Bacterial cytoskeleton is an exciting and relatively new field of research. Recent findings have proven that microbes are well-organized and neatly structured organisms. In this study we have shown that intermediate filament-like proteins with a characteristic rod domain architecture of coiled coil segments separated by non-coiled coil linkers, are widely spread among bacteria. We identified and characterized an intermediate filament-like protein (named FilP after filamentous protein) in Streptomyces coelicolor. It shares the characteristic biochemical property of eukaryotic intermediate filaments of formation of spontaneous filaments in vitro without requiring any energy or co-factor. We have provided here a preliminary model of its assembly in vitro. FilP also forms in vivo filaments in S. coelicolor hyphae, which are strongest at the sub-apical location of growing vegetative hyphae. We have proposed that FilP cytoskeletal network provides rigidity to the hyphae, especially at the growing tips, by interacting with an essential coiled coil protein DivIVA and possibly other partner elements, yet to be found. S. coelicolor is a well-studied model organism with a complicated life cycle. It germinates from a spore and spreads by forming branched vegetative hyphae. Lack of nutrients in the environment initiates formation of aerial hyphae in the air, perpendicular to the vegetative ones. The aerial hyphae differentiate into spore chains and eventually grey-pigmented dispersed individual spores are released. The signals involved in sporulation including cell division and chromosome segregation are not clear yet. We characterized here a novel locus consisting of two genes: a small putative membrane protein with no defined function, named SmeA and a member of the SpoIIIE/FtsK family, called SffA. The expression of this locus appears to be dependent on whiA and whiG-whiH-whiI pathways. This finding is intriguing as it can provide insight to the relationship between two apparently unrelated pathways, both leading to the same function of septation and maturation during sporulation.

<p>Glucose is a monosaccharide and fuel for body, it cannot pass through membrane by simple diffusion so, integral transmembrane proteins named glucose transporters (Gluts) are involved in the regulation of the movement of glucose between the extracellular and intracellular spaces within the body. GLUT1 and GLUT3 have previously been shown by cold detergent extraction methods to reside in distinct plasma membrane domains in non-polarized mammalian cells, with GLUT1, but not GLUT3, residing  in detergent-resistant membrane (DRM) domains. To confirm this observation under less invasive conditions, molecular fusion tags are inserted in the first external loop in Glut1 with biotin ligase acceptor peptide (BLAP) between Ser-55 and Ile-56 and in Glut3 with Acyl carrier peptide (ACP) in between Val-57 and Leu-58 respectively. These Glut fusion proteins will be used in order to confirm these observations by fluorescence recovery after photobleaching (FRAP) and single molecule fluorescence microscopy in live cells. hGLUT1-EGFP, hGLUT1 (<em>AgeI</em>)-EGFP recombinants were constructed and transfected human embryonic kidney cells (HEK-293) quantum dot images supports the fact that EGFP transfected cells uniformly and is distributed in the cell cytoplasm, hGLUT1-EGFP transfected cells and is localized to the cell membrane and hGLUT1 (<em>AgeI</em>)-EGFP transfected cells and located to the plasma membrane with high intensity.</p>

As global meat consumption is rising, the negative impact the animal husbandry sector has on the environment will increase. Greenhouse gas emissions have increased by 40 % during the last 200 years, and the animal husbandry sector is today responsible for 18 % of the total greenhouse gas emissions from food production. More environmentally friendly protein sources, such as soy and pea, must therefore be developed. Pisum sativum can (unlike the most popular meat alternative – soy) be grown all over Europe and might thus be a good alternative that allows for locally sourced alternatives to meat protein. Identifying genes with important agricultural properties might aid the development of pea cultivars with a more reliable protein content. One such gene was hypothesised to be the OMT gene, which is strongly expressed during the embryonic development of P. sativum and seems involved in functions such as seed storage and protein synthesis. Thirty-one accessions of P. sativum were tested to see if different improvement types differed from each other regarding protein content and seed weight, but no such differences were found. DNA was extracted from all accessions, sequenced, and successful sequences were tested to determine if variation in the gene correlated with protein content. Two haplotypes were identified, but there was no correlation between them and protein content found. Based on the results of this study, there is little evidence that the OMT gene correlates with protein content in the studied accessions.

Trichome formation in Arabidopsis lyrata is a naturally occurring trait with phenotypic polymorphisms within wild populations. In Swedish accessions of A. lyrata, three genetic polymorphisms situated in the coding region of GL1, an important transcription factor in trichome production, have been identified, and these are candidates for being the cause of a glabrous phenotype. In this study a complementation test has been performed to clarify which mutation/mutations that are detrimental for trichome formation. A set of constructs has been transformed into A. thaliana, a close relative to A. lyrata, and subsequent generations of plants were examined for phenotype, genotype and gene expression. A SNP (Single Nucleotide Polymorphism) in the R3 MYB domain of GL1, resulting in a change of an alanine to aspartic acid, was identified as the critical polymorphism. The other two mutations, two indels, were harmless to protein function. The inserted constructs were under control of the native GL1 promoter. Plants that, because of the SNP, lacked trichome production, became totally glabrous.

Hydrogen peroxide (H2O2), a reactive oxygen specie (ROS), is most commonly associated with oxidative stress causing cytotoxic effects on living cells. Oxidative stress has been implicated in various conditions including neurodegenerative diseases, autoimmune diseases and cancer. In addition H2O2 is produced as a defense mechanism against pathogens, as being released by activated phagocytes. In recent years, H2O2 has become established as an important regulator of signal transduction in eukaryotic cells. Hydrogen peroxide is generated both intracellularly and extracellularly in response to various stimuli including cytokines and growth factors. There are different mechanisms by which H2O2 is generated, facilitating signal transduction in cells; through NOX-system in miyochondria, via singlet oxygen, receptor/ligand interaction or by redox active metal ions. The HIV glycoprotein 120 (gp120) is associated with HIV dementia and it is known as a neurotoxin that causes neuronal damage. It has been proposed that free radicals may be involved in the pathogenesis caused by gp120. In addition the truncated form of thioredoxin (Trx80) is known to stimulate HIV replication in HIV infected cells, however, the exact mechanism is not known. A possible way both proteins may mediate their activity is by inducing H2O2 production. The aim of this study was to investigate H2O2 production induced by the proteins gp120 and Trx80. In order to detect H2O2 production an assay based on the fluorescent compound Amplex Red, was established. The assay was used to detect H2O2 released by gp120 and Trx80 in a cell-free environment, in a cell-system and in the presence of metal ions (copper ions) with a physiological reductant (ascorbate). We did not detect H2O2 production induced by gp120 and Trx80 respectively, using our assay, however, other ROS such as hydroxyl radicals may have been generated although they were not detectable with our method. Hence, further studies are needed in order to fully understand how gp120 and Trx80 mediate their activity.

L1 syndrome is an X-linked recessive disorder, characterised by congenital hydrocephalus, adducted thumbs, spastic paraplegia, agenesis of the corpus callosum and mental retardation. The disease is caused by mutations in the L1CAM gene, encoding a protein predominantly expressed in the nervous system. This protein has been implicated in a variety of processes including neuronal migration, neurite outgrowth and fasciculation, myelination, and long-term memory formation. L1 syndrome was suspected at genetic counselling of a family with a boy suffering from congenital hydrocephalus and mental retardation. Complete sequencing of L1CAM, performed by an external laboratory, revealed a novel mutation in the family, including a boy, affected with L1 syndrome, his sister, his mother and his maternal grandmother. To verify this mutation and to be able to detect mutations in the L1CAM gene locally in the future, a method for mutational analysis of this gene was set up using PCR optimisation and cycle sequencing. Sequencing of L1CAM was then performed on DNA samples from the four family members and the mutation was verified. A point mutation (c.3458-1G&gt;C) in the 5’ splice site of exon 26 was detected in all of them. This new, not previously described, mutation is supposed to cause a deletion of the 26th exon and a frameshift in the part of the protein encoded by exons 27 and 28. This means that almost the entire cytoplasmic domain of the protein would have a loss of function, explaining the symptoms affecting the boy.

Klorat som avfallsprodukt från pappersindustrin har lett till miljöproblem på grund av klorats toxiska verkan på växter och alger, och har även lett till bekymmer för människohälsan där klorat avfallet har kommit i kontakt med dricksvatten. För att åtgärda detta så har mycket forskning gjorts på bakterier med förmågan att reducera klorat till syrgas och kloridjoner, en anaerob process som vissa naturligt förekommande bakterier kan utföra. Med ökad kunskap om regleringen av denna kloratreducerande process, kan dessa bakterier utgöra en effektiv reningsprocess av pappersbrukens avloppsvatten.  Ideonella dechloratans är en sådan bakterie, den har ett genkluster som kodar för de kloratreducerande enzymerna. Nedströms för dessa finns en arsR-sekvens som tros att koda för en transkriptionsfaktor; och ytterligare information om denna transkriptionsfaktor kan möjligen bidra till förståelse av genuttrycket hos den kloratreducerande funktionen. Syftet med detta arbete är att transformera expressionsceller med förmågan att uttrycka arsR, så framtida försök kan göras för att identifiera potentiella bindningssäten för ArsR-proteinet. arsR-sekvensen amplifierades med primrar specifika för ändarna hos arsR, sekvensen ligerades i en pET-21a(+) vektor från Novagen och transformerades med BL21 (DE3) expressionsceller. Med en IPTG inducering kunde ett stort uttryck av olösligt ArsR observeras. Komplikationer med resultatet och framtida tillvägagångsätt diskuteras. / Chlorate as a waste product from the paper industry has caused environmental problems due its toxic effect on plants and algae and is also a concern for human health where chlorate has contaminated the tap water supplies. To address this issue, a great deal of research has been carried out on naturally occurring bacteria that can anaerobically reduce chlorate to oxygen and chloride ions. With additional knowledge of how this chlorate-reducing process is regulated, these bacteria may one day provide an effective purification process of wastewater from paper mills. Ideonella dechloratans is such a bacterium that has a gene cluster which encodes the chlorate-reducing enzymes. Downstream of this cluster is an arsR-sequence believed to encode a transcription factor, which could aid in the understanding of the gene expression for the chlorate-reducing operon. The goal of this research is to transform expression cells with the ability to express the arsR-sequence so that future trials can be made to identify any potential binding sites for the ArsR-protein. The arsR-sequence was amplified with primers specific to the ends of the arsR-sequence. The sequence was then ligated into a pET-21a(+) vector from Novagen and transformed with BL21 (DE3) expression cells. By IPTG inducing these transformants it was possible to observe a significant expression of insoluble ArsR. Complications with the outcome and future approaches are discussed.

Peripheral neuropathy includes a wide range of diseases affecting millions around the world, and many of these diseases have unknown etiology. Peripheral neuropathy in diabetes represents a large proportion of peripheral neuropathies. Nerve damage can also be caused by trauma. Peripheral neuropathies are a significant clinical problem and efficient treatments are largely lacking. In the case of a transected nerve, different methods have been used to repair or reconstruct the nerve, including the use of nerve conduits, but functional recovery is usually poor. Autophagy, a cellular mechanism that recycles damaged proteins, is impaired in the brain in many neurodegenerative diseases affecting animals and humans. No research, however, has investigated the presence of autophagy in the human peripheral nervous system. In this study, I present the first structural evidence of autophagy in human peripheral nerves. I also show that the density of autophagy structures is higher in peripheral nerves of patients with chronic idiopathic axonal polyneuropathy (CIAP) and inflammatory neuropathy than in controls. The density of these structures increases with the severity of the neuropathy. In animal model, using Goto-Kakizaki (GK) rats with diabetes resembling human type 2 diabetes, activation of autophagy by local administration of rapamycin incorporated in collagen conduits that were used for reconnection of the transected sciatic nerve led to an increase in autophagy proteins LC3 and a decrease in p62 suggesting that the autophagic flux was activated. In addition, immunoreactivity of neurofilaments, which are parts of the cytoskeleton of axons, was increased indicating increased axonal regeneration. I also show that many proteins involved in axonal regeneration and cell survival were up-regulated by rapamycin in the injured sciatic nerve of GK rats four weeks after injury. Taken together, these findings provide new knowledge about the involvement of autophagy in neuropathy and after peripheral nerve injury and reconstruction using collagen conduits.

This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4. The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s-1, the folding rate was found to be unusually slow for a protein of this size. The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre-transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H2O. In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (&lt; 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl. Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour.

Bacterial antibiotic resistance is an increasing global health problem, leaving few therapeutic options available for the treatment of pathogenic infections. The development of new antibiotics has been slow since their discovery more than 8 decades, therefore, monitoring the extent and distribution of antibiotic resistance is of great importance. The aim of this study was to determine the presence of antibiotic resistance genes in sludge samples obtained from three wastewater treatment plants (WWTPs) in Sweden. Samples were collected and analyzed for the presence of nalidixic acid (NA), chloramphenicol (CHL), and tetracycline (TC) resistance genes using polymerase chain reaction (PCR). The DNA extracted from Eskilstuna and MälarEnergi sludge showed the presence of NA and TC resistance genes, whereas Örebro sludge was found to have resistance for TC antibiotic genes. To validate the results, PCR detection for resistance genes was performed on Escherichia coli isolates from the sludge samples. Antibiotic susceptibility testing was used to confirm the genetic analysis for antibiotic resistance genes detection in these E. coli. The PCR results for TC resistance genes correlated between sludge PCR analysis and bacterial isolates for all 3 WWTPs. Based on the results obtained from the genotypic analysis of sludge and E coli, incomplete compatibility in regards to NA, and CHL were observed. However on the basis of antibiotic susceptibility testing, E coli isolates from MälarEnergi sludge samples unveiled the majority presence for antibiotic resistance genes. The results suggest that extra monitoring for the wastewater treatment facilities are vital to minimize the rising incidence of antibiotic resistant bacteria.