The eukaryotic nuclear envelope (NE), separates the nucleoplasm from cytoplasm and is made up of two concentric lipid membranes, the outer and the inner nuclear membranes (ONM and INM), the nuclear pore complexes (NPCs) and an underlying filamentous nuclear lamina. The INM contains hundreds of unique transmembrane proteins of which only a handful have been characterized. In this thesis, I aimed to understand the functional organization of proteins in the nuclear envelope and I focused on investigating the functions of a recently identified INM transmembrane protein, Samp1. We have developed a novel and robust approach, MCLIP, to identify specific protein-protein interactions taking place in live cells. Using MCLIP, we have shown that Samp1 interacts with proteins of the LINC complex, the nuclear lamina and components of the mitotic spindle. Samp1's specific interactions with a variety of binding partners, suggest that Samp1 plays important roles both in interphase and in mitosis.  We have also shown that Samp1 can provide a binding site at the INM for the GTPase Ran, a master regulator of protein interactions in interphase and in mitosis. Furthermore, we have also investigated the role of Samp1 in cell differentiation using two independent model systems. In human iPSCs, ectopic expression of Samp1 promoted differentiation despite pluripotent culture conditions. In C2C12 myoblast, depletion of Samp1 completely blocked differentiation into myotubes. The two studies complement each other and suggest that Samp1 has a strong differentiation promoting activity. Taken together, the findings in this thesis, give insights on the unexpected and unforeseen roles played by a transmembrane protein in different fundamental cellular process. / <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At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.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. Paper 5: Manuscript.</p>

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Accepted.</p>

A sophisticated evolutionary conserved innate immune system has evolved in insects to fight pathogens and to restrict damage in harmful (danger) situations including cancer. A significant amount of knowledge about different infection models in Drosophila has been generated in past decades, which revealed functional resemblances and implications for vertebrate systems. However, how Drosophila responds towards multicellular parasitic nematodes and in danger situations is still little understood. Therefore, the aim of the thesis was to characterize multiple aspects of the host defense in the two important contexts mentioned above. We analyzed the transcriptome profiles of nematode-infected Drosophila larvae with uninfected samples. For this we employed the entomopathogenic nematode Heterorhabditis bacteriophora with its symbiont Photorhabdus luminescence to infect Drosophila larvae. We found 642 genes were differentially regulated upon infection. Among them a significant portion belonged to immune categories. Further functional analysis identified a thioester containing protein TEP3, a recognition protein GNBP-like 3, the basement membrane component protein Glutactin and several other small peptides. Upon loss or reduced expression of these genes hosts showed mortality during nematode infections. This study uncovers a novel function for several of the genes in immunity. Furthermore, we investigated the cellular response towards nematodes. When we eliminated hemocytes genetically (referred to as hml-apo) in Drosophila, we found hml-apo larvae are resistant to nematodes. Subsequent characterization of hml-apo larvae showed massive lamellocyte differentiation (another blood cell type which is rare in naïve larvae), emergence of melanotic masses, up- and down-regulation of Toll and Imd signaling respectively suggesting a pro-inflammatory response. Moreover, a striking defective leg phenotype in adult escapers from pupal lethality was observed. We identified nitric oxide (NO) as a key regulator of these processes. We also showed that imaginal disc growth factors 3 (IDGF3): (a) protects hosts against nematodes, (b) is a clotting component and (c) negatively regulates Wnt and JAK/STAT signaling. To follow larval behavior in the presence or absence of nematodes we monitored Drosophila larval locomotion behaviors using FIMtrack (a recently devised automated method) to elucidate evasive strategies of hosts. Finally, we characterized host defenses in three Drosophila leukemia models with and without nematode infection. Taken together, these studies shed light on host responses in two crucial circumstances, nematode infections and danger situations. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Manuscript.</p>

Osteoarthritis is an inflammation-related disease that progressively destroys joint cartilage. This disease causes pain and stiffness of the joints, and at advanced stages, limitations to the movement or bending of injured joints. Therefore, it often restricts daily activities and the ability to work. Currently, there is no cure to prevent its progression, although certain damaged joints, such as fingers, knees and hips, can be treated with joint replacement surgeries. However, joint replacement surgeries of larger joints are very invasive operations and the joint replacements have a limited lifetime. Cell-based therapies could offer a way to treat cartilage injuries before the ultimate damage of osteoarthritis on articular cartilage. The development of novel treatments needs both a good knowledge of articular cartilage biology and tissue engineering methods. This thesis primarily investigates the effects of mechanical cyclic stretching, a 5% low oxygen atmosphere and the Rho-kinase inhibitor, Y-27632, on protein responses in chondrocytic human chondrosarcoma (HCS-2/8) cells. Special focus is placed on Rho-kinase inhibition, relating to its potential to promote and support extracellular matrix production in cultured chondrocytes and its role in fibroblast cells as a part of direct chemical cellular differentiation. The means to enhance the production of cartilage-specific extracellular matrix is needed for cell-based tissue engineering applications, since cultured chondrocytes quickly lose their cartilage-specific phenotype. A mechanical 8% cyclic cell stretching at a 1 Hz frequency was used to model a stretching rhythm similar to walking. The cellular stretching relates to stresses, which are directed to chondrocytes during the mechanical load. The stretch induced changes in proteins related, e.g., to certain cytoskeletal proteins, but also in enzymes associated with protein synthesis, such as eukaryotic elongation factors 1-beta and 1-delta. Hypoxic conditions were used to model the oxygen tension present in healthy cartilage tissue. Long-term hypoxia changed relative amounts in a total of 44 proteins and induced gene expressions of aggrecan and type II collagen, in addition to chondrocyte differentiation markers S100A1 and S100B. A short-term inhibition of Rho-kinase failed to induce extracellular matrix production in fibroblasts or in HCS-2/8 cells, while its long-term exposure increased the expressions of chondrocyte-specific genes and differentiation markers, and also promoted the synthesis of sulfated glycosaminoglycans by chondrocytic cells. Interestingly, Rho kinase inhibition under hypoxic conditions produced a more effective increase in chondrocyte-specific gene expression and synthesis of extracellular matrix components by HCS-2/8 cells. The treatment induced changes in the synthesis of 101 proteins and ELISA analysis revealed a sixfold higher secretion of type II collagen compared to control cells. The secretion of sulfated glycosaminoglycans was simultaneously increased by 65.8%. Thus, Rho-kinase inhibition at low oxygen tension can be regarded as a potential way to enhance extracellular matrix production and maintain a chondrocyte phenotype in cell-based tissue engineering applications.

The use of a surface plasmon biosensor fills a missing link in kinetic studies of enzymes, since it measures directly the interaction between biomolecules and allows determination of parameters that are determined only indirectly in activity assays. The present thesis deals with kinetic and dynamic aspects of ligand binding to two viral enzymes: the human cytomegalovirus (HCMV) protease and the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). The improved description of interactions presented herein will contribute to the discovery and development of antiviral drugs. The biosensor method provided new insights into the interaction between serine proteases and a peptide substrate, as well as substrate-induced conformational changes of the enzymes. The direct binding assay served as a tool for characterising the binding mechanism of HCMV protease inhibitors. Kinetic details of the interaction between HIV-1 RT and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were unravelled. The recorded sensorgrams revealed several forms of complexity. A general binding model for the analysis was derived from the data, describing a two-state mechanism for the enzyme and a high- and a low-affinity interaction with the inhibitor. Interaction kinetic constants were determined for the clinically used NNRTIs and several investigational inhibitors. The established method was applied to investigate the mechanism of resistance against NNRTIs. Amino acid substitutions in the NNRTI-binding site resulted in both decreased association rates and increased dissociation rates for the inhibitors. The K103N and the L100I substitution also interfered with the formation of the binding site, thereby facilitating inhibitor binding and unbinding. Finally, thermodynamic analysis revealed that, despite the hydrophobic character of the interaction, NNRTI binding was mainly enthalpy-driven at equilibrium. Large entropy contributions in the association and dissociation indicated that binding is associated with a dynamic effect in the enzyme.

Starting with the development of an enzymatic assay, where an enzyme in solution hydrolysed a solid-phase bound peptide, a model for the kinetics of enzyme action was introduced. This model allowed the estimation of kinetic parameters and enzyme activity for a system that has the peculiarity of not being saturable with the substrate, but with the enzyme. In a derivation of the model, it was found that the sensitivity of the signal to variations in the enzyme concentration had a transient increase along the reaction progress with a maximum at high substrate conversion levels. The same behaviour was derived for the sensitivity in classical homogeneous enzymatic assays and experimental evidence of this was obtained. The impact of the transient increase of the sensitivity on the error structure, and on the ability of homogeneous end-point enzymatic assays to detect competitive inhibition, came into focus. First, a non-monotonous shape in the standard deviation of progress curve data was found and it was attributed to the random dispersion in the enzyme concentration operating through the transient increase in the sensitivity. Second, a model for the detection limit of the quantity Ki/[I] (the IDL-factor) as a function of the substrate conversion level was developed for homogeneous end-point enzymatic assays. It was found that the substrate conversion level where the IDL-factor reached an optimum was beyond the initial velocity range. Moreover, at this optimal point not only the ability to detect inhibitors but also the robustness of the assays was maximized. These results may prove to be relevant in drug discovery for optimising end point homogeneous enzymatic assays that are used to find inhibitors against a target enzyme in compound libraries, which are usually big (&gt;10000) and crowded with irrelevant compounds.

This thesis investigates the anti-leukemic activity of terpenoids isolated from medicinal mushrooms and marine sponges, as well as their possible targets and mechanisms of action. In the first section, we focused on studying the triterpenoidal components of three triterpenoid-enriched medicinal mushrooms Antrodia cinnamomea, Ganoderma lucidum, and Poria cocos, which have been used in folk medicine for centuries and also developed into several contemporary marketed products. We isolated the major and characteristic triterpenoids from these mushrooms, together with six new lanostanoids (II-1–II-6). The anti-leukemic activity of the isolates was evaluated in vitro using MTT proliferative assay and seven of them exhibited potential anti-leukemic effect. The active lead compounds were further subjected to computational analyses utilizing the ChemGPS-NP tool. We established a database for the anti-leukemic relevant chemical space of triterpenoids isolated from these three medicinal mushrooms, which could be used as a reference database for further research on anti-leukemic triterpenoids. Our results indicated that the anti-leukemic effect of the active lead compounds was mediated not only through topoisomerases inhibition but also through inhibiting DNA polymerases. The second and third sections focused on isolation of anti-leukemic sesterterpenoids from sponges. The investigation of Carteriospongia sp. led to the isolation of two new scalarane-type sesterterpenoids (III-1 and III-2) and one known tetraprenyltoluquinol-related metabolite (III-3). All isolates exhibit an apoptotic mechanism of action against Molt 4 cells, found to be mediated through the disruption of the mitochondrial membrane potential (MMP) and inhibition of topoisomerase IIα expression. Detailed investigation of the apoptotic mechanism of action using molecular docking analysis revealed that compound III-1 might target Hsp90 protein. The apoptotic-inducing effect of III-3 was supported by in vivo experiment by suppressing the volume of xenograft tumor growth (47.58%) compared with the control. In the final section of this thesis we studied manoalide and its derivatives, sesterterpenoids isolated from the sponge Luffariella sp.. Manoalide has been studied as a potential anti-inflammatory agent for the last thirty years with more than 200 publications and 40 patents. However, the configurations at positions 24 and 25 were never revealed. In the current study, ten manoalide-type sesterterpenoids (IV-1–IV-10) were isolated from Luffariella sp. and their stereoisomers at positions 24 and 25 were identified and separated for the first time. The configuration at positions 24 and 25 showed to have a significant effect on the anti-leukemic activity of manoalide derivatives, with the 24R,25S-isomer exhibiting the most potent anti-leukemic activity. The apoptotic mechanism of action of compound IV-7 against Molt 4 cells was investigated, and the compound was found to trigger MMP disruption and intracellular reactive oxygen species (ROS) generation. Compound IV-7 also inhibited activity against both human topoisomerases, I and II. The in vivo experiment further supported the anti-leukemic effect of IV-7 with a 66.11% tumor volume suppression compared to the control.

Many different chemicals are used in the everyday life, like detergents and pharmaceuticals. However, their production has a big impact on health and environment as much of the raw materials are not renewable and the standard ways of production in many cases includes toxic and environmentally hazardous components. As the population and as the life standard increases all over the planet, the demand for different important chemicals, like pharmaceuticals, will increase. A way to handle this is to apply the concept of Green chemistry, where biocatalysis, in the form of enzymes, is a very good alternative. Enzymes do not normally function in industrial processes and needs modifications through protein engineering to cope in such conditions. To be able to efficiently improve an enzyme, there is a need to understand the mechanism and characteristics of that enzyme. Acyloins (α-hydroxy ketones) are important building blocks in the synthesis of pharmaceuticals. In this thesis, the enzyme alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber has been in focus, as it has been shown to display a wide substrate scope, also accepting aryl-substituted alcohols. The aim has been to study the usefulness of ADH-A as a biocatalyst towards production of acyloins and its activity with aryl-substituted vicinal diols and to study substrate-, regio-, and enantioselectivity of this enzyme. This thesis is based on four different papers where the focus of the first has been to biochemically characterize ADH-A and determine its mechanism, kinetics and its substrate-, regio-, and enantioselectivity. The second and third paper aims towards deeper understanding of some aspects of selectivity of ADH-A. Non-productive binding and its importance for enantioselectivity is studied in the second paper by evolving ADH-A towards increased activity with the least favored enantiomer through protein engineering. In the third paper, regioselectivity is in focus, where an evolved variant displaying reversed regioselectivity is studied. In the fourth and last paper ADH-A is studied towards the possibility to increase its activity towards aryl-substituted vicinal diols, with R-1-phenyl ethane-1,2-diol as the model substrate, and the possibility to link ADH-A with an epoxide hydrolase to produce acyloins from racemic epoxides.

Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost. Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell. The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization. In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water. Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy in both men and women, and one of the leading causes of cancer-related deaths worldwide. One frequently mutated pathway involved in oncogenesis in CRC is the RAS/RAF/MAP kinase pathway. Oncogenic activation of KRAS and BRAF occur in 30‒40% and 5‒15% of all CRCs, respectively, and the mutations are mutually exclusive. Even though KRAS and BRAF are known to act in the same pathway, KRAS- and BRAF-mutated CRCs have different clinical and histopathological features. For example, BRAF mutation in CRC is tightly linked to microsatellite instability (MSI) and a CpG island methylator phenotype (CIMP), which is not seen in KRAS-mutated tumours. BRAF-mutated CRCs are also more often found in right-sided tumours. However, the underlying molecular reasons for these differences have not yet been defined. The overall aim of this thesis was to investigate molecular differences between KRAS- and BRAF-mutated CRCs to understand how KRAS and BRAF mutations differentially affect tumour progression. We used an in vitro cell culture system to explore molecular differences between KRAS- and BRAF-mutated CRCs and verified our findings using CRC tissue specimens from the Colorectal Cancer in Umeå Study (CRUMS). We found that BRAF mutation, but not KRAS mutation, was associated with expression of the stem cell factor SOX2. Furthermore, SOX2 was found to be correlated to a poor patient prognosis, especially in BRAF-mutated cancers. We further investigated the role of BRAF in regulation of SOX2 expression and found that SOX2 is at least partly regulated by BRAF in vitro. We continued by investigating the functional role of SOX2 in CRC and found that SOX2-expressing cells shared several characteristics with cancer stem cells, and also had down-regulated expression of the intestinal epithelial marker CDX2. There was a strong correlation between loss of CDX2 expression and poor patient prognosis, and patients with SOX2 expression were found to have a particularly poor prognosis when CDX2 levels were down-regulated. In conclusion, in these studies we identified a subgroup of BRAF-mutated CRCs with a particularly poor prognosis, and having a cancer stem cell-like appearance with increased expression of SOX2 and decreased expression of CDX2. Tumour progression is regulated by interactions with cells of the immune system. We found that BRAF-mutated CRCs were more highly infiltrated by Th1 lymphocytes than BRAF wild-type tumours, while the opposite was true for KRAS-mutated CRCs. Interestingly, we found that part of this difference is probably caused by differences in secreted chemokines and cytokines between KRAS- and BRAF-mutated CRCs, stimulating different arms of the immune response. Altered levels of expression of miRNAs have been seen in several malignancies, including CRC. We found that BRAF- and KRAS-mutated CRCs showed miRNA signatures different from those of wild-type CRCs, but the expression of miRNAs did not distinguish KRAS-mutated tumours from BRAF-mutated tumours. In summary, our findings have revealed possible molecular differences between KRAS- and BRAF-mutated CRCs that may explain some of the differences in their clinical and histopathological behaviour.