Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.

Bacteria of the genus Streptococcus are common asymptomatic colonisers of humans and animals. As opportunistic pathogens they can however, depending on their host’s immune status and other circumstances, cause mild to very severe infections. Streptococci are highly intertwined with specific host species, but can also cause zoonosis or anthroponosis in more uncommon hosts. Prolonged and reoccurring infections require immune evasion strategies to circumvent detection and eradication by the host’s immune defence. A substantial part of the immune defence against bacterial pathogens is mediated by immunoglobulins. This thesis is based on work to identify and characterise immunoglobulin degrading enzymes secreted by different Streptococcus species as a means to sabotage and evade antibody-mediated immune responses. Stoichiometric and kinetic analysis of the IgG degrading enzyme IdeS from the important human pathogen S. pyogenes revealed that IdeS cleaves IgG, opposed to previous publications, as a monomer following classical Michaelis-Menten kinetics. The IdeS homologue of S. suis, IdeSsuis, did however not cleave IgG, but was highly specific fo rporcine IgM. S. suis was found to possess yet another protease, IgdE, capable of cleaving porcine IgG. Both of these proteases were shown to promote increased bacterial survival in porcine blood during certain conditions. IgdE is the founding member of a novel cysteine protease family (C113). Novel streptococcal members of this protease family were shown to specifically degrade certain IgG subtypes of the respective Streptococcus species’ main host. The observed substrate specificity of IgdE family proteases reflects the host tropism of these Streptococcus species, thereby giving insights into host-pathogen co-evolution. The abundance of immunoglobulin degrading enzymes among Streptococcus species indicates the importance of evasion from the antibody mediated immune responses for streptococci. These novel identified immunoglobulin degrading enzymes of the IdeS and IgdE protease families are potential valid vaccine targets and could also be of biotechnological use.

In the work presented this thesis, two different conditions that are classified as protein misfolding diseases: Alzheimer's disease and lysozyme amyloidosis and proteins that could have a beneficial effect in these diseases, have been studied using Drosophila melanogaster, commonly known as the fruit fly. The fruit fly has been used for over 100 years to study and better understand fundamental biological processes. Although the fruit fly, unlike humans, is an invertebrate, many of its central biological mechanisms are very similar to ours. The first transgenic flies were designed in the early 1980s, and since then, the fruit fly has been one of the most widely used model organisms in studies on the effects of over-expressed human proteins in a biological system; one can regard the fly as a living, biological test tube. For  most proteins, it is necessary that they fold into a three-dimensional structure to function properly. But sometimes the folding goes wrong; this may be due to mutations that make the protein unstable and subject to misfolding. A misfolded protein molecule can then aggregate with other misfolded proteins. In Alzheimer's disease, which is the most common form of dementia, protein aggregates are present in the brains of patients. These aggregates are composed of the amyloid-β (Aβ) peptide, a small peptide of around 42 amino acids which is cleaved from the larger, membrane-bound, protein AβPP by two different enzymes, BACE1 and γ-secretase. In the first part of this thesis, two different fly models for Alzheimer’s disease were used: the Aβ fly model, which directly expresses the Aβ peptide, and the AβPP-BACE1 fly model, in which all the components necessary to produce the Aβ peptide in the fly are expressed in the fly central nervous system (CNS). The two different fly models were compared and the results show that a significantly smaller amount of the Aβ peptide is needed to achieve the same, or an even greater, toxic effect in the AβPP-BACE1 model compared to the Aβ model. In the second part of the thesis, these two fly models for Alzheimer’s disease were again used, but now to investigate whether lysozyme, a protein involved in our innate immune system, can counteract the toxic effect of Aβ generated in the fly models. And indeed, lysozyme is able to save the flies from Aβ-induced toxicity. Aβ and lysozyme were found to interact with each other in vivo. The second misfolding disease studied in this thesis is lysozyme amyloidosis. It is a rare, dominantly inherited amyloid disease in which mutant variants of lysozyme give rise to aggregates, weighing up to several kilograms, that accumulate around the kidneys and liver, eventually leading to organ failure. In the third part of this thesis, a fly model for lysozyme amyloidosis was used to study the effect of co-expressing the serum amyloid P component (SAP), a protein that is part of all protein aggregates found within this disease class. SAP is able to rescue the toxicity induced by expressing the mutant variant of lysozyme, F57I, in the fly's CNS. To further investigate how SAP was able to do this, double-expressing lysozyme flies, which exhibit stronger disease phenotypes than those of the single-expressing lysozyme flies previously studied, were used in the fourth part of this thesis. SAP was observed to reduce F57I toxicity and promote F57I to form aggregates with more distinct amyloid characteristics. In conclusion, the work included in this thesis demonstrates that: i) Aβ generated from AβPP processing in the fly CNS results in higher proteotoxicity compared with direct expression of Aβ from the transgene, ii) lysozyme can prevent Aβ proteotoxicity in Drosophila and could thus be a potential therapeutic molecule to treat Alzheimer’s disease and iii) in a Drosophila model of lysozyme amyloidosis, SAP can prevent toxicity from the disease-associated lysozyme variant F57I and promote formation of aggregated lysozyme morphotypes with amyloid properties; this is important to take into account when a reduced level of SAP is considered as a treatment strategy for lysozyme amyloidosis.

The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV. By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided. Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease. This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays. Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor. Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen. The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.   Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.

Endocytosis is a fundamental cellular process by which cells transport material from the outside to the inside of the cell through the formation of membrane invaginations that bud off from the plasma membrane. This process is important for nutrient uptake, regulating cell surface receptors and the overall plasma membrane composition. Cells have several different types of endocytic pathways where clathrin- mediated endocytosis is the most studied. Importantly, pathogens and secreted virulence factors bind to cell surface receptors and hijack the endocytic pathways in order to enter host cells. Depending on their size and molecular composition, pathogens and virulence factors are thought to make use of distinct endocytic pathways into the cell. This thesis focuses on early host cell interactions with virus, bacterial membrane vesicles and a pore-forming toxin, with a particular emphasis on endocytic mechanisms and plasma membrane repair. During entry of pathogens, it is thought that interactions with specific cell surface molecules drive the recruitment of endocytic proteins to the plasma membrane. Viruses possess a very defined molecular composition and architecture, which facilitate specificity to these interactions. We found that Adenovirus 37, a human ocular pathogen, binds to αVβ1 and α3β1 integrins on human corneal epithelial cells and that this interaction is important for infection. In contrast to viruses, membrane vesicles shed from Helicobacter pylori are heterogeneous in size and molecular composition. These vesicles harbour various adhesins and toxins that may facilitate binding to the cell surface and recruitment of different endocytic pathways. We developed a quantitative internalization assay and showed that the H. pylori vesicles were internalized mainly via clathrin-mediated endocytosis but were also capable of exploiting other endocytic pathways. Damage to the plasma membrane disrupts cellular homeostasis and can lead to cell death if not repaired immediately. Although endocytic mechanisms have been shown to be important for plasma membrane repair, little is known about their specific role. Listeriolysin O (LLO) is a bacterial toxin that can form pores in the plasma membrane and disrupt cellular homeostasis. We developed a reporter system for real-time imaging of the endocytic response to LLO pore formation. We found that two clathrin-independent endocytic pathways were important for plasma membrane repair. However, they were not directly involved in removing LLO pores from the plasma membrane. Our data suggests that these endocytic systems might rather influence membrane repair by their ability to regulate the plasma membrane composition, shape and tension. In conclusion, this thesis describes how pathogens and their virulence factors make use of specific mechanisms to enter host cells as well as revealing new insights on the role of the endocytic pathways in plasma membrane repair.

Infection of barley (Hordeum vulgare) leaves with the rust fungus, Puccinia hordei, causes changes in the host protein synthesis. This thesis analyses these changes in the barley cultivar Triumph following inoculation of 7-day-old leaves with either a virulent or an avirulent race of P. hordei. The initial approach was to isolate membrane-bound polysomes from infected leaves, translate them in vitro and analyse the translation products. These products include the integral membrane proteins which were expected to be involved in the response of the host to the pathogen. A method based on differential centrifugation in the presence of a ribonuclease-inhibiting buffer was developed for separating membrane-bound polysomes from the rest of the cytoplasmic polysomes. Membrane-bound polysomes were found to comprise one fifth of the total polysomes in the leaves. Analysis of the translation products of membrane-bound polysomes by SDS-PAGE showed them to be of higher average molecular weight than those from free polysomes. Comparison of polypeptides produced by membrane-bound polysomes from healthy and inoculated plants showed some differences however the low yield of membrane-bound polysomes made it difficult to obtain conclusive results. Thus it was decided to isolate total polysomes by including 1% Triton X-100 in the extraction buffer. Polysomes were extracted from 12 to 72 h after inoculation. Infection caused a decline in yield of polysomes during this period when compared with healthy leaves of the same age. Polysomes isolated 16 h after inoculation with the virulent race were 20% less efficient at translation than polysomes from control leaves. In contrast polysome isolated from leaves inoculated with the avirulent race were 20% more efficient. Analysis of the labelled translation products by SDS-PAGE and fluorography showed relative increases in the synthesis of some proteins by 16 h after inoculation with either race when compared to products from healthy leaves. Protein synthesis in the infected plants was further analysed by in vivo labelling and one- and two-dimensional PAGE. The fluorographs revealed increased synthesis of a group of proteins from 58 to 116 kDa starting 12 h after inoculation with either race of P. hordei; confirming the results from the polysome translations. Two polypeptides with molecular weights of about 66 kDa were found to increase following infection only with the virulent race. By three days after inoculation with either fungal race the most obvious change in protein synthesis was a marked decrease in the synthesis of the two most prominent polypeptides with molecular weights of 15 and 51 kDa which were considered to be the subunits of ribulose bisphosphate carboxylase. The elicitor hypothesis, in attempting to explain cultivar-specific resistance in plants, postulates that resistance is controlled by the interaction of specific fungal elicitors and plant receptors and that this interaction which only occurs between resistant hosts and avirulent pathogens triggers specific gene expression leading to resistance. This hypothesis does not fit the situation in the barley-P. hordei interaction as protein synthesis showed similar changes following infection with either a virulent or an avirulent race.

This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system. The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence. Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA > dG > dT > dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.

The detection and analysis of biomolecules, such as proteins, are of great interest since these molecules are fundamental for life and our health. Due to the complexity of biological processes, there is a great advantage of studying proteins in their natural context, for example by using bioimaging. The objective of this doctoral thesis has been to develop, implement and evaluate techniques for the use of proteinspecific affinity reagents in diverse bioimaging platforms for analysis of protein expression in situ in cells and tissues. To be able to visualize a desired protein in situ using affinity reagents, reporter labels are needed. A novel technique for labeling of antibodies on solid phase was developed. This method offers simultaneous purification, concentration and labeling of an antibody sample, giving highly predictable and reproducible results, in a miniaturized format. Another study demonstrates the use of an alternative affinity reagent, the Affibody molecule, in bioimaging as well as other immunoassays. As a relevant proof-of-principle, an Affibody molecule binding the HER2 receptor was site-specificly labeled and employed for analysis of HER2 protein expression in cells and tissue using immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation and flow cytometry. Furthermore, it is shown how antibody-based bioimaging approaches can be applied for systematic analysis of protein expression in terms of subcellular localization and expression levels in cell lines. The systematic subcellular localization of nearly 500 proteins was performed using IF and confocal microscopy. Global analysis of expression levels of nearly 2000 proteins in a panel of cell lines using IHC and automated image analysis, revealed that most proteins are expressed in a cell size dependent manner. Two normalization approaches were evaluated and found to allow for protein profiling across the panel of morphologically diverse cells, revealing patterns of protein over- and underexpression, and proteins with stable as well as with lineage specific expression were identified. Finally, the value of antibody-based, bioimaging proteomics as a platform for biomarker discovery is demonstrated. The identification and in depth study of a candidate biomarker for colorectal cancer, SATB2, is described using both IHC and IF bioimaging. Results from extended analyses of tumor biopsies showed that detection of SATB2 protein using IHC provides a clinically relevant diagnostic tool with high specificity and sensitivity to aid in diagnosis of colorectal cancer. Furthermore, the study demonstrated a potential prognostic role of SATB2, as decreased expression was associated with a significantly shorter overall survival in patients with advanced colorectal cancer. / QC 20100824

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.