Cervical cancer is the second most common cancer in women worldwide. The principal cause of cervical cancer is infection with human papillomavirus (HPV). HPV-16 is a high-risk virus and it is responsible for a high portion of all HPV-caused cancers. The HPV-16 genome consists of early and late genes. The virus initially infects basal cells of the cervix epithelium and in these cells early genes are expressed, whilst late genes, L1 and L2, are only expressed in the upper cell layers of the epithelium. Proteins encoded by the late genes are highly immunogenic, thus it is speculated that expression of the late genes earlier in the virus life cycle could lead to clearance of the virus due to interference of the immune system.     The aim of this study was to treat reporter cell lines with three different small molecule drugs to see if they had the ability to induce HPV-16 late gene expression. The reporter cell lines used in this study had been previously created by transfecting HeLa-cells with plasmids representing the HPV-16 genome. In these plasmids, L1 is replaced with a CAT reporter gene that encodes the CAT protein, which can be easily quantified using a sandwich ELISA.     Upon treating the reporter cell lines with TPA, a significant induction of late gene expression was detected. Furthermore, treatment with valproic acid showed some induction of late gene expression. In conclusion, TPA and valproic acid was deemed to have potential to act as a candidate drugs for treatment of HPV infections.

High stromal PDGF receptor B expression was shown to have strong prognostic value in a studyinvolving over 600 breast cancer patients however, the molecular role of the receptor in tumordevelopment remains unclear. In this project we studied the spatial distribution and expressionlevels of a panel genes and markers associated with PDGF signaling, in breast cancer tumormicroenvironment (TME) using a newly developed technique -in situ sequencing. The techniquerelies on padlock probes which we validated with corresponding RNA sequencing, microarray,and immunohistochemistry data. Our results showed that high PDGF receptor B mRNA colocalizedwith markers of two pathways, TGFβ and Hedgehog signaling; this suggests that theymight contribute to the PDGF-receptor B-driven tumor growth. We also showed that stromalPDGF signaling is stimulated predominantly by tumor cells. Finally, further expression profilingof each individual gene revealed that CXCL14 was mainly expressed in the stroma, ACTA2expression was enriched in the tumor/stroma boundary while the stem-cell marker, OCT3, wasexpressed in the interior of the tumor cells.

Trapezius myalgia is a pain condition that usually develops in people with repetitive and stressful work tasks, which can lead to chronic widespread pain (CWP). This work compares protein expression levels in healthy women with those in women who have chronic widespread pain, including pain in the trapezius muscle, by using a proteomic approach.  Twodimensional gel electrophoresis and silver staining with a subsequent digital quantification of protein spots was used to detect spots which had significantly higher protein levels in either group. Preparative gels were made and stained with SYPRO Ruby, the protein spots that were significantly different between the groups were picked from the SYPRO Ruby gels and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF. The optical density of seven protein spots were significantly decreased in the trapezius muscle of the CWP subjects; however the standard deviations were notably high. Five of the seven proteins could be identified as desmin, creatine kinase B-type, serum albumin, heat shock protein beta-1 and slow skeletal muscle troponin T. Apart from serum albumin, all these proteins can possibly be responsible for pain in the trapezius muscle in CWP. In conclusion, this study demonstrates that two-dimensional gel electrophoresis in combination with mass spectrometry is a powerful tool to identify potential biomarkers of musculoskeletal pain in subjects with CWP. The results may provide new insights into the mechanisms and patho-physiology of trapezius myalgia.

The serotonergic system is involved in a broad range of functions in both vertebrates and invertebrates and is highly conserved across taxa. Serotonin is an important monoamine acting in the brains of humans and animals, and has large and varying influences on many aspects of an individual’s life. For example, in humans, serotonin modulates feelings of happiness and in fruit flies, higher levels of serotonin increase aggression. In humans, an abnormal serotonergic system can result in health issues, such as depression and obsessive compulsive disorders, for which medications have been developed, including selective serotonin reuptake inhibitors (SSRI). Because the serotonin system has a large influence on human health, understanding how it functions is of great interest to researchers. Using comparative studies to explore differences in the serotonin system across taxa can provide insight into the mechanistic details of the system. To investigate if the serotonin system is comparable between vertebrates and invertebrates, a literature study with particular focus on receptors and proteins involved was performed. In addition, this report takes part in an experimental study investigating the effect of the SSRI fluoxetine in Mediterranean field crickets.  Fluoxetine reduced exploration propensity of crickets, which was reversed, compared to what was anticipated and compared to effects seen in vertebrates. The literature review suggests that serotonin receptors are quite similar, but that proteins differ more when comparing vertebrates and invertebrates. This offers a likely explanation as to why results of studies on these different groups of animals may differ.

Cells are encapsulated by a biological membrane in order to separate the cell interior from the surrounding environment. Different lipids and proteins compose the membrane and present a semi-permeable barrier for the diffusion of ions and molecules across the lipid bilayer. Membrane proteins also mediate the passage of signals between the interior and the exterior of the cell.   To ensure the proper functioning of membrane proteins, it is essential that nascent membrane proteins are correctly integrated into the lipid bilayer to be able to fold and oligomerize.  In this thesis, an engineered protein containing two natural transmembrane segments followed by an additional test segment, has been used as a model protein to study (i) sequence requirements for translocon-mediated insertion of the test segment, (ii) dynamics of nascent membrane proteins undergoing translocon-mediated insertion and (iii) to carry out an extensive mutagenesis scan to identify critical residues in the mammalian arrest peptide Xbp1 that enhances translational stalling in the ribosome. This provides a toolbox of arrest peptides with different stalling strengths that will be useful for force measurements on nascent protein chains. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Osteoarthritis and osteochondral defects are ailments that are increasing in frequency as the lifespan of the population increases and sedentary lifestyle becomes more common. Osteoarthritis is an inflammatory disease that causes the progressive degeneration of articular surfaces and the underlying bone. Accidents and injuries can cause osteochondral defects similar to osteoarthritis. In both cases the structure of the articular cartilage fails, leading to pain and disability. Articular cartilage has a naturally poor ability to regenerate since there is no vasculature and it is aneural. The sparse chondrocytes mainly act to maintain the healthy extracellular matrix. Once the defect is severe enough, a surgical intervention becomes necessary. For small defects and young patients, a cell-based treatment can be used, whereas for larger defects and severe osteoarthritis a partial or whole joint arthroplasty is performed. Methods to repair osteochondral defects have been improving over the years as the inter-disciplinary understanding of joints, and what is required to repair them, has increased. However, there are still issues to solve in order to achieve consistently good results in both joint replacement and repair of cartilage. The main issue faced with current techniques used for joint replacement is poor integration of the artificial joint, leading to loosening at the bone interface over time, while cartilage repair techniques face the problem of generating mechanically inferior fibrocartilage. It is known that surface chemistry and structures at micro- and nanoscale influence cell behaviour, which can be utilised to guide their attachment, proliferation and phenotype. Scaffold-free approaches and mechanical stimulation have previously given promising results in generating articular neocartilage. This thesis aims at exploring tools and solutions to the problems involved in implant integration, chondrocyte expansion and neocartilage tissue engineering. We hypothesised that 1) ultra-short pulsed laser deposition can be used to create biocompatible coatings; 2) micropillars with nanoscale features can improve the maintenance of the chondrocyte phenotype in culture and 3) hypergravity can aid in the production of more native-like neocartilage constructs. Our studies showed that ultra-short pulsed laser ablation can be used to create various surfaces for studying cell behaviour. Cell viability was slightly higher on a rough titanium oxide, whereas the cell area was significantly smaller on rough titanium oxide, indicating a lower amount of focal adhesions. Nanopatterned microstructures were not capable of maintaining the chondrocyte phenotype in culture, but they were not disadvantageous either. Hypergravity might help in creating a native-like distribution of collagen and proteoglycans. The constructs were more uniform in shape, but biomechanically the constructs were not different from non-centrifuged controls.

The aim of the studies included in the thesis is to better understand the interplay between neuronal activity-dependent gene regulation and the bioactive vitamin A metabolite all-trans-retinoic acid (RA) during postnatal development, refinement and maintenance of precise neuronal connectivity using the olfactory sensory neuron (OSN) in the olfactory epithelium (OE) of genetically modified mice as a model. We show that: Inhibition of RA receptor (RAR)-mediated transcription in OSNs reduces expression of the olfactory cyclic nucleotide-gated (CNG) ion channel, which is required for odorant receptor (OR)-mediated stimulus transduction. This, results in increased OSN death and errors in precise connectivity. The increased cell death may be a consequence of reduced intrinsic excitability and/or reduced influx of Ca2+ ions while the errors in connectivity may be due to altered OR-dependent expression of axonal guidance proteins, such as Kirrel-2 and Neuropilin-1. Expression of the RA catabolic enzyme Cyp26B1 in OSNs is positively regulated by RAR-mediated transcription as well as sensory stimulation in a CNG channel-dependent manner. This shows that neuronal activity and local vitamin A metabolism are parts of novel regulatory feedback loop controlling precise connectivity and neuronal survival. The feedback loop may be a form of homeostatic plasticity in response to global changes in neuronal activity. BACE1, an enzyme is implicated in Alzheimer´s disease, and Cyp26B1 are inversely regulated by CNG channel-dependent sensory stimulation. Cyp26B1 expression is switched on at birth, forms a topographic expression gradient in OE and inhibits BACE1 expression into an inverse counter gradient. Taken together these results reveal a novel neuronal activity-dependent mechanism by which sensory stimuli can shape spatial gene expression via altered RA bioavailability. Increased Cyp26B1 expression stimulates turnover of OSNs during adult neurogenesis by a non-cell-autonomous mechanism. The gradient of Cyp26B1 expression correlates with spatially-regulated diversification of OSNs into subpopulations that express different subsets of OR genes. Cyp26B1 expression influences spatial OR diversification of OSNs by two different mechanisms. In the ventrolateral OE, Cyp26B1 inhibits OR expression by blocking OSN differentiation at a stage that may be associated with the cell intrinsic mechanism regulating OR gene choice. In the dorsomedial OE the expression frequency of some ORs is unaltered while other increases, presumably as a consequence of neuronal activity-dependent competition. A probable function of graded and activity-dependent Cyp26B1 expression is to form a topographic partitioning of the olfactory sensory map into functional domains, which gradually differ from each other with regard to experience-driven plasticity and neurogenic potential along the dorsomedial-ventrolateral axis of OE.

Messenger RNAs are transcribed and co-transcriptionally processed in the nucleus, and transported to the cytoplasm. In the cytoplasm, mRNAs serve as the template for protein synthesis and are eventually degraded. The removal of intron sequences from a precursor mRNA is termed splicing and is carried out by the dynamic spliceosome. In this thesis, I describe the regulated splicing of two transcripts in Saccharomyces cerevisiae. I also describe a study where the mechanisms that control the expression of magnesium transporters are elucidated. The pre-mRNA retention and splicing (RES) complex is a spliceosome-associated protein complex that promotes the splicing and nuclear retention of a subset of pre-mRNAs. The RES complex consists of three subunits, Bud13p, Snu17p and Pml1p. We show that the lack of RES factors causes a decrease in the formation of N4-acetylcytidine (ac4C) in tRNAs. This phenotype is caused by inefficient splicing of the pre-mRNA of the TAN1 gene, which is required for the formation of ac4C in tRNAs. The RES mutants also show growth defects that are exacerbated at elevated temperatures. We show that the temperature sensitive phenotype of the bud13Δ and snu17Δ cells is caused by the inefficient splicing of the MED20 pre-mRNA. The MED20 gene encodes a subunit of the Mediator complex. Unspliced pre-mRNAs that enter the cytoplasm are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway, which targets transcripts that contain premature translation termination codons. Consistent with the nuclear retention function of the RES complex, we find that NMD inactivation in the RES mutants leads to the accumulation of both TAN1 and MED20 pre-mRNAs. We also show that the cis-acting elements that promote RES-dependent splicing are different between the TAN1 and MED20 pre-mRNAs. The NMD pathway also targets transcripts with upstream ORFs (uORFs) for degradation. The ALR1 gene encodes the major magnesium importer in yeast, and its expression is controlled by the NMD pathway via a uORF in the 5’ untranslated region. We show that the ribosome reaches the downstream main ORF by a translation reinitiation mechanism. The NMD pathway was shown to control cellular Mg2+ levels by regulating the expression of the ALR1 gene. We further show that the NMD pathway targets the transcripts of the vacuolar Mg2+ exporter Mnr2p and the mitochondrial Mg2+ exporter Mme1p for degradation. In summary, we conclude that the RES complex has a role in the splicing regulation of a subset of transcripts. We also suggest a regulatory role for the NMD pathway in maintaining the cellular Mg2+ concentration by controlling the expression of Mg2+ transporters.

Fish has for a long time been a very important source of protein for human kind and with the world population at an all-time high, 7.5 billion and rapidly growing, the demand for fish as a food source is also at an all-time high and rapidly increasing. This has in turn led to overexploitation of many of the fish stocks of the world ocean’s and in many cases to depletion of fish stocks. The demand for sustainable food sources and sustainable usage of the world ocean’s fish stocks is therefore a subject with great deal of interest today. Much of the fish caught today are used for production of fish meal for usage as fish food at fish farms, which also increases the depletion of fish stocks around the globe. One way of dealing with this problem is to replace the fish meal as protein source in fish feed with protein from agricultural crops which in many cases are done today by usage of soy bean protein. This however poses another problem as the agricultural crops take up vast amount of land, in many cases obtained by diminishing the rainforests in the area. Another usage for the soybean would be as a direct human food source. Agricultural products are also dependent on environmental conditions to ensure reasonable production. The problems related to production of fish meal and soy has sparked the idea of using microorganisms for production of Single cell protein for usage as protein source in fish feed. Single cell protein can be produced in closed fermentation vessels and can be produced at a controlled rate and under controlled manners, while taking up negligible land space. During this thesis, the production of single cell protein from spent sulfite liquor using the filamentous fungi Paecilomyces variotii was examined. The aim of the project was to examine the effect of cultivation parameters (i.e., pH, temperature and nutrients) on the production of biomass as well as the protein content of the biomass. The correlation of the biomass growth and protein content have also been examined. The project was carried out by performing several experiment cultivations using spent sulfite liquor provided by Domsjö Fabriker in Örnsköldsvik. This process enables the utilization of a residual stream from the pulp industry which gives this process a huge environmental upside compared to similar processes as for example the commercial production of Quorn (a Single cell based food product) which utilizes pure glucose. The results showed that the protein content will steadily decrease as the biomass production increases hence it is desirable to keep the cultivation time at a minimum while maximizing biomass production during that time frame. It also points towards that the highest protein content is present in the young cell mass. The key conclusion from this thesis is however that it is possible to lower the pH of the cultivation from pH 6 down to pH 4.5 while still maintaining the biomass production and increasing the protein content. The highest obtained protein content was 62.7% at pH 4.5. The high protein content might be due to a slightly longer lag phase in the beginning of the cultivation which yields a higher number of younger cells in the final broth thus increasing protein content. Running the process at a lower pH is a huge advantage for industrial implementation as this on large scale means significant lower amounts of chemicals needed for pH adjusting of the spent sulfite liquor which renders the process much more economical. This is because pH adjustment today is one of the most costly process steps in the production of bioethanol from spent sulfite liquor.

The increasing emergence and spread of antibiotic-resistant bacteria is a serious threat to public health. Of particular concern are Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant to a large number of antibiotics and thus our treatment options are rapidly declining. In addition to the increasing number of antibiotic-resistant bacteria, a major problem is that many of the antibiotics at our disposal are ineffective against Gram-negative bacteria. This is partly due to the properties of the outer membrane (OM) which prevents efficient uptake. The overarching goal of this thesis was to investigate how the OM of the Gram-negative bacterium E. coli could be weakened to improve the activity of antibiotics. In the first two papers of my thesis (paper I + II), I investigated the periplasmic chaperone network which consists of the two parallel pathways SurA and Skp/DegP. This network is essential for the integrity of the OM and strains lacking either SurA or Skp are defective in the assembly of the OM, which results in an increased sensitivity towards vancomycin and other antimicrobials. We identified a novel component of the periplasmic chaperone network, namely YfgM, and showed that it operates in the same network as Skp and SurA/DegP. In particular, we demonstrated that deletion of YfgM in strains with either a ΔsurA or Δskp background further compromised the integrity of the OM, as evidenced by an increased sensitivity towards vancomycin. In the remaining two papers of my thesis (paper III + IV), the goal was to characterize small molecules that permeabilize the OM and thus could be used to improve the activity of antibiotics. Towards this goal, we performed a high-throughput screen and identified an inhibitor of the periplasmic chaperone LolA, namely MAC-13243, and showed that it can be used to permeabilize the OM of E. coli (paper III). We further demonstrated that MAC-13243 can be used to potentiate the activity of antibiotics which are normally ineffective against E. coli. In the last paper of my thesis (paper IV), we undertook a more specific approach and wanted to identify an inhibitor against the glycosyltransferase WaaG. This enzyme is involved in the synthesis of LPS and genetic inactivation of WaaG results in a defect in the OM, which leads to an increased sensitivity to various antibiotics. In this paper, we identified a small molecular fragment (compound L1) and showed that it can be used to inhibit the activity of WaaG in vitro. To summarize, this thesis provides novel insights into how the OM of the Gram-negative bacterium E. coli can be weakened by using small molecules. We believe that the two identified small molecules represent important first steps towards the design of more potent inhibitors that could be used in clinics to enhance the activity of antibiotics. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>