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No description available.

The amygdala is essential in the process of learned aversive signals. Its implication in processing an innately aversive threat remains to be decoded. This implies asking questions on how a learned and an innate aversive threat is processed. Learned aversive threat involves changes in synaptic plasticity while the responses to the innate aversive threat are hard-wired in the animal’s brain. A way to answer this question is to visualize the neurons activated by both behaviors in the same animal. To do so, a set of behavioral experiments were done with the help of a cross of Rosa td Tomato and TRAP2-Cre mice in combination with c-Fos staining. Following this, a combination of techniques including behavioral studies, immunohistochemistry and apotome microscopy for cell quantification were being used. This was intended to find the number of TRAP cells and c-Fos cells in the areas of LA, BA, CeL and CeM respectively. A heat map was further being plotted to measure the density of the cells in each area. The percentage of cells which got activated when a specific stimulus was being repeated twice were also being quantified using the Fiji software. The results section discusses the analysis of shock-shock and loom-loom experiments wherein the freezing percentages of the first set of mice (aversive threat measurement experiments) and the looming behavior of the second set of mice were being analyzed. It was observed that the freezing rate of all the mice used for the aversive threat measurement experiments increased steadily with time. The responses of the mice to the looming stimulus were also significant. The quantification of the number of cells which got activated when the mice were exposed to the shock stimulus twice indicated that Lateral amygdala (50%) exhibited the maximum percentage of cells in the case of shock-shock experiments and the central amygdala in the case of loom-loom experiments (30-50%). This helped us to identify some of the primary areas involved when the mice were being exposed to a shock or loom stimulus. It also helped us to infer the percentage of cells which got activated when the same stimulus was being repeated twice. These experiments serve as a foundation for the future experiments to learn about the specific cell types involved in each area and whether these cell populations get activated when the same stimulus is being repeated twice.

Sepsis, defined by organ dysfunction caused by an adverse immune response of the host to an infection, comes with considerable cost in human lives and as a substantial burden financially. Significant upgrades have been made over the past two decades when diagnosing and treating sepsis but still with room for improvements. Early detection is a cornerstone in the fight against sepsis, and the focus on strengthening diagnostics is in the forefront of modern research. The implementation of biomarkers may be the path of progression in this objective. This study aimed at establishing procedural foundations when using microRNAs as potential biomarkers. The study conducted looked at: (1) Isolation procedure, of microRNA from human plasma, of three kits: Total RNA Purification Kit (Norgen Biotech), miRNAeasy Serum/Plasma Kit (Qiagen), and miRNeasy Serum/Plasma Advanced Kit (Qiagen). (2) Amplification of miRNA through two Reverse Transcription Quantitative PCR methods: Two-tailed RT-qPCR (TATAA Biocenter), and miRCURY LNA miRNA PCR (Qiagen). (3) Developing a normalization strategy by identifying miRNA reference targets in a geNorm pilot experiment. Qubit analysis revealed that the two isolation kits from Qiagen performed similar, and better that the Norgen kit. The Two-tailed RT-qPCR failed to amplify miRNA samples, whereas the miRCURY LNA miRNA PCR showed consistent amplification across samples with a high call rate. The geNorm analysis concluded that hsa-miR-425-5p and hsa-miR-93-5p was the optimal reference target set. The study demonstrated that the isolation kits from Qiagen coupled with the miRCURY LNA miRNA PCR is a viable option for future miRNA biomarker studies.

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The nucleotide-binding oligomerization domain–like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a group of multi-protein complex that mediates immune responses through the production of biologically active IL-1β and IL-18. Dysregulation of NLRP3 inflammasome has been linked to various diseases associated with infection, inflammation, and cancer. However, the molecular mechanisms of ligand binding that result in the formation of the NLRP3 inflammasome are not fully understood. Potential therapeutics for NLRP3 inflammasomes related diseases are relatively nonspecific, have low efficacy, and may cause unexpected side effects. Therefore, this study aimed to understand if BABAM1 can serve as a potential target for treating NLRP3 inflammasome related diseases. This was done by attempting to knock out BABAM1 gene in THP-1 monocyte-like cell line using the CRISPR-Cas9 gene-editing technology. The cDNA prepared from THP-1 cells in which an attempt was made to knock out BABAM1 gene were amplified using qPCR. The result showed no biologically relevant difference in BABAM1 gene expression level between the target and control THP-1 cells. Additionally, this study found that the expression of the reference gene, ACTB, was not stable as the cycle threshold values for untransfected cells were lower when compared to cells transfected the plasmid DNA. In conclusion, successful attempts were made in this study to understand the role of BABAM1 in regulating the activation of NLRP3 inflammasome and that further research are needed if BABAM1 is to be considered as a potential target for treating NLRP3 inflammasome related diseases.

No description available.

No description available.

No description available.

This thesis presents two techniques in the field of biocatalysis: An enzyme immobilisation method based on the His6-tag for attachment on modified silica oxide beads, and it’s employment in aqueous and organic medium for synthesis applications. The method functions as a one step extraction and immobilisation protocol. An equilibrium displacement system which enables complete conversion in reactions with ω-transaminases where isopropylamine is the donor, a route for synthesis of pharmaceutically interesting enantiopure amines. Biocatalysis is predicted to be a paramount technology for an environmentally sustainable chemical industry, to which every newly developed method represents a small but important step. The work done here is aimed to be a part of this development. / I denna avhandling presenteras två tekniker inom ämnet biokatalys: En metod för immobilisering av His6-enzym på modifierad kiseloxid, och användning av detta konstrukt för kemiska synteser i vatten och organiska lösningsmedel. Detta system fungerar även som en snabb extraherings- och immobiliseringsmetod. Ett jämviksförskjutningssystem som möjliggör fullständig omsätt-ning i reaktioner med ω-transaminaser där isopropylamin är amino-donator, en syntesväg för tillverkning av farmakologiskt intressanta kirala aminer. Biokatalys förutspås att bli en ovärderlig teknologi i en miljömässigt hållbar kemisk industri, i vilken varje ny metod är en liten men dock viktig del. Detta arbete är menat som en del i denna utveckling. / QC 20100519