Seminar Synopsis Essay

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Gastelum, Gilberto
The objective of this project was to understand the initial mechanistic step that results in the induced-phagocytosis of apoptotic cells triggered by the soluble molecule C1q. The molecule C1q is a subcomponent of the C1 complex commonly found in the pathway of complement activation, which helps drive the ability of antibodies to interact with cellular and foreign components that can potentially pose a danger to cells. Individuals with a deficiency in C1q are affected by the chronic autoimmune disorder Lupus, a condition where necrotic cells produce antigen-specific antibodies that induce antibody-generating complexes, which in part precipitate and become deposited in tissues leading to inflammatory responses. The hypothesis proposed for this experiment was that C1q serves as bridge between a dead cell and a phagocyte; the researcher performed seven experiments to help understand the role of C1q in mouse macrophages. The first experimental set up consisted of mouse macrophages; a control set with human serum albumin (HSA) and a treatment in the presence of C1q for 4-5 hours. They observed that in the treatment, phagocytosis was a time dependent process, which indicated that the presence of C1q was relaying gene transcription to produce phagocytosis gene-products. To further support the hypothesis and the latter results, another experiment was performed in which protein production was blocked with the use of a chemical known as cyclohexamine. This second experiment consisted of a control set of macrophage cells in the presence of HSA, cyclohexamine, and in the absence of C1q, which did not undergo apoptosis. In addition, they exposed cells to C1q and no cyclohexamine and another set of cells with both C1q and cyclohexamine, which resulted in apoptosis in absence of cyclohexamine and no apoptosis in presence of cyclohexamine. The results from this experiment further supported the idea that C1q was playing a role in gene regulation for the activation of apoptosis-related gene products. Their next goal was to look for C1q-upregulated protein(s), which they did by synthesizing cDNA from harvest RNA during macrophage cells exposed to C1q for 4-18 hours that was ultimately used in a microarray assay. They found Mer tyrosine kinase (MTK) and GAS6 to be upregulated; these two genes have been shown to be involved during apoptotic activity. For their next experiment, they wanted to test whether MTK and GAS6 were required for C1q dependent phagocytosis. To determine if MTK was required for C1q-dependent phagocytosis, they designed an experiment that included macrophage cells with C1q and MTK and a control of macrophage cells with C1q and no MTK. They found that samples in the absence of MTK were unable to engulf apoptotic cells, whereas those with MTK did. Thus, this indicated that C1q stimulates the expression of MTK, which was capable of binding to GAS6 to help induce the engulfment of apoptotic cells. To further help them understand the mechanism activated by C1q they performed another microarray assay from which they subtracted out any signal they believed came from a homologous 6-amino acid sequence in C1q tails also found in the mannose-binding protein (MBL) because they knew from another experiment that they did not activate the pathway. With the help of a bioinformatics team, they found that C1q upregulated the Adiponectin signal pathway, which is a pathway that relies on AMP kinases. What they observed was that C1q was inducing the phosphorylation of this pathway that was maintained for at least 300 min after stimulation with C1q. For their last experiment, they wanted to determine if AMP kinase was required for C1q-dependent phagocytosis; to test this out, they silenced AMP kinases using siRNAs. They observed that when this happened, C1q was no longer able to trigger MTK and in addition, the pathway suggested that Ca2+ signals upstream of AMK kinase were involved in the process. Thus, from their
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