RNA interference (RNAi) is an important mechanism of gene silencing in Caenorhabditis elegans, in which short RNAs direct sequence-specific silencing of gene expression mediated by Argonaute proteins. RNAi in C. elegans can be sorted into exogenous RNAi, where the short RNAs originate from foreign RNA sequences, and endogenous RNAi, where the short RNAs originate from RNA sequences in the genome. One endogenous RNAi pathway is the ERGO-1 pathway, which is active in the germline and in embryos. Mutants deficient in the ERGO-1 pathway show an increased response to exogenous RNAi, which is thought to be due to competition between exogenous and endogenous silencing pathways.  FUBL-1 is an RNA-binding protein found in C. elegans, which has three predicted functional isoforms: isoforms a, b and c. Prior research by the Hinas group has indicated that FUBL-1 may play a role in the ERGO-1 pathway of RNAi. FUBL-1 deletion mutants show an increased response to exogenous RNAi similar to ERGO-1 mutants, and also an upregulation of ERGO-1 target genes. They have also found that FUBL-1 is broadly expressed in somatic tissues, but germline expression has not been confirmed. The function of the three different isoforms has not yet been examined. The aim of this study was to further investigate the function of FUBL-1 by assessing the function of the different isoforms through RT-qPCR of C. elegans with mutations affecting the different isoforms, to use immunofluorescence staining to see whether FUBL-1 is expressed in the germline, and to identify FUBL-1 RNA targets through CLIP-seq. Preliminary RT-qPCR results indicated that the upregulation of ERGO-1 targets is less in isoform mutants than in FUBL-1 deletion mutants, indicating partial redundancy of the isoforms. Immunofluorescence staining showed that FUBL-1 is expressed in the germline nuclei. Growth protocols have been optimized and crosslinking has been performed on worms, but the full CLIP-seq protocol has not been performed yet.

Density functional theory is employed to study the reaction mechanisms of different epoxide-transforming enzymes. Calculations are based on quantum chemical active site models, which are build from X-ray crystal structures. The models are used to study conversion of various epoxides into their corresponding diols or substituted alcohols. Epoxide-transforming enzymes from three different families are studied. The human soluble epoxide hydrolase (sEH) belongs to the α/β-hydrolase fold family. sEH employs a covalent mechanism to hydrolyze various epoxides into vicinal diols. The Rhodococcus erythrobacter limonene epoxide hydrolase (LEH) constitutes a novel epoxide hydrolase, which is considered the founding member of a new family of enzymes. LEH mediates transformation of limone-1,2-epoxide into the corresponding vicinal diol by employing a general acid/general base-mediated mechanism. The Agrobacterium radiobacter AD1 haloalcohol dehalogenase HheC is related to the short-chain dehydrogenase/reductases. HheC is able to convert epoxides using various nucleophiles such as azide, cyanide, and nitrite. Reaction mechanisms of these three enzymes are analyzed in depth and the role of different active site residues is studied through in silico mutations. Steric and electronic factors influencing the regioselectivity of epoxide opening are identified. The computed energetics help to explain preferred reaction pathways and experimentally observed regioselectivities. Our results confirm the usefulness of the employed computational methodology for investigating enzymatic reactions. / QC 20101108

The present thesis is based upon two papers concerning the core-valence double onization of molecular oxygen and mutual neutralization of H+ and H- ions at low collision energies. The former of these processes has been studied for the first time using a magnetic bottle time-of-ight electron coincidence spectrometer in combination with ab initio electronic structure calculations. The core-valence photoelectron spectra have been interpreted by comparing with the calculated double ionization energies, as well as the conventional valence band spectrum. Based on this comparison, some general features of the process are discussed and assignments for several of the dicationic states proposed. The latter process has been studied by means of a molecular close coupling approach in which both the nuclei and the electrons have been treated at a quantum mechanical level of theory. Accurate ab initio potential energy curves and non-adiabatic couplings have been used to calculate the neutralization cross section in the collision energy region 0.001 to 100 eV. Special emphasis has been put on the energy region below a few eV from which the low temperature rate coe_cient is evaluated. In this region, the calculated neutralization cross section is in good agreement with several other theoretical studies, but is a factor of two to three lower than the only published experimental data. / QC 20101123

No description available.

In this study, 20 well-known Chinese medicinal plants are included. Traditionally, these medicinal herbs were consumed for different diseases but have one application in common, treatment of rheumatism arthritis. Peptides are pharmacologically attractive substances and the reason behind this work as there is only one study on peptide content of included herbs. Commonly, plant-derived peptides are cysteine-rich peptides.  The plants were extracted in series by 60%, 30% and 10% AcN in H2O and FA. Then, the combined extracts were purified by SPE and SEC. Additionally, reduction by DDT, alkylation by IAM, and digestion by trypsin performed. UPLC-QToF was used for separation and identification of potential peptides.  The investigation resulted in finding peptides in Coix lacryma-jobi L, Atractylodes lancea, Astragalus membranaceus. Peptides found in these species are mainly in the range 2,5-5 kDa. This finding raises new questions about the function of peptides in these species and the possibility of having pharmacological activity.

Viruses are biological entities that constantly affect the world around us and in many cases they can harm the health of humans and negatively impact food production, in agriculture andfisheries. The project concept is that, through the structural studies of viruses that infectsimple hosts, it is possible to point out structural features that are unique and conserved inviruses, that infect human, other animals, and crops, in the same phylogenetic lineage. Theseunique features have functionally been acquired, and are therefore potential drug and vaccinetargets. Standing on this concept, we aim to study three groups of pathogenic viruses: (i)Protozoan/Yeast Totiviridae and metazoan totivirus-like viruses, (ii) Invertebrate flavi-likeand vertebrate Flaviviridae viruses, and (iii) Invertebrate Mesoniviridae and vertebrateCoronaviridae viruses.First, we focused on acquired capsid structures of the totivirus-like Omono River Virus(OmRV). Unlike yeast Totiviridae viruses, the totivirus-like OmRV capsid presents surfaceprotrusion proteins, which are expected to be crucial for its extracellular transmission. Theinteraction between the capsid and the protrusion proteins is hypothesized to be largelymediated by amino acid residue T365 of the protrusion proteins. To clarify the function ofthese protrusion proteins we have successfully developed molecular tools, which are arecombinantly expressed protrusion protein and an OmRV infectious DNA clone with aT365A mutation. Although the excess of the protrusion proteins does not remarkably affectthe infectivity and binding capabilities of the OmRV particles, the OmRV with the T365Amutation shows a significantly reduced infectivity. Assuming that the infectivity was partiallyeliminated by the mutation, a putative transmission mechanism is associated with theprotrusion proteins. However, it is still under debate whether the protrusion proteins are anessential factor for extracellular transmission. Additionally, a new method was preliminarilyused for observing the interaction between the OmRV capsid and protrusion proteins usingdifferential scanning fluorimetry. The findings and the established methods can contribute tothe understanding of molecular mechanisms present in other pathogenic totivirus-like virusesthat affect fish, such as the salmon piscine myocarditis virus (PMCV).Secondly, we focused on acquired surface structures of vertebrate Flaviviridae andCoronaviridae viruses. Unlike the invertebrate flavi-like and Mesoniviridae viruses, thevertebrate viruses have acquired surface structures that are expected to be critical for evadinghosts’ adaptive immunity. To discover these acquired surface structure, it is necessary todetermine the surface structure of the invertebrate flavi-like and Mesoniviridae viruses. Herewe report the successful expression and purification of the invertebrate flavi-like Southernpygmy squid flavivirus (SpsFV) precursor/membrane-envelope (prM-E) protein and theMesoniviridae Nam Dinh virus (NDiV) p2a spike protein. The established expression andpurification system can be further used to resolve their structures using cryo-EM singleparticle analysis in future studies.

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.