Heated ghee on membranes

Piper occurring in Karnataka Region

Oncogenes and tumor suppressor genes play a key role in cancer induction and progression. They directly or indirectly regulate critical metabolic pathways, phosphatidylinositol-3 kinase pathway being frequently activated pathway in cancer. The catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), p110α, is the most frequently mutated kinase in human cancer, E542K, E545K, and H1047R mutations being the most common. Expression of hepatic E545K and H1047R p110α mutants in vivo shows marked and rapid increase in hepatic lipid and glycogen accumulation in mice with developmental (chronic) liver-specific deletion of p110α, which was not seen in mice when wildtype p110α is overexpressed. To investigate the logical pathways that could explain the lipid accumulation in mutant expressing mice, RNA sequencing from wildtype, knockout and mutated mouse livers was performed. Read alignment and count quantification was done using the Rsubread package and the statistical analyses are performed using the DeSeq2 package. Differentially expressed genes were identified with adjusted p-value of 0.05. Gene ontology analysis was performed on the differentially expressed genes using clusterProfiler, an R package to identify several key pathways which were upregulated and downregulated among the different sample groups. Signaling pathways related to cell cycle processes were mainly upregulated in the mutated samples when compared with the wildtype as well as knockout samples while signaling pathways related to many metabolic processes seem to be downregulated in mutated samples, even though these mutants showed upregulated metabolism by accumulation of lipids and glycogen physiologically. To confirm the results of gene expression data the results have to be cross validated with the gold standard quantitative Real Time Polymerase Chain Reaction.

Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

Obesity leading to Type-2-diabetes is a major health issue all over the world. Obesity characterized by expansion of adipose tissue, in particular white adipose tissue (WAT) which controls the metabolic physiology in the body by secreting proteins like adiponectin and resistin. Adiponectin has a protective role against diabetes development whereas resistin causes insulin resistance. Protein folding, maturation and translocation is performed by the Endoplasmic reticulum using calcium ions. The calcium homeostasis is maintained by calcium pumps and channels, chief of this is sarco-/endoplasmic reticulum (SR/ER) Ca2+ ATPase pump (SERCA) which restores calcium back to the ER. To study the effect of SERCA2 on adiponectin and resistin secretion in different adipose tissue depots using an adipocyte specific tamoxifen-inducible SERCA2 Knock-out mice, short term secretion experiments were performed. Chemical inhibition of SERCA2 and ER stress was performed in in-vitro experiments using adipocyte like 3T3-L1 cell line. The experiments revealed that SERCA2 dysfunction led to decrease in adiponectin and resistin secretion in normal and stimulant conditions in both male and female mice. In-vitro experiments revealed that ER stress led to misfolded protein accumulation affecting exocytotic events of adiponectin containing vesicles. Therapeutic agents can be formulated to tackle the SERCA2 dysfunction and to maintain calcium homeostasis by identifying these key mechanisms for diabetes and related metabolic disorders.

Tuberculosis (TB) disease is a world-wide spread disease caused by Mycobacterium tuberculosis which affects a significant percentage of the world population. The disease is often localized in the lungs, but can further develop and spread to other organs and is then called extrapulmonary TB (EPTB). The diagnosis of TB by culture is time consuming and requires specialised facilities. The use of DNA amplification is therefore an advantageous and rapid diagnostic tool. One such tool is the GeneXpert system which also can detect resistance to rifampicin (Rif) in samples. Although the system is designed to mainly analyse unprocessed sputum samples, there is a possibility to also analyse formalin-fixed paraffin-embedded (FFPE) tissue. The project's aim was to compare different pretreatment methods for processing FFPE tissue samples along with the application of Xpert MTB/Rif Ultra assay to improve EPTB case diagnosis. Tissue sections from eleven different TB negative FFPE tissue samples were used to assess three different pretreatment methods. Tissue sections from seven TB positive FFPE tissue samples were later used to validate the application of Xpert MTB/Rif Ultra assay usage. The current lab developed pretreatment method were after statistical testing and handling evaluation chosen and applied for the TB positive FFPE tissue samples. Results from the seven historical patient samples gave a 100 % positive Mtb detection result. The project's results show that the pretreatment of FFPE tissue can be applied before the Xpert MTB/Rif Ultra assay cartridges for processing on the GeneXpert instrument.

The NLRP3 inflammasome is a multiprotein complex that controls caspase-1 activation and the development of the pro-inflammatory cytokines IL-1 and IL-18, as well as pyroptosis. The NLRP3 inflammasome in murine macrophages is normally triggered by pathogen-associated molecular patterns as well as a variety of structurally unrelated stimuli. The exact mechanism of NLRP3 activation by such a diverse set of stimuli is unknown, although many signaling processes have been proposed, including cytosolic efflux and the inflow of certain ions. As a result, numerous studies have suggested that anion channels play a role in NLRP3 inflammasome formation, although no direct evidence of their participation has been found. This thesis project aims to measure the expressions level of CLIC1 before and after LPS treatment. The measurement was done with the help of reference genes. Using qPCR, potential reference genes were tested for their stability and evaluated by GenEx. The study identified GUSB and TBP as the most stable genes. Using the delta delta cq method, data from qPCR were normalized by reference genes. The results from this analysis showed an upregulation in the expression levels of CLIC1. These results showed that CLIC1 an anion channel plays a role in the activation and formation of the NLRP3 inflammasome and other inflammatory disorders such as oxidative stress. The study identified GUSB and TBP as reference genes in LPS stimulated THP-1 macrophages and an upregulation in the expression levels of CLIC1, leading to speculation that LPS stimulation leads to the translocation of CLIC1 to the plasma membrane and suggests the possibility to target CLICs to treat NLRP3-driven diseases.

Sepsis is a condition of the host’s extreme response to an infection affecting multiple physical functions which can lead to septic shock and death. Currently, the method for diagnosing sepsis is time-consuming and relies on multiple non-specific biomarkers such as Procalcitonin, lactate and C-reactive protein, with blood culturing being the golden standard. Recently proposed and possible sepsis-specific biomarkers are the microRNAs, which are short, ~22 nucleotide long non-coding RNAs that regulate gene expression post transcriptionally. MicroRNA expression levels have the advantage of being more specific to certain disease types, making them promising biomarkers for sepsis diagnostics. The aim of this study was to evaluate the method and performance of microRNA extraction, using microRNA spike-ins to increase concentration of said extractions, and microRNA amplification by using the novel two-tailed RT-qPCR method developed by TATAA Biocenter. Blood gathered from self-assessed healthy donors wascentrifuged to collect plasma, from which microRNA extraction was performed using the miRNeasy Serum/Plasma Advanced kit (Qiagen). Each sample was spiked with the synthetic microRNAs miR-223 or miR-let7b either before or after RNA extraction. The two-tailed RT-qPCR method was used to amplify the spiked-in microRNAs miR-223 and miR-let7b present in the extractions, and absolute quantification was used to determine the amount of those microRNA. The results conclude that spiked-in microRNA from plasma could be amplified using the twotailed RT-qPCR, and the results depended on which synthetic microRNA had been added, at what concentration and at what stage of the extraction process.

Inflammation is the body's response to infection or injury and is mediated by the innate immune system. The NLRP3 inflammasome is a multi-protein complex that is a major contributor to many inflammatory disorders. Emerging evidence suggests the involvement of the Endoplasmic reticulum stress with the NLRP3 inflammasome. The endoplasmic reticulum stress is a series of stress signals that can activate the unfolded protein response and usually accompanies inflammation and eventually causes cell death. Recently, a localized endoplasmic reticulum micro-protein called the chloride clic like-1 channel was found to be involved in the endoplasmic reticulum homeostasis. Recent evidence suggests the involvement of endoplasmic reticulum stress in the inflammation pathways of the NLRP3 inflammasome. The relationship between the ER and the NLRP3 inflammasome has not been clearly described. This study aimed at investigating the expression levels of the microprotein CLCC1 to shed a light on the relationship between the endoplasmic reticulum stress and the NLRP3 inflammasome. The expression levels of CLCC1 were analyzed by qPCR in cultured monocytes under different time points of Lipopolysachaaride immuno-stimulation. The stability of expression in candidate reference genes was investigated for normalization purposes. This study reported the regulation of CLCC1 as a novel finding under prolonged LPS exposure of monocytes and stable reference genes such as GUSB and ACTB were identified. The relationship between CLCC1 and NLRP3 inflammasome priming by LPS indicated that CLCC1 is regulated and may be involved in the inflammatory mechanisms of endoplasmic reticulum stress and NLRP3 inflammasome inflammatory diseases, contributing to a potential therapeutic target in the endoplasmic reticulum and inflammasome related diseases.

The recent advancement in the field of stem cell research gave rise to high expectations from both general and scientific audiences, and indeed, stem cells and stem cell-derived cells have an enormous potential to forward the fields of medical research and regenerative medicine. Nevertheless, there are still many obstacles and limitations associated with stem cell technology. One of those limitations is that currently no method to derive fully mature cardiomyocytes from human pluripotent stem cell is available. Instead, stem cell-derived cardiomyocytes only show basic characteristics of their adult counterparts and are thus only of limited use. However, there is experimental evidence that those cells may increase in maturity during extended culture times. To improve the understanding of those processes, a characterisation of the transcriptional changes in human embryonic stem cell-derived cardiomyocytes over two weeks was made. Using standard bioinformatics methods, including differential and enrichment analysis as well as basic machine learning technologies, changes in mRNA and miRNA transcription and several functions related to those changes could be identified. The analyses indicated increasing structural organisation in the cell cultures, increasing expression of cardiac ion channels and decreasing proliferative capacity in the cardiomyocyte cultures. Furthermore, potential dysregulations of important signalling pathways were observed. The results of this project may aid in developing protocols for differentiating stem cells into cardiomyocytes with features that are more mature than those of the currently available stem cell derived-cardiomyocytes.