Many regions of the world are facing the problem with arsenic toxicity. Arsenic contamination has become a considerable threat to the environment triggering various big health issues for every life in that contaminated environment. Lysinibacillus sphaericus (B1-CDA) is an arsenic tolerant strain of bacteria that has been reported and characterized before by the researchers of the University of Skövde, Sweden. The bacteria were found to contain many arsenic responsive genes such as arsB, arsC, and arsR which are responsible for arsenic tolerance in the bacterium. The main focus of the current study was to characterize one of the arsB genes (gene-4251) of Lysinibacillus sphaericus B1-CDA by in silico and in vitro analyses in order to determine the molecular function of this gene. The in silico studies conducted by using the Iterative Threading Assembly and Refinement (I-TASSER) server predicted the tertiary structure of the ArsB protein and suggested that this protein is an intrinsic component of the membrane which primarily helps in the binding of metal ions and liberation of metabolic energy. To validate this predictive results, several in vitro experiments were performed. For complementation studies, the arsB gene was cloned from L. sphaericus B1-CDA and transferred to an arsB knock-out mutant of Escherichia coli JW3469-1. Both, the transgenic and mutant strains were grown under the arsenic stress of 50 mM for 96 hrs followed by measuring their growth and arsenic tolerance after every 24 hrs. Statistical analysis confirmed that there was a significant difference in growth between the transgenic and the mutant E. coli strains. The ICP-MS (Inductive Coupled Plasma-Mass Spectroscopy) analysis revealed that after 24 hrs of culture, the arsenic content in the cell-free broth of transgenic strain was reduced from 50 mM to 9.10 mM (81.8%), whereas the reduction in arsenic content by the mutant strain was from 50 mM to 9.80 mM (80.2%). These results suggest that the arsB gene is partly involved in the accumulation of arsenic inside the cells and this feature could be used for a large scale removal of arsenic from the contaminated environment.

Hair is important for thermoregulation, physical protection, sensory activity, seasonal camouflage and social interactions. Hair is produced in hair follicles (HFs), complex mini-organs in the skin devoted to this task. HFs are formed during embryonic development (morphogenesis) and new hair is continuously generated throughout life since the postnatal HF goes through cycles of regression (catagen), quiescence (telogen) and growth (anagen). The transcriptional regulation of this process is not well understood. The LIM-homeodomain transcription factor Lhx2 has previously been shown to be critically involved in epithelial-mesenchymal interactions during development of various organs and a potent regulator of stem cell function. We therefore elucidated the expression pattern and function of Lhx2 during hair formation. Lhx2 is expressed during both morphogenesis and anagen in cells scattered in the outer root sheath and in a subpopulation of the matrix cells in the proximal part of the hair bulb. Matrix cells are proliferating progenitor cells that differentiate into the components of the HF including the hair shaft. Expression is turned off during telogen, however Lhx2 expression reappears in the secondary hair germ immediately prior to initiation of the anagen stage. In contrast to previously published results Lhx2 appears to be expressed by progenitor cells distinct from those in the stem cell niche in the bulge region. The developmental-, stage- and cell-specific expression pattern of Lhx2 suggests that Lhx2 is involved in the generation and regeneration of hair. To test our hypothesis we used different genetically modified mouse strains. First we studied the effect of over-expression of Lhx2 in the HFs using a mouse model where transgenic Lhx2 expression could be induced in dorsal skin. Using this model we could show that Lhx2 expression is sufficient to induce anagen. To analyze the consequence of lack-of-function of Lhx2 we developed a mouse model where it is possible to conditionally inactivate Lhx2 and a mouse strain harbouring a hypomorphic allele of Lhx2. Mice where Lhx2 was conditionally inactivated in postnatal HFs were unable to regrow hair on a shaved area whereas all controls did regrow their hair. The mutant HFs initiated anagen but were unable to produce normal hair shafts. Thus Lhx2 is required for postnatal hair formation. We used the mouse strain carrying a hypomorphic allele of Lhx2 to study the role of Lhx2 during HF morphogenesis. Embryos homozygous for the hypomorphic allele form significantly less HFs compared to control embryos, and the HFs that do form in the mutant embryos appear to be developmentally arrested. These results suggest that Lhx2 is also important during HF morphogenesis. Thus, Lhx2 is an essential positive regulator of hair generation and regeneration.

Large-scale culturing of algae with the aim of production of economically valuable substances is a growing industrial sector. A common problem of such industrial algae cultures is that they are prone to suffer from fungal infections causing growth inhibition of the algae and reduced production of the target substances. Having sensitive and target specific methods for the early detection of these infections are of great economic importance since in later stages infections may be very hard to treat. There are several potential methods that can be used for infection detection such as visual detection through microscopy or DNA amplification based methods like PCR. This report presents a novel protocol for the early detection of the parasitic fungi Paraphysoderma sedebokerense infecting the green micro algae Haematococcus pluvialis. DNA from infected frozen algae cultures collected from the Gustavsberg production plant of Astareal AB was extracted using ZR Fungal /Bacterial DNA MiniPrep kit. Conventional PCR and SYBR Green quantitative PCR were further compared to each other and optimized to be more sensitive in detecting parasitic DNA. The annealing temperature and primer concentration were optimized. In addition, two different SYBR Green PCR kits and the use of fresh versus frozen algae cultures were compared. The quantitative PCR method was able to detect as low as 300 DNA copies accurately but occasionally was capable to give a signal corresponding to as little as 4 DNA copies in a reaction. Meanwhile the conventional PCR method could accurately detect as low as 600 DNA copies and occasionally 60 DNA copies. This report shows the possibility of improving the detection of P. sedebokerense infection in industrial H. pluvialis cultures by PCR technique, emphasizes the differences between conventional PCR and quantitative PCR and presents a working protocol for the quantitative PCR based monitoring of P. sedebokerense infections.

The natural occurrence of the non-mevalonate (also called MEP after the compound methyl-erythriol phosphate) pathway in the model cyanobacterium Synechocystis sp. PCC 6803 allows for biosynthesis of various high-value terpenoid compounds. An important co-factor of this pathway is thiamine pyrophosphate (TPP), coenzyme to the 1- deoxy-D-xylulose-5-phosphate synthase (DXS) reaction in the initial step of the MEP pathway. Concurrently, TPP biosynthesis derives partially from 1-deoxy-D-xylulose phosphate, the product of DXS. This makes TPP a potentially significant measure of MEP pathway activity, and thus terpenoid productivity. The implementation of a molecular biosensor for TPP could be a promising approach towards on-line assessment and feedback regulation of MEP pathway activity and this application is therefore investigated in this work. Riboswitches have been suggested as versatile RNA-based tools for biotechnological applications in bacteria, including various cyanobacterial species. However, TPP-responsive riboswitches have not been addressed in cyanobacteria thus far. This project therefore aims at the evaluation and implementation of TPP-responsive riboswitches in Synechocystis, using a yellow fluorescent reporter protein as quantitative readout of translational regulation. Native putative OFF-switches from two cyanobacterial species are investigated along with one synthetic ON-switch, originally based on the native riboswitch from E. coli. The induction effects are assessed on both RNA and protein level for both TPP and its precursor thiamine. The synthetic riboswitch is found to be effective in Synechocystis and is further examined for its dynamic range. Several protocols for fluorescence and transcript level experiments are developed. Several continuation experiments are suggested, including further investigation of the cyanobacterial OFF-switches.

Plants produce a large diverse array of organic compounds that may function in protection against pathogens. Diverse antifungal compounds were reported to exist in barley (Hordeum vulgare L.); the indole alkaloid, gramine, and the pathogenesis-related proteins are some of them. Both the N-methyltransferase that is involved in gramine biosynthesis and PR-17a were studied in barley upon infestation by the bird cherry-oat aphid (Rhopalosiphum padi). The effect of infestation by R. padi on induction of PR-17a and N-methyltransferase was investigated in different barley lines, susceptible and resistant. The gene expression of PR-17a was down-regulated in the susceptible cv. Golf and to some extent up-regulated at the first days in var. Lina and then down-regulated. The PR-17a was induced by the aphid infestation in the resistant line CI16145; the gene expression was stronger in the infested plants than in the controls. The different responses in resistant and susceptible lines indicate that the induced PR-17a may play a role in the resistance against aphid infestation. PR-17a was up-regulated systemically in the base in barley after infestation by R. padi. In the susceptible varieties Lina and Golf, the accumulation of N-methyltransferase did not increase with time from 1 day to 7 days after infestation, as determined by western blots with antibody raised against NMT from barley. The NMT-gene was down-regulated after 7 days infestation in both variety Lina and Golf both locally in the first leaf and in the base. Barley line CI16145 had no accumulation of NMT as was seen by western blotting. There was no induction of NMT in barley upon aphid infestation.

The goal of this project is to express both the normal CYP26 B1 and the spliced CYP26 B1 from human in Escherichia coli (E.coli) cells for further crystallization. This will be achieved by cloning in the DNA fragments into the Champion pET SUMO vector that is later transformed into E.coli cells. The CYP26 B1 contains a hydrophobic helix at the N-terminal of the protein, making both protein expression and crystallization difficult. Two variants of both full-length CYP26 B1 and the spliced variant will therefore be made, one with the trans-membrane helix present and one without the helix. The SUMO-vector will produce a fusion protein that will make CYP26 B1 more hydrophilic and improve the purification of the two proteins.

There is ample evidence that both diabetes as well as obesity leads to various metabolic disturbances that leads to oxidative stress. Oxidative stress has been shown  to  be  associated  with  congenital  malformations  of  which  neural  tube defects  and  cardiac  malformations  are  more  common.  The  cellular  and molecular  mechanisms  through  which  oxidative  stress  induces  these  defects during the developmental stage are not well known. Previous work in this field suggests   that   oxidative   stress   results   in   lipid   peroxidation   and   altered expression  of  genes  that  have  key  roles  in  the  developmental  processes.  The present study aimed to investigate gene alterations in embryos from pregnant diabetic  or  obese  rats.  Embryos  and  adipose  tissue  obtained  from  the  locally bred  diabetic  and  obese  Sprague-Dawley  inbred  rat  strain  were  subjected  to Total  RNA  extraction  and  were  quantified  using  Real  time  PCR  for  relative gene expressions analysis. The present study showed that maternal diabetes as well  as  obesity  diminishes  the  antioxidative  defense  mechanisms  by  down regulating the gene expressions of the key reactive oxygen species scavenging enzymes   copper   zinc   superoxide   dismutase   and   manganese   superoxide dismutase  in  day  10  rat  embryos.  There  was  also  altered  embryonic  gene expression  for  several  developmental  genes  due  to  maternal  diabetes  at gestational day 11 and 13 in rat embryos.

The presented work describes construction and analysis of recombinantly produced forms of Aleuria aurantia lectin (AAL). The binding properties of the produced AAL forms were studied using techniques such as tryptophan fluorescence, hemagglutination analysis, ELISA and surface plasmon resonance analysis. Lectins are proteins that are ubiquitous in nature with the ability to bind specifically to different types of carbohydrates. The physiological function of different lectins is not always known, but they are involved in many recognition events at molecular and cellular levels. In research, lectins are widely used for structural and functional studies of complex carbohydrates, and they are also used to detect changes in the carbohydrate pattern on glycoproteins in different diseases. With the use of recombinant technology it is now possible to refine properties of lectins such as decreasing the valency and alter specificity and affinity. This may be a way of constructing more suitable reagents for use in diagnostic glycosylation analysis assays. AAL has been extensively used in different types of research for its ability to bind the monosaccharide fucose and to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. AAL was expressed recombinantly (rAAL) and its properties was investigated. These studies reveled that one of the binding sites in rAAL had unusually high affinities towards fucose and fucosecontaining oligosaccharides with Kd-values in the nanomolar range. This binding site is not detected in AAL that have been exposed to fucose during its purification, and therefore we proposed that this site may be blocked with free fucose in commercial preparations of AAL. Normally lectin-oligosaccharide interactions are considered to be of weak affinity, so the finding of a high affinity site was interesting for the future study of recombinant forms of AAL. The next step was to produce recombinant AAL forms with decreased valency. This was done using site-directed mutagenesis. First a monomeric form of AAL (mAAL) was constructed and then a monovalent form of AAL, containing only one fucose-binding site (S2-AAL) was constructed. Both of these forms had retained ability to bind fucose. The binding characteristics of mAAL were similar to that of rAAL, but mAAL showed decreased hemagglutinating activity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and did not bind to sialylated fuco-oligosaccharides such as sialyl-LewisX. This study shows that molecular engineering techniques could be important tools for development of reliable and specific diagnostic and biological assays for carbohydrate analysis.

SAD-C, a gene belonging to the small short-chain alcohol dehydrogenase-like protein (SAD) gene family, is up-regulated in Pisum sativum (pea) when the plant is exposed to UV-B (280-320 nm) radiation. SAD-C has a molecular weight of about 28 kDa and adopts a tetrameric structure. The aim of this work was to purify the protein SAD-C from Pisum sativum when overexpressed in E. coli strain BL21 StarTM (DE3) One Shot®. The purification was facilitated by the presence of a His-tag consisting of six histidine residues at the C-terminal end of the protein. The purification trials of SAD-C were faced with problems since the sample fractions contained several other proteins as well. Several purification steps seem to be necessary for future trials. A crystallization trial was still set up and crystals were formed, but the crystals formed were probably not of SAD-C.

Introduction: Cancer is a group of about 200 different types of diseases. Cancer can appear in different cells and tissues in the body when abnormal cells grow in an uncontrolled manner and invade other tissues. A multistep process that occurs over a long period eventually leads to the onset of cancer. Kinases are enzymes that catalyze phosphorylation, that is, the reaction in which a phosphate group from ATP is transferred to another molecule. Kinase phosphorylation is an important mechanism for modifying proteins and thus regulating various cellular functions. Overactivity of kinases can lead to loss of control of signal transducer cascades controlled by kinases and it produces harmful effects such as cardiovascular disease, diabetes and cancer. Chronic myeloid leukemia (CML) is a rare blood cancer in which an abnormal BCR-ABL fusion protein occurs in neoplastic cells. This causes leukocytes to be produced abnormally and damages the bone marrow and blood. Dasatinib is a tyrosine kinase inhibitor indicated for the treatment of CML. Dasatinib inhibits the activity of the BCR-ABL kinase, but since many kinases are similar, it also binds to other kinases. Some of the kinases that dasatinib inhibits and that we analyzed in this study are ABL1, PTK6, MYT1 and STK10. Purpose: The purpose of this study was to explain with the help of the structures of the proteins ABL1, PTK6, MYT1 and STK10 why dasatinib binds well to some proteins but less well to others. Methods: Using the structures of the proteins, an analysis of interactions between dasatinib and the selected proteins has been performed. We then built a model for each protein with only the amino acids that binds to the drug and finally used these structures to estimate the binding energy ΔGb with the PM7 theoretical method. The PM7 method was used to calculate the energy of molecules. The results correspond to the enthalpy of formation ΔfH. The Gibbs energy is estimated by including the solvent, which also accounts for entropy changes, most of which are mediated by the solvent. The equation used to calculate Gibbs energy for protein-drug binding was: ΔGb = ΔfG(complex) - [ΔfG(drug) + ΔfG(protein)]. Results: The results show that there is a link between the number of dasatinib-protein interactions and Kd. Some proteins had several different structures in the same crystal. In such cases there was an extensive variation in the number of interactions between the different structures and dasatinib. The connection between the number of interactions and Kd was not perfect. Using the number of interactions we were able to distinguish between the proteins that bind best to dasatinib, which were ABL1 and PTK6, and the proteins that bind worst which were MYT1 and STK10. We could however not explain which protein binds best based on the number of interactions. The theoretical calculation of ΔGb follows the same trend (the more interactions there are in the structures, the lower is ΔGb). Discussion and conclusion: A relatively quick and easy calculation using a built model (dasatinib with protein binding interaction) could be used to distinguish between the best and worst binders. When it comes to the difference between ABL1 and PTK6, our analysis failed. A large difference between the calculated ΔGb values ​​for different structures of the same protein makes it difficult to choose the right structure for an analysis. A factor that could explain the discrepancy in the analysis is that the structure of the protein changes when it binds to the drug and such a change requires an investment of Gibbs energy. In general, experimental binding energies were higher than the calculated ones. For example, experimental ΔGb for MYT1 was -9 kcal/mol but the calculated ΔGb was -33.2 kcal/mol. This may be because the calculation with PM7 is too simplified.