Posttranslational modification (PTM) of proteins is essential to maintain homeostasis and viability in all eukaryotic cells. Hence, besides the sequence and 3D folding of a polypeptide, modification by multiple types of PTMs, ranging from small molecular groups to entire protein modules, adds another layer of complexity to protein function and regulation. The ubiquitin-like modifiers (UBLs) are such a group of evolutionary conserved protein modifiers, which by covalently conjugating to target proteins can modulate the subcellular localization and activity of their targets. One example of such a UBL, is the Ubiquitin related modifier 1 (Urm1). Since its discovery in 2000, Urm1 has been depicted as a dual function protein, which besides acting as a PTM, in addition functions as a sulfur carrier during the thio-modification of a specific group of tRNAs. Due to this dual capacity, Urm1 is considered as the evolutionary ancestor of the entire UBL family. At present, it is well established that Urm1, with help of its dedicated E1 enzyme Uba4/MOCS3, conjugates to multiple target proteins (urmylation) and that Urm1 thus plays important roles in viability and the response against oxidative stress. The aim of this thesis has been to, for the first time, investigate the role of Urm1 and Uba4 in a multicellular organism, utilising a multidisciplinary approach that integrates Drosophila genetics with classical biochemical assays and proteomics. In Paper I, we first characterized the Drosophila orthologues of Urm1 (CG33276) and Uba4 (CG13090), verified that they interact physically as well as genetically, and that they together can induce urmylation in the fly. By subsequently generating an Urm1 null Drosophila mutant (Urm1n123), we established that Urm1 is essential for viability and that flies lacking Urm1 are resistant to oxidative stress. Providing a molecular explanation for this phenotype, we demonstrated an involvement of Urm1 in the regulation of JNK signaling, including the transcription of the cytoprotective genes Jafrac1 and gstD1. Besides the resistance to oxidative stress, we have moreover (Manuscript IV) made an in-depth investigation of another phenotype displayed by Urm1n123 mutants, an overgrowth of third instar larval neuromuscular junctions (NMJs), a phenotype which is shared also with mutants lacking Uba4 (Uba4n29). To increase the understanding of Urm1 in the fly, we next employed a proteomics-based approach to identify candidate Urm1 target proteins (Paper II). Using this strategy, we identified 79 Urm1-interacting proteins during three different stages of fly development. Of these, six was biochemically confirmed to interact covalently with Urm1, whereas one was found to be associated with Urm1 by non-covalent means. In Manuscript III, we additionally identified the virally encoded oncogene Tax as a target of Urm1, both in Drosophila tissues and mammalian cell lines. In this study, we established a strong correlation between Tax urmylation and subcellular localization, and that Urm1 promoted a cytoplasmic accumulation and enhanced signalling activity of Tax, with implications for a potential role of Urm1 in Tax-induced oncogenesis. Taken together, this thesis provides a basic understanding of the potential roles and targets of Urm1 in a multicellular organism. The four studies included cover different aspects of Urm1 function and clearly points towards a highly dynamic role of protein urmylation in fly development, as well as in adult life.

Transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways are involved in many physiological processes during embryonic and adult life. TGF-β promotes epithelial to mesenchymal transition (EMT). We identified a gene target of TGF-β signaling, encoding the salt-inducible kinase 1 (SIK1). A potential substrate of this kinase, the polarity protein Par3, is an established regulator of tight junction assembly. SIK1 associates with Par3, can potentially phosphorylate Par3 and leads to its degradation, contributing to tight junction disassembly. Glioblastoma multiforme (GBM) is a common malignancy in the central nervous system, characterized by high heterogeneity, invasiveness, and resistance to therapy. One of the causes of heterogeneity and therapy-resistance is the existence of glioblastoma stem cells (GSCs). TGF-β signaling promotes self-renewal while BMP signaling induces differentiation of GSCs. Snail is a potent inducer of the EMT in carcinomas. However, in the context of GBM, Snail induces BMP signaling and represses TGF-β signaling through interaction with SMADs, the signaling mediators of TGF-β and BMP. In conclusion, Snail differentially regulates the activity of the opposing BMP and TGF-β pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs. Although profound changes in cell polarity is a hallmark of invasive malignancies, little is known about the role of the polarity machinery in tumor suppression. Patient transcriptomic data suggested low Par3 expression, correlating with poor survival of the GBM patients. Par3 silencing decreased the GSC self-renewal capacity and enhanced their invasiveness. Transcriptomic analysis indicates that loss of Par3 leads to downregulation of genes encoding mitochondrial enzymes that generate ATP. These results support a novel role of Par3 in GBM, beyond its contribution to junctional contacts between cells. Another regulator of TGF-β and BMP signaling is the liver kinase B1 (LKB1). According to GBM patient mRNA analysis, high levels of LKB1 correlate with poor prognosis. Silencing of LKB1 in GSCs impairs invasion and self-renewal capacity due to downregulation of genes involved in these processes. Moreover, loss of LKB1 induces mitochondrial dysfunction, leading to decreased ATP levels. Collectively, this thesis has delivered a group of novel regulatory pathways that control critical aspects of cancer cell polarity, invasion and stemness.

ZC3H11A (ZC3) protein has been reported to be part of the TREX (TRanscription-EXport) nuclear export system for mammalian cells. According to our previous publication, ZC3 not only plays an unelucidated role in the TREX complex, but also supports the growth of several human nucleus replicating virus, such as influenza virus, adenovirus (HAdV), herpes simplex virus and HIV. We thought to further elucidate the role of ZC3 in immunological stress based on previous observations that ZC3 was upregulated in stress condition. Our previous experiment tested the effect of knocking out ZC3 in HeLa cell then stimulating the cells with IL-1β to induce immunological stress. It showed that IL-1β stimulated ZC3 knockout Hela cells produce more than double fold IL6 compared to IL-1β stimulated HeLa Cas 9 wild type. Since IL-6 is downstream of NFkB signalling pathway, we aimed to explore a possible role of ZC3 protein in mammalian cell’s NFkB pathway. Our primary results showed that NFkB pathway might be more upregulated in ZC3 KO cells than in wild type HeLa Cas9 cells. This up-regulation was found to be correlated to defective IkBα inhibitory mRNA biogenesis in knockout cells. Our results indicate that ZC3 might play a role in IkBα inhibitory mRNA biogenesis, process, and/or export. Further work is needed to describe the exact role of ZC3 in IKBα mRNA biogenesis.

Panarthropoda is a monophyletic group comprised of arthropods and lobopods, molting animals with a segmented body, paired appendages, dorsal brain, and ventral nerve cords. Evolutionary Developmental Biology (EvoDevo) is an interdisciplinary field that seeks to understand how changes in development form the basis for variations in morphology and phenotypic evolution, including the genetic network underlying these processes. To study the evolution of panarthropods from such an EvoDevo perspective, one typically uses standard molecular techniques. A first step here is to investigate the expression of a gene of interest in order to find out where and when it is transcribed during development. A hallmark of EvoDevo studies is its comparative character, often with respect to model organisms such as the fruit fly Drosophila melanogaster. Recently developed single-cell RNA sequencing technologies allow the profiling of a plethora of gene expression on the level of individual cells, and thus provide a much more detailed insight into gene expression. In Paper I, I applied standard molecular techniques used in EvoDevo research such as PCR, gene cloning, probe synthesis and whole mount in situ hybridization, to investigate the embryonic expression patterns of the tiptop/teashirt (tio/tsh) and spalt (sal) genes in a range of arthropods representing all main groups of this phylum, and an onychophoran. In the arthropod model Drosophila, these genes act as trunk-specifiers, and the objective of my work was to find out if this is conserved in Arthropoda or even Panarthropoda as a whole. I provide comprehensive data on arthropod tio/tsh and sal expression, including the first data from an onychophoran. The results support the idea that tio/tsh genes are involved in the development of ‘trunk’ segments by regulating limb development. In addition, my data suggest that the function of Sal is unlikely to be conserved in trunk vs head development. Early expression of sal, however, is in line with a potential homeotic function of this gene, at least in Arthropoda. In Paper II, I provide an embryonic tissue dissociation protocol for embryos of the common house spider Parasteatoda tepidariorum that I developed and that I successfully applied for single-cell RNA sequencing. In addition, I report on the progress of this experiment, and provide and discuss preliminary results.

Sclerotinia sclerotiorum is a pathogenic fungus that infects around 400 species of host    plants. Stem rot disease caused by this fungus is economically disastrous for Brassica napus cultivators in Sweden. Due to the lack of disease resistant cultivars, disease management has been solely dependent on fungicide application. The current disease  prediction models are not scientifically accurate and take into account factors such as   weather, previous disease incidence, and conomic effects which often result in unnecessary and excessive use of fungicides by cultivators. Real-Time Polymerase Chain Reaction has proven to be the fastest, most accurate and reliable technique for detecting plant pathogens as it gives an idea about disease severity by measuring pathogen concentration in environmental samples. Reproducible and able qPCR assays have the potential of being the main principle on which more scientifically accurate plant disease prediction and management models an be developed. The aim of this study was to validate a previously established qPCR assay to detect S. sclerotiorum. An absolute quantification experiment     was performed by using plasmid DNA cloned with a target gene as template. Further,   three different qPCR machines  were compared  to make a plausible conclusion regarding    their sensitivity and efficiency in detecting minuscule amounts of DNA from the   environment. While a solid conclusion could not be reached regarding the sensitivity of    each of these machines, this study pointed out some basic trends about each machine    that may help researchers in selecting the most efficient qPCR system when working with detection of plant pathogens.

Fragment-screening by X-ray crystallography (XFS) is an expensive and low throughput fragment drug discovery screening method, and it requires a lot of optimization for each protein target. The advantages with this screening method are that it is very sensitive, it directly gives the three-dimensional structure of the protein-fragment complexes, and false positives are rarely obtained. The aim of this project was to help Sprint Bioscience assess if the advantages with XFS outweigh the disadvantages, and if this method should be used as a complement to their differential scanning fluorimetry (DSF) screening method. An XFS campaign was run using the oncoprotein vaccinia related kinase 1 (VRK1) as a target protein to evaluate this screening method. During the development of the XFS campaign, a diverse fragment library was created which consisted of 298 fragments that were all soluble in DMSO at 1 M concentration. The crystallization of the protein VRK1 was also optimized in this project to get a robust, high throughput crystallization set up which generated crystals that diffracted at higher resolution than 2.0 Å when they were not soaked with fragments. The soaking protocol was also optimized in order to reduce both the steps during the screening procedure and mechanical stress caused to the crystals during handling. Lastly, the created fragment library was used in screening VRK1 at 87.5 mM concentration with XFS. 23 fragment hits could be obtained from the X-ray crystallography screening campaign, and the mean resolution of the crystal structures of the protein-fragment complexes was 1.87Å. 11 of the 23 fragment hits were not identified as hits when they were screened against VRK1 using DSF. XFS was deemed as a suitable and efficient screening method to complement DSF since the hit rate was high and fragments hits could be obtained with this method that could not be obtained with DSF. However, in order to use this screening method a lot of time needs to be spent in optimizing the crystal system so it becomes suitable for fragment screening. Sprint Bioscience would therefore need to evaluate the cost/benefit ratio of using this screening method for each new project.

Antibodies are the gold standard molecular recognition elements and a cornerstone of molecular biology. They are used in immunoassays to precisely measure a specific analyte, but certain targets are especially challenging. Difficult targets include small molecules and molecules that do not induce an immune response. Aptamers are short oligonucleotides that can form 3-dimensional structures and bind targets with high specificity. Aptamers are smaller and more flexible than antibodies and could therefore solve this problem. In contrast to antibodies, aptamers are synthetically produced, so they can have affinity for molecules that do not induce an immune response. This also makes them cheaper, faster and more ethical to produce. They are also easily modified and have the ability to renature and can therefore be reused.  Our conclusions are that aptamers can outperform antibodies, especially for small molecule targets, and that the synthetic production of aptamers gives them a further advantage over antibodies. Our report compares several different types of detection methods that use aptamers and we conclude that fluorescence-based methods are the most easy to use with basic lab equipment, can be made similar to the ELISA kits in addition to giving highly sensitive detection. We describe a variety of fluorescence-based detection strategies but the optimal method will depend on the specific aptamer and target. The report also includes an ethical analysis where antibodies and aptamers are compared. This report is commissioned by Mercodia AB, a company that develops, manufactures and distributes immunoassays for biomarkers within the field of metabolic disorders. They commissioned this report in order to give an overview of how aptamers interact with their target, and also to compare aptamer-based detection strategies with sensitivity prioritized over selectivity.  This was done by literature research.