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.

Prevalence of type 2 diabetes mellitus (T2DM) is still rising and even so in the juvenile population. Obesity is highly associated with increased risk for developing T2DM. The development has been related to elevated fasting concentrations of the pancreatic islet hormones insulin and glucagon as well as to an increase in plasma lipids that occurs during obesity. Specifically, research has indicated that chronic exposure to high levels of saturated free fatty acids cause dysfunction in islet alpha- and beta-cells. Fatty acids can affect islet cells by various mechanisms one of which is the G-protein coupled receptor FFAR1/GPR40. The role of the receptor in the effects of fatty acids on pancreatic islet-cell function is not clear. The aim of this thesis was to clarify the role of FFAR1 in how fatty acids, and more specifically the long-chain saturated fatty acid palmitate, affect insulin and glucagon secretion. In children and adolescents with obesity elevated fasting levels of insulin and glucagon were positively correlated with lipid parameters. Specifically, plasma triglycerides and free fatty acids were positively correlated with insulin and glucagon at fasting as well as with visceral adipose tissue volume. Elevated glucagon levels at fasting were associated with worsening of glucose tolerance in the same population. In in vitro studies of isolated human islets palmitate stimulated basal insulin and glucagon secretion as well as mitochondrial respiration at fasting glucose levels. The effect was mediated by FFAR1 and fatty acid beta-oxidation. At higher glucose concentrations the receptor was involved in the potentiation of insulin secretion from isolated human islets and insulin-secreting MIN6 cells. Furthermore, we found that the effects of palmitate on hormone secretion were associated with enhanced mitochondrial respiration mediated by FFAR1 Gαq signaling and PKC activity as well as increased intracellular metabolism induced by the fatty acid. When islets were exposed to palmitate for long time periods and in the presence of FFAR1 antagonist, normalized insulin and glucagon secretion during culture and insulin response to glucose after culture were observed. In MIN6 cells chronic palmitate treatment increased mitochondrial uncoupling irrespective of FFAR1 involvement. However, FFAR1 antagonism during palmitate exposure resulted in elevated respiration and reduced apoptosis. In conclusion, children and adolescents with obesity have elevated fasting concentrations of insulin and glucagon that correlate with free fatty acids and fatty acid sources. High glucagon levels are linked to worsening of glucose tolerance in these subjects. In vitro the combination or synergy of FFAR1 activation and intracellular metabolism caused by palmitate is decisive for both the short-term enhancement effects and the negative chronic effects on insulin and glucagon secretion.

Background: Tendinosis is a painful chronic, degenerative condition characterized by objective changes in the tissue structure of a tendon. Hallmark features in tendinosis tendons include increased number of cells (hypercellularity), extracellular matrix (ECM) degradation and disorganized collagen. The progression of these pathological changes seen in tendinosis is neither well characterized nor fully understood. Studies have suggested that there are biochemical and mechanical elements involved in tendinosis. From a biochemical perspective, studies have shown that the tendon cells, tenocytes, produce a number of neuronal signal substances/neuromodulators, such as substance P (SP) and acetylcholine (ACh), traditionally thought to be confined to the nervous system. Furthermore, it has been shown that the expression of these neuromodulators is elevated in tendinosis tendons as compared to normal healthy tendons. Interestingly, studies on other tissue types have revealed that both SP and ACh can induce tissue changes seen in tendinosis, such as hypercellularity and collagen disorganization. From a mechanical angle, it has been suggested that overload of tendons, including extensive strain on the primary tendon cells (tenocytes), causes the degenerative processes associated with tendinosis. In vivo studies have shown that in overloaded tendons, the presence of neuromodulators is elevated, not least SP, which also precedes the development of the tissue changes seen in tendinosis. This further supports the importance of combining biochemical factors and mechanical factors in the pathogenesis of tendinosis. Hypotheses: In this thesis project, we hypothesize: 1) that neuromodulators, such as SP and ACh when stimulating their preferred receptors, the neurokinin 1 (NK-1 R) and muscarinic receptors (mAChRs), respectively, can cause increased tenocyte proliferation; 2) that the effects of SP and ACh on tenocyte proliferation converge mechanistically via a shared signalling pathway; 3) that mechanical loading of tenocytes results in increased production of SP by the tenocytes; and 4) that SP enhances collagen remodelling by tenocytes via NK-1 R. Model system: In vitro studies offer insight into the function of healthy tendon matrix and the etiology of tendinopathy. Using a cell culture model of human primary tendon cells, highly controlled experiments were performed in this thesis project to study a subset of biological and mechanical parameters that are implicated in tendinosis. The FlexCell® Tension System was used to study the influence of mechanical loading on tenocytes. As well, a collagen gel contraction assay was used to examine the intrinsic ability of tenocytes to reorganise type I collagen matrices under the influence of the neuromodulator SP. Results: The studies showed that exogenous administration of SP and ACh results in increased tenocyte proliferation that is mediated via activation of the ERK1/2 mitogenic pathway when the preferred receptors of SP and ACh, the NK-1 R and mAChRs, respectively, are stimulated. Furthermore, the studies resulted in the novel finding that SP and ACh both converge mechanistically via transforming growth factor (TGF)-β1 and that a negative feedback mechanism is present in which TGF-β1 downregulates the expression of mAChRs and NK-1 R. The studies also showed that SP can increase collagen remodelling and upregulate expression of genes related to tendinosis. Finally, it was established that tenocytes are mechanoresponsive by showing that cyclic mechanical loading increases the expression of SP by human tenocytes. Conclusions: This thesis work concludes that stimulation of NK-1 R and mAChRs results in proliferation of human tenocytes, which both involve the ERK1/2 signalling pathway. It also shows that SP and ACh converge mechanistically via TGF-β1 in their contribution to tenocyte proliferation. The role of hypercellularity in tendinosis tissue is unknown. Possibly, it has different roles at different stages of the disease. The findings also show that SP increases collagen remodelling, suggesting that increased SP not only results in hypercellularity but also contributes to the collagen morphology in tendinosis.

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.

Anti-TNF alpha antibodies were among the first approved antibody drugs and now belongs to the best-selling drugs. Today, several companies are developing biosimilars to those drugs which will increase the access of medications and potentially reduce health care costs. There is a great demand for pharmacokinetic assays for anti-TNF-alpha drugs and the bridging assay format is a potential tool, mostly due to its high serum tolerance. This project at Gyros Protein Technologies AB aimed to investigate the properties of the solid phase on the Gyrolab and to utilize this to optimize the bridging assay to be used as a pharmacokinetic assay for a human antibody in the presence of serum. The solid phase was optimized by incorporating three reagents with increasing molecular weight and examining the column profiles generated. Furthermore, the capture reagent was titrated with b-BSA to avoid cross-binding of both arms of the antibody to the capture reagent. Since the background was relatively high, further optimization was done to reduce background and increase the signal to noise ratio. The performance of the optimized bridging assay was compared to alternative PK assay formats. The estimated sensitivity of the bridging assay was 5 ng/ml compared to 250 ng/ml for the indirect antibody assay and 2.5 ng/ml for the bridging assay using an anti-idiotypic antibody as detect. The optimized bridging assay performed well without dilution in buffer and was therefore used for affinity determination of Humira in neat serum. Variable concentrations of TNF-alpha were added to a fix concentration of Humira to compete with the interaction. Calculated KD-values were similar regardless of whether the measurements were performed in neat serum or after dilution in Rexxip buffer.

The innate immune system are a cooperation of many components – receptors being one of them. Both membrane-bound and cytosolic receptors play a large role in the defence system against pathogens and danger. NLRP3 is a receptor which assembles a protein complex called inflammasome in response to cytosolic stress and is responsible for many autoimmune diseases if it malfunctions. The activation of the NLRP3 inflammasome leads to secretion of inflammatory cytokines and in many cases to programmed cell death. The structure, function and activation of the NLRP3 inflammasome is still not fully understood and the urge to understand the mechanisms behind are important for future medical improvements. The aim was to anchor the NLRP3 inflammasome by the cell membrane - By Overlap PCR, the NLRP3 cDNA was fused extracellular and trans-membrane parts of the TLR4 cDNA to anchor the NLRP3 to the membrane and in turn analyse the inflammasome with LPI™ technology. Multiple primers and a TLR4 nucleotide were designed and the NLRP3 was amplified with specific overhangs by PCR. The fusion protein was successfully linked together by Overlap PCR but not confirmed by sequencing. The gene fusion demands high quality primers for amplification and further evaluation must be made to the details of the laboratory. To anchor the protein complex to the cell membrane, continue to be of full importance and can be an asset in many structural studies and biopharmaceuticals trials.