Arsenic is a toxic metalloid existing everywhere in the nature. It is toxic to most organisms and considered as human carcinogen. Arsenic contamination leads to severe health problems with diseases like damage of skin, lung, bladder, liver and kidney as well as central nervous system. It is very likely that too much chemicals such as cadmium and arsenic in the consumed foods can also lead to increased birth defects like spinal bifida. In some regions of South-East Asia, like Bangladesh, Burma, Thailand and India, arsenic contamination of human population via either food chain or drinking water is now considered as a national threat for mankind. As arsenic can be found everywhere in nature it may come in contact with food chain very easily through either water or cultivated crops. In South-East Asia the major cultivated crop is rice and it is the staple food for people in many countries like Bangladesh, Burma and Thailand. Cultivation of rice plants requires water either from rainfall or irrigation. Irrigated water in some regions of South-East Asia is highly contaminated with arsenic and by this way arsenic is accumulated in the rice corn which consumed not only by human but also by animals, birds and fishes. In order to avoid arsenic contamination in human food it is essential to find out a way to inhibit arsenic uptake in cultivated plants. Alternatively, we can also find out a way to metabolize arsenic "in plant". In my experiment I have used Arabidopsis thaliana as a model plant to isolate an arsenic reductase (ACR2) gene. This gene has been reported to be involved in metabolism, transport and sequestration of arsenic in plants. My thesis works include studies of the ACR2 gene based on characterization of the corresponding SALK mutants. All plants were exposed to arsenics under in vitro conditions. It was observed that the SALK mutants were more sensitive to arsenics in comparison with the wild type control plants. ACR2 gene was cloned from the genomic DNA of A. thaliana by using Phire Plant Direct PCR kits using database sequences as primers. The amplified product was first ligated to the vector pKOH152 and then transferred to E. coli DH5α competent cells. Recombinant bacterial colonies were screened by colony PCR to confirm the insertion of ACR2. The band (1.3 kb) obtained in gel image indicates that the ACR2 gene was cloned successfully. For further confirmation of these results the cloned gene should be sequenced.

Inherited retinal diseases are considered as a leading cause of vision loss in a young population. Neither a permanent cure nor long-term treatment has yet been discovered. However, treating congenital visual impairment by utilizing a sequence-specific nuclease gene editing tool, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9), has appeared to have a positive impact in restoring eyesight. The following systematic review was implemented to gain more knowledge about the safety and efficiency features of CRISPR/Cas9 technology when used in clinical trials to treat inherited retinal diseases (IRDs). The review focuses on trials with Leber Congenital Amaurosis and Autosomal Dominant Retinitis Pigmentosa, common childhood IRDs. The studies were synthesized in a proportional meta-analysis using MedCalc software, and supplemented with a narrative literature review, considering both qualitative and quantitative data. The review covers different aspects related tothe use of CRISPR/Cas9s and provides an overview of IRDs and future treatment methods. In conclusion, CRISPR/Cas9, indeed, is seen as a potential technique to treat IRDs. However, different complications do arise, and researchers need to be reminded of the side effects and downsides of CRISPR/Cas9. So, they can further enhance this innovative gene-editing technique in exchange for ultimately achieving long-term treatments for blindness and other inherited diseases. / <p>Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.</p>

Barley loose smut caused by the fungal pathogen Ustilago nuda is a global concern with detrimental effects on barley production. Early detection of this infection is vital for effective disease supervision. However, current seed health testing protocols suffer from limitations in terms of time and efficiency. The present research work aimed to produce a method using qPCR for simultaneous screening of barley and U. nuda. A set of primers, 1F and 1R, was employed for the detection of rRNA operon internal transcribed spacer 1 sequence from U. nuda and a part of the COX1 gene, present in barley seeds, was selected as an internal control for comparison with U. nuda. A specific 79 bp target amplicon from a part of the COX1 gene was successfully amplified using COX1 F and COX1 R primers, and cloned into a vector for standard curve generation. However, attempts to replicate the previously published qPCR method by bachelor researcher for U. nuda internal transcribed spacer 1 sequence detection using 1F and 1R primers were unsuccessful. Several efforts were made to reproduce the results, but amplification was not observed. Further optimization, including literature review, primers and probe optimization is required to improve this method. The successful amplification of a part of the COX1 gene in both normal and infected samples underscore its potential as a reliable internal control. However, further research is necessary to refine the detection of U. nuda. This study underscores the need for continuous advancements in disease screening methodologies to meet global market demands.

Rapeseed is a versatile crop with significant economic value as fuel, food, and feed, contributing to farmers' income. However, its cultivation is often hindered by the devastating plant pathogen Sclerotinia sclerotiorum, which infects numerous plant species, including rapeseed. Stem rot disease caused by this fungus has historically caused substantial yield losses, ranging from 30 to 70% in Sweden, Germany, and the UK. The absence of disease-resistant cultivars poses a challenge for effective disease control. A real-time PCR is one of the several techniques used in laboratory for the detection of infection which has many benefits over conventional methods. But this study aimed to develop a technique for the detection of disease by confirming if MinION Nanopore Sequencing can be used for such purpose to save time, resources, and the environment as compared to real-time PCR and other conventional methods. Rapeseed leaves samples were collected from three naturally infested fields in Skaraborg; DNA was extracted and ITS regions which are commonly used marker or barcodes for identification of fungi were amplified with three different pair of primers. Amplicons were sequenced with MinION and hundreds of thousands of reads were recovered to Ascomycota and Basidiomycota fungi division while Saccharomycodes ludwigii fungi was the most abundant species recovered. Many pathogens were successfully detected while no single read of S. sclerotiorum could be recovered in the study. MinION is concluded to provide a fast and efficient method for the detection of plant pathogens however, in this study S. sclerotiorum were unable to be identified.

No description available.

This thesis describes the improvement of two radically different cellulose materials, paper and artificial blood vessels, constructed from two diverse cellulose sources, wood pulp and Acetobacter xylinum. The improvement of both materials was possible due to the natural affinity of the hemicellulose xyloglucan for cellulose. Chemical and mechanical pulps were treated with xyloglucan in the wet-end prior to hand sheet formation or by spray application of dry hand sheets, loading a comparable amount of xyloglucan. The tensile strength increases for the wet-end treatment and spray application were 28% and 71% respectively for bleached soft wood, compared to untreated sheets (20.7 Nm/g). The corresponding strength increases for hand sheets made of thermo-mechanical pulp were 6% and 13% respectively compared to untreated sheets (42.4 Nm/g). The tendency for chemical pulp to be superior to mechanical pulp with respect to strength increase was valid even for tear strength and Scott-Bond. These results suggest, in agreement with other studies, that adhesion of xyloglucan to wood fibres is dependent on their degree of surface lignification. Also, a method was developed to increase the blood compatibility of artificial blood vessels constructed of bacterial cellulose. Xyloglucan was covalently linked to the endothelial cell adhesion motif (Arg-Gly-Asp). To obtain this, new solid-phase coupling chemistry was developed. Xyloglucan oligosaccharides (XGO) were transformed into XGO-succinamic acid via the corresponding XGO--NH2 derivative prior to coupling with the N-terminus of the solid-phase synthesised Gly-Arg-Gly-Asp-Ser peptide. The resin-bound glyco-peptide was then cleaved and enzymatically re-incorporated into high molecular weight xyloglucan. The glyco-peptide was further adsorbed onto bacterial cellulose scaffolds, increasing the adhesion and proliferation of endothelial cells and therefore blood compatibility. / QC 20101102

Enzymes are successfully employed in the synthesis of different types of polymers. Candida antarctica lipase B is a highly efficient catalyst for the synthesis of polyesters by ring opening polymerization. ω-Pentadecalactone is an interesting lactone due to the unique proprieties of its polymer (poly-pentadecalactone). These polymers have not been applied in any industrial application due to the difficulties to reach them by chemical polymerization. Enzymatically, poly-pentadecalactone macromonomers can be obtained to high conversion. In this investigation we synthesized difunctionalized poly-pentadecalactone with different functional groups. Taking advantage of the selectivity of Candida antarctica lipase B, we introduced different functional end groups. α,ω-Difunctionalized poly-pentadecalactone macromonomers with two thiol ends, two (meth)acrylate ends or with one thiol and one acrylate end were obtained with a high degree of functional ends. We have improved the difunctionalization procedure to a single-step route for the synthesis of α,ω-functionalized poly-pentadecalactones. This procedure has a great potential for industrial applications due to the simplicity of the process and the clean products afforded. Macromonomers with functionalized ends can be used to obtain new polymer architectures with novel proprieties. We also show how the use of enzymes could have some limitations when using an initiator with a cleavable ester bond. 2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the ring opening polymerization (eROP) of ε-caprolactone and ω-pentadecalactone aiming for methacrylate functional polyester. However, the lipase catalyzed not only the ring opening polymerization but also the cleavage of the HEMA moiety resulting in a mixture of polymer products with various end groups. A kinetics study of the eROP and the transesterification processes when using HEMA showed that the transesterification processes occurs at moderate frequency at low monomer concentration, it becomes dominant at longer reaction times. We showed that fully difunctionalized polymers can be obtained when using HEMA as initiator for the eROP of lactones by adding a proper end capper. / QC 20101124

As life expectancy increases globally, so does the prevalence of age-related diseases, some of which are more difficult to adapt to and accommodate for than others. In particular, neurodegenerative disorders are among those for which adaptations are more complex, often requiring long-term care. Alzheimer’s disease is a neurodegenerative disorder linked with atrophy of certain cognition related brain regions, causing severe memory, and cognitive function loss. A major hypothesis behind Alzheimer’s disease, upon which most pharmaceutical therapies are based, proposes its cause as the degeneration of cholinergic neurons. Nerve growth factor is a biomolecule found to stimulate the generation, protection, and regeneration of cholinergic neurons. Synthesis of nerve growth factor has been found to be promoted by hericenones and erinacines, bioactive compounds originally extracted from the mycelium of the Lion’s Mane Mushroom (Hericium erinaceus); however, direct supplementation with H. erinaceus has also yielded positive results. In animal models H. erinaceus has enhanced Nerve growth factor levels, increased neuronal survival, promoted hippocampal neurogenesis, decreased amyloid plaque build-up, and improved behavioural outcomes. Human trials showed improvements in cognitive function scores, short-term memory, and visual contrast sensitivity. Phytotherapeutic remedies such as these have long been used across a multitude of cultures, however, now with quantitative scientific evidence supporting their benefits, their implementation into clinical therapies is being explored. Though there is still room for further research, H. erinaceus shows a promising future as a potential pharmaceutical therapy for Alzheimer’s disease and other cognitive impairments.

Thermo Fisher Scientific in Uppsala manufactures in vitro tests for allergy diagnostics by producing recombinant allergens. However, the absence of an effective method for quantification of recombinant protein post-fermentation, challenges the production since protein yield cannot be determined. This study is made upon request by Thermo Fisher Scientific in Uppsala as a response to this issue with the aim to provide a recommendation of  a method to implement. Thermo Fisher Scientific uses histidine-tagged recombinant proteins in their production which allows purification through immobilized metal ion affinity chromatography (IMAC). Hence, this study mainly surveys quantification methods that utilize the histidine-tag. This project provides insight into the following methods: SDS-PAGE, Western blotting, ELISA, Simple Western and FluoroHis-PAGE. The methods have been evaluated based on three key aspects: cost, analysis time, and method performance.  The recommended method for implementation is FluoroHis-PAGE, as it was found to be the most appropriate method for Thermo Fisher Scientific’s current process. FluoroHis-PAGE utilizes fluorescent Ni-NTA or antibody conjugates, which demonstrated high performance in the quantification of His-tagged recombinant proteins at a low cost and analysis time.

Cancer utgör ett betydande globalt hälsoproblem och är den näst vanligaste dödsorsaken globalt sett. Transforming growth factor-beta (TGF-β) är en tillväxtfaktor som signalerar huvudsakligen via transkriptionsfaktorerna SMAD2 och SMAD3 som fosforyleras och aktiveras av TGF-β receptorerna. I normala celler fungerar TGF-β som tumörsuppressor men forskning har visat att mutationer i TGF-β- signaleringen ofta uppstår, vilket leder till motsatta effekter i senare stadier av cancer. I stället för att hämma tumörtillväxt kan TGF-β stimulera metastasering och invasion i senare stadier av cancer. Orsaken och mekanismer bakom detta är fortfarande oklara därför behövs det mer forskning inom TGF-β signalering för att kunna förstå orsaken bakom mutationerna som kan uppstå. Cdc42 är ett GTPase-protein som tillhör Rho GTPases-familjen och är en av de tre klassiska Rho GTPases i däggdjur celler. Flera studier har visat att detta GTPase-protein påverkar signaleringen av andra tillväxtfaktorer såsom VEGFA och EGF. Därför är syftet med denna studie att undersöka hur Rho GTPaset Cdc42 påverkar TGF-β SMAD-signalering i cancerceller. I denna studie odlades MDA-MB-231-celler och därefter transfekterades de med en Cdc42-specifik siRNA för att nedreglera Cdc42-uttrycket. Efter detta stimulerades cellerna med TGF-β och den relativa fosforyleringsnivån och aktiveringen av SMAD analyserades med western blot. Resultaten visade att nedreglering av Cdc42 inte påverkade nivån av fosforylering av SMAD2 i TGF-β-signaleringen jämfört med kontrollerna. Dock observerades en lägre fosforylering av SMAD3 vid nedreglering av Cdc42. Sammanfattningsvis visar denna studie ingen påverkan på fosforyleringen av SMAD2 i TGFβ-signaleringen, men det kan finnas en roll för fosforyleringen av SMAD3. Ytterligare forskning behövs för att bekräfta detta.