People are, in their daily lives, exposed to a number of airborne foreign compounds that do not normally affect the body. However, depending on the nature of these compounds, dose and duration of exposure, various airway symptoms may arise. Early symptoms are often manifested as upper airway mucosal inflammation which generates changes in protein composition in the airway lining fluid. This thesis aims at identifying, understanding mechanisms and characterizing protein alterations in the upper airway mucosa that can be used as potential new biomarkers for inflammation in the mucosa. The protein composition in the mucosa was studied by sampling of nasal lavage fluid that was further analyzed with a proteomic approach using twodimensional gel electrophoresis and mass spectrometry. Additionally, by studying factors on site through environmental examination, health questionnaires and biological analyses, we have tried to understand the background to these protein alterations and their impact on health. Respiratory symptoms from the upper airways are common among people who are exposed to irritative and microbial agents. This thesis have focused on personnel in swimming pool facilities exposed to trichloramine, metal industry workers exposed to metalworking fluids, employees working in damp and moldy buildings and infants diagnosed with respiratory syncytial virus infection. The common denominator in these four studies is that the subjects experience upper airway mucosal inflammation, which is manifested as cough, rhinitis, phlegm etc. In the three occupational studies, the symptoms were work related. Notably, a high prevalence of perceived mucosal symptoms was shown despite the relatively low levels of airborne irritants revealed by the environmental examination. Protein profiling verified an ongoing inflammatory response by identification of several proteins that displayed altered levels. Interestingly, innate immune proteins dominated and four protein alterations occurred in most of the studies; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin. Similarly, these proteins were also found in nasal fluid from children with virus infection and in addition a truncated form of SPLUNC1 and two other S100 proteins (S100A7-like 2 and S100A16), not previously found in nasal secretion, were identified. Altogether, the results indicate the potential use of a proteomic approach for identifying new biomarkers for the upper respiratory tract at an early stage in the disease process after exposure to irritant and microbial agents. The results indicate an effect on the innate immunity system and the proteins; SPLUNC1, protein S100A8 and S100A9 and alpha-1-antitrypsin are especially promising new biomarkers. Moreover, further studies of these proteins may help us to understand the molecular mechanisms involved in irritant-induced airway inflammation.

Viral proteases are important targets for anti-viral drugs. Discovery of protease inhibitors as anti-viral drugs is aided by an understanding of the interactions between viral protease and inhibitors. This thesis addresses the characterization of protease-inhibitor interactions for application to drug discovery and model system development. The choice of a relevant target is essential to molecular interaction studies. Therefore, full-length NS3 protein of hepatitis C virus (HCV) was obtained, providing a more relevant target and a better model for the development of HCV protease inhibitors. In addition, resistance to anti-viral drugs, a serious problem in the treatment of AIDS, prompted the investigation of resistant variants of human immunodeficiency virus (HIV) protease. Drug resistance was initially explored by characterization of the interactions between a series of closely related inhibitors and resistant variants of HIV protease, using an inhibition assay to determine the inhibition dissociation constants (Ki). The relationship between structure, activity and resistance profiles was not clarified, indicating that the effect of structural changes in the inhibitors and the protease are not predictable and must be analyzed case wise. It was proposed that additional kinetic characterization of the interactions was required and a biosensor-based method allowing for determination of affinity, KD, and interaction rate constants, kon and koff, was adopted. The increased physiological relevance of this method was confirmed, and the affinity data have better correlation with cell culture data. In addition, interactions between clinical inhibitors of HIV protease and enzyme variants indicate that increased dissociation rates (koff) are associated with the development of resistance. Thermodynamic characterization of the interactions between HIV-1 protease and clinically relevant inhibitors revealed distinct energetic characteristics for inhibitors. The resolution of the energetics of association and dissociation identified an inhibitor with unique interaction characteristics and confirmed the validity of using this method for further characterization of molecular interactions. This work resulted in the development of model systems for the analysis of kinetics, resistance and thermodynamic characteristics of protein-inhibitor interactions. The results give increased understanding of the biomolecular interactions and can be applied to drug discovery.

Hepatitis C is a serious disease that affects about 200 million people worldwide. No anti-HCV vaccine or specific anti-viral drugs are available today. Non-structural protein 3 (NS3) of HCV is a bifunctional serine protease/helicase, and the protease has become a prime target in the search for anti-HCV drugs. In this work, the complete HCV NS3 gene has been cloned and expressed, and the protein has been purified using affinity chromatography. An assay for measuring the protease activity of full-length NS3 protease has been developed and used for inhibition studies. A series of peptide-based inhibitors of NS3 protease varying in length, the composition of the side-chain and the N- and C-terminal groups have been studied. Potent tetra-, penta- and hexapeptide inhibitors of the NS3 protease were discovered. Hexapeptides with an acyl sulfonamide C-terminal residue were the most potent inhibitors of the NS3 protease, having nanomolar Ki-values. The selectivity of the inhibitors was assessed using other serine and cysteine proteases. NS3 protease inhibitors with electrophilic C-terminal groups were non-selective while those comprising a C-terminal carboxylate or acyl sulfonamide group were selective. All inhibitors with a small hydrophobic P1 side-chain residue were non-selective for the NS3 protease, being good inhibitors of human leukocyte elastase. This result highlights the importance of the P1 residue for inhibitor selectivity, which stems from the major role of this residue in determining substrate specificity of serine proteases. Electrophilic inhibitors often cause slow-binding inhibition of serine and cysteine proteases. This was observed with other proteases used in our work but not with NS3 protease, which indicates that mechanism of inhibition of NS3 protease by electrophilic inhibitors may not involve formation of a covalent bond. The structure-activity relationships obtained in this work can be used for improvement of peptide-based inhibitors of HCV NS3 protease towards higher inhibitory potency and selectivity.

A variety of HIV-1 protease inhibitors and their interactions with the enzyme have been characterized in order to identify novel and improved drugs against AIDS. The investigated inhibitors were represented by clinical and non-clinical inhibitors, active site and allosteric inhibitors, transition-state analogues and metal-ions. In addition, different enzyme variants were used to investigate the contribution of different amino acid residues to the interaction with different ligands. The problem of resistance has been addressed by exploring novel types of inhibitors, and resistant mutants of HIV-1 protease. A study resolving the inhibition of HIV-1 protease by Cu2+ showed that the enzyme can be allosterically inhibited and that copper inhibition is a result of an interaction with His-69 and a subsequent conformational change. Several types of transition-state analogues were analyzed with respect to their inhibition of wild-type and resistant mutants of HIV-1 protease. Unfortunately cyclic compounds were not found to be better than linear compounds. Moreover, it was not possible to identify structure-activity relationships that clearly correlated with efficacy towards mutants and a biosensor based method for more detailed kinetic studies was therefore adopted. By cross-linking the immobilized enzyme on the biosensor matrix, a stable surface was obtained and kinetic rate constants could be determined for the interaction between the enzyme and inhibitors. Additional improvements in the methodology involved identification of a more representative interaction model, allowing more detailed studies of interactions with resistant mutants and varying conditions. Finally, absorption to lipid membranes and interaction with human serum albumin and α1-glycoprotein by clinical drugs were studied in a simplified ADME model system for improvement of the earlier stages of drug development. These studies have revealed important characteristics of these drugs that can potentially be modeled into new compounds that have improved efficacy of both wild-type and resistant mutants of HIV-1 protease.

The present study has focused on characterising protease-ligand interactions in the context of drug discovery. The proteases that have been studied are human matrix metallopeptidase 12 (MMP-12), HIV-protease and Hepatitis C virus (HCV) NS3/NS4A protease. These studies have involved kinetic characterisation of protease-inhibitor interactions using biosensor technology, as well as determination of inhibition and activity regulation by using activity assays. The regulation of MMP-12 activity by calcium was proposed, based on the study of the calcium dependence of MMP-12 activity. Furthermore, it was shown that the high affinity of hydroxamate-based inhibitors of MMP-12 were due to slow dissociation of the enzyme-inhibitor complex by using a new biosensor assay for the study of interactions between MMP-12 and ligands. A study of the pH-dependency of protease-inhibitor interactions revealed that the interaction kinetics of HIV-protease inhibitors differed with pH in a way that could be related to the inhibitor structures. This suggested that the forces of interaction are different in the association and dissociation phases of an interaction. Furthermore, it demonstrated the usefulness of pH as a variable in characterising protein-ligand interactions. Results applicable in the discovery of drugs against Hepatitis C were obtained, with the analysis of structure-activity relationships of novel inhibitors. Furthermore, the mode of binding imposed by key functional groups of the inhibitors was explored by investigating the effect of pH on the interactions with NS3. The results show the importance of using appropriate model systems for drug discovery by selecting relevant targets and assay conditions. Furthermore, the usefulness of kinetic rate information in drug discovery is demonstrated. Thus, by contributing to the knowledge of protease-ligand interactions, applicable to both protease inhibitor interactions and protease activity regulation, this thesis is expected to have an impact on the field of protease inhibitor development and drug discovery in general.

This work is a comparison of a well-established and a novel, "green" and efficient technique to separate peptides of pharmaceutical interest. An attempt is made to derive the chromatographic retention behaviour from these techniques to a number of property descriptors derived from the linear sequence of amino acids. A set of therapeutic peptides were carefully chosen to be experimentally evaluated using in silico-based descriptor calculations. A principle component analysis was performed to assess the distribution of calculated descriptors for including peptides with variable properties. A diluent optimization study was also included to find the optimal diluent for peptides with minimal diluent effects and peak splitting phenomena. The results showed that the solvents tert-butanol and methanol performed best between 20-30 and 50 volumetric percent water as additive in SFC and HPLC, respectively. These diluents were then used for the peptides within the set to evaluate the retention and selectivity in HPLC and SFC. SFC performed well in terms of resolving power. Inparticular, SFC was able to separate Leuprolide and Triptorelin while HPLC was not. A comparison was also made in between the two stationary phases CN and XT, where a global selectivity was shown to be higher for CN. This work does also assess a novel method for determining solubility of analytes in supercritical fluid. The method was evaluated using the pharmaceutical compounds caffeine and aspirin and then used to determine solubility of Leu-Enkephalin in 20% (v/v%) methanol. The solubility of caffeine was determined to be 0.45 mg ml-1 in pure SF-CO2 under 140 bar pressure and 3.9 mg ml-1 for aspirin in 2.4% methanol. Both values correlated well with measurements from four acknowledged papers within this field. Leu-Enkephalin was found to have a solubility of 1.90 mg ml-1 using a solvent corresponding to the initial phase condition of the gradient used for peptide analysis in SFC. Further experimental work is required before the method can be implemented as a useful tool in preparative chromatography, however the results presented here show the compatibility of assessing biomolecules in both pure SF-CO2 and mixed with modifier. The possibility to determine solubility with additional modifier infers an important step of including and evaluating these compounds creating a solid support to subsequent large scale separation.

Inflammasomes are a group of protein complex that regulate inflammation throughcomplex signal transduction, although their specific mechanisms and structures have notbeen fully described. As the protein that kickstarts assembly of a type of inflammasome,NLRP3 is a key regulator of inflammation and may play a relevant role in the developmentof inflammatory diseases. In this project it has been attempted to perform a Gene Fusionbetween a segment of NLRP3 and regions of Toll-Like Receptor 4 by means of overlapextensionPCR, a technique that employs hybrid primers to create an overlap between bothsequences that can be filled by a polymerase, causing them to merge. Results suggest GeneFusion was successful, however cloning and expression of the construct have not beenachieved so far. If expressed as a fusion protein, the added transmembrane domain willanchor two domains of NLRP3 to the membrane, allowing more precise study of thecomposition and functionality of the inflammasome. Removal of the terminal domain ofNLRP3 will help determine its implication and relevance in the assembly process of theprotein complex.

There is a continuous search for novel enzymes to complement the abilities of today’s commercially available enzyme and find tailor-fit alternatives to suit the diverse array of bio-based industries. One application could be to increase biogas yield by finding substrate degrading proteases that can be added to the anaerobic digestion process and survive degradation themselves. A novel enzyme identified as a hypothetical dipeptidyl peptidase III, a zinc dependent metallo-protease, was found by a metaproteogenomics approach to be produced by the microorganisms of a thermophilic biogas process. The aim of this study was to express and refold a recombinant variant of the novel DPP III to its active form after production in inclusion bodies in Escherichia Coli. Assaying of refolding conditions was performed by stepwise dialysis and drip dilution. Nine attempts were performed based on findings in literature, although no other variant of DPP III has earlier been successfully refolded from inclusion bodies. The study resulted in a limited set of conditions of temperature, volumes, metal ions, salts and other additives being tested in the refolding buffers. Enzyme refolding and activation was monitored by the hydrolysis of the DPP III fluorescent substrate Arg-Arg β-naphthylamide trihydrochloride, alongside with measurements of protein concentration and SDS-PAGE. The novel DPP III was successfully purified but no definite strategy of producing correctly folded protein was found.

Fluorescens In Situ Hybridisering (FISH) är en cytogenetisk teknik som kan detektera genetiska sjukdomar och avvikelser i Deoxyribonukleinsyra (DNA) och Ribonukleinsyra (RNA). FISH börjar med kromosomutvinning av önskat analyspreparat, därefter får en direkt- eller indirekt fluorescensinmärkt probe (15-30 baspar lång) binda in till sin genetiska målsekvens via hybridisering. Preparatet kan sedan analyseras i fluorescensmikroskop för att bedöma om proben bundit in till sin målsekvens. I kursen ”Fördjupad laboratoriemetodik” för Biomedicinska analytikerprogrammet, Linnéuniversitetet utförs en FISH-laboration där resultaten varit otydliga och ej reproducerbara. Syftet med examensarbetet var att förbättra kurslaboration FISH som ges under kursen ”Fördjupad laboratoriemetodik”. Adherenta celler odlades till ≥ 3 x 106 i antal och till viss konfluens; primära endotelcell-linjen HCMEC till konfluenserna 60 % och 80 % och cancercell-linjerna VMM1 och H1915 till 80 % konfluens. Därefter skördades cellerna och dess respektive kromosomer utvanns. Kromosomerna undersöktes sedan med metoderna G-bandsfärgning, DNA-FISH och Multicolor-FISH (24X-probe) för tydliga och reproducerbara resultat. G-bandsfärgningen av HCMEC visade många hela interfasceller och få fria kromosomer för celler i 60 % konfluens. Både G-bandsfärgningen och DNA-FISH visade att HCMEC odlade till 80 % konfluens visade fria kromosomer från celler i metafas (celldelningsfas) där det fanns en svag signal för X-kromosomen. Multicolor-FISH-analys av VMM1 och H1915 gav tydliga resultat i fluorescensmikroskop där fria kromosomer var Multicolor-probeinmärkta; blå/aqua, röd och grön. Konklusionen är att vid kromosomutvinning från odlade adherenta celler bör dessa vara 80 % konfluenta. Detta för att ge tydliga och reproducerbara probeinfärgningar av kromosomer i metafas vid analys med Multicolor-FISH. Analys av 80 % konfluenta celler och användning av Multicolor-FISH-analys är en klar förbättring av kurslaborationen. / Fluorescens in situ hybridization (FISH) is used to detect cytogenetic aberrations and abnormalities of Deoxyribonucleic acid (DNA) and Ribonucleic Acid (RNA). The FISH begins with chromosome extraction of the desired cell preparation then a direct or indirect fluorescently labeled probe (15-30 base pair long) is hybridized to its genetic target sequence. The preparation can thereafter be analyzed in fluorescence microscope to see bound probe at chromosome level. In the course “Advanced laboratory methodology” for the Biomedical Scientist program, Linnaeus University, a FISH laboratory experiment is conducted where results have not been clear nor reproducible. The aim of this study was to improve the laboratory experiment FISH. Human Cardiac Microvascular Endothelial Cells (HCMEC) was grown to 60 % and 80 % confluence, to an estimated number of ≥ 3 x106, and analyzed by G-band staining and DNA-FISH. G-band staining showed many cells in interphase and few free chromosomes of cells with 60 % confluence. G-band staining and DNA-FISH showed that cells grown to 80 % confluence showed more free chromosomes from metaphase. The cancer cell lines VMM1 and H1915 were therefore grown to 80 % confluence and ≥ 3 x106. Multicolor-FISH on VMM1 and H1915 showed results from all painting probes blue/aqua, red and green. The conclusion is that in chromosomal extraction from cultured adherent cells should be 80 % confluent to give clear and reproducible probe staining of chromosomes in metaphase when assayed with Multicolor FISH. Analysis of 80 % confluent cells and the use of Multicolor FISH technology is a clear improvement to the “Advanced laboratory methodology” course.

Acute lymphoblastic leukemia, one of the most malignant cancer forms in children is commonly treated with the thiopurine 6-mercaptopurine (6-MP) in combination with a high dose of methotrexate (MTX). 6-Mercaptopurine is in the body metabolized by the enzyme thiopurine S-methyltransferase (TPMT). Polymorphic variants of TPMT express different catalytic activities, and for this reason the dosage of 6-MP needs to be individualized. In order to better optimize the treatment it is important to understand how mutations in TPMT affect its enzymatic activity. In this thesis we have investigated how the wild type and two variants of TPMT interact with different ligands using fluorescence and isothermal titration calorimetry. Experiments with MTX, ANS and furosemide resulted in a similar binding strength for the wild type and the variant TPMT*8, while the other variant TPMT*6 showed a slightly weaker binding. A binding affinity for polyglutamated MTX to TPMTwt was also determined which resulted in an almost twice as strong binding compared to MTX. Today’s methods to determine enzymatic activity are either based on radioactivity, time consuming or expensive. As an alternative the use of a spectrophotometric assay using 5-thio-2-nitrobenzoic acid (TNB) was investigated. The method showed positive results and could hopefully be adapted to plate readers in future experiments. Using 5.5’-dithiobis-(2-nitrobenzoic acid) (DTNB, also known as Ellman’s reagent) the amount of accessible thiol groups on the protein was estimated. This revealed a similar relationship between TPMTwt and TPMT*6, while the result for TPMT*8 was inconclusive.