<p>Heparan sulfate (HS) belongs to the glycosaminoglycan family of polysaccharides and is found attached to protein cores on cell surfaces and in the extracellular matrix. The HS backbone consists of alternating hexuronic acid and glucosamine units and undergoes a number of modification reactions creating HS chains with alternating highly and low modified domains, where high degree of modification correlates with high negative charge. Fibroblast growth factors (FGFs) and their receptors (FRs) both bind to HS, which affect formation of the FGF–FR complexes on the cell surfaces. Activated FRs can trigger several intracellular signaling pathways leading thereby to diverse cellular responses. </p><p>Work presented in this thesis focuses on the effect of HS and its structures on FGF–FR complex formation and FGF-induced signaling. Studies with short, highly modified oligosaccharides and FGF1 and 2 combined with FR1c, 2c, 3c or 4 showed a correlation between the overall degree of modification and amount/stability of FGF–FR complexes. Our findings imply that several HS structures, differently modified but with the same negative charge density are equal in their ability to support complex formation. Co-application of oligosaccharides with FGF2 to HS-deficient cells and investigation of the thereby induced cell signaling confirmed our findings with a cell-free system. The oligosaccharide with the highest modification degree displayed the biggest impact on cell signaling, which was FGF2 concentration dependent. Studies with long HS polysaccharides with preserved high and low modified domains suggest that the proportion between these two types of domains and also the structure of the low modified domains are of importance for the FGF–HS–FR complex formation and cell activation capacity. </p><p>This work illuminates several aspects in how HS structure influences the interplay between FGFs and FRs and contributes to the understanding of what factors affect a cell’s response following FGF stimulation.</p>

<p>Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates. </p><p>Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates. </p><p>Several multivariate analysis tools, <i>e.g.</i> principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.</p>

<p>The chromatin integrity in sperm cells is vital for successful pregnancy. In this</p><p>study DNA-damage was evaluated in sperm cells from 50 men attending In Vitro Fertilization</p><p>(IVF) or Intra Cytoplasmic Sperm Injection (ICSI) treatment. Male semen samples were</p><p>purified with a two-shift gradient before the sperm cells were treated with the Halosperm® Test</p><p>Kit and evaluated for DNA-damage. The samples were divided in two groups according to DNAFragmentation</p><p>Index (DFI) of 30 % and the results correlated with fertilization, cleavage and</p><p>pregnancy rate. Men with DFI ≥ 30 % had a higher fertilization and pregnancy rate and a lower</p><p>cleavage rate compared to men with DFI ≤ 30 %. The conclusions were that fertilization in vitro</p><p>may be independent of the degree of DNA-damage, the embryonic development could be</p><p>seriously disrupted by damaged sperm cells, and the pregnancy rate showed no correlation to a</p><p>DFI threshold of 30 %.</p>

<p>Food allergy is an IgE-mediated immunological disease, which affects almost 4% of the adult population and up to 6% of children. Proteins from milk, egg, peanuts, soybean, wheat, fish and nuts are the main cause of food allergies. A less common allergen is pea protein. The National Food Administration analyses undeclared pea protein and contaminations of pea protein in foods using rocket immunoelectrophoresis and immunodiffusion. For both methods an antiserum against pea protein is needed. The aim of this study has been to characterize a newly developed rabbit-antiserum against pea protein. It is important to know if the antiserum is specific against peas, the detection as well as the quantification limits before it can be taken into use. The results of the study show that the antiserum was not absolutely specific, since it cross-reacted with chickpeas, fenugreek and lenses. However there is an "in-house" established PCR-method that can distinguish between chickpeas, fenugreek and peas and that method can be used as a complement to the rocket immunoelectrophoresis. The PCR-method cannot be used alone because it is not quantitative. Rocket immunoelectro¬phoresis detects 0,003% pea protein with purified IgG-antibodies from the antiserum.</p>

The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.

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.

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.