Tuberculosis remains a major public health problem and one of the leading causes of death worldwide. Human genetic factors determine susceptibility to M. tuberculosis (M. tb) infection and predispose to clinical TB. Genome-wide association studies (GWAS) aim to discover human genes associated with susceptibility to TB. Recently, a GWAS conducted by our lab identified a new TB-associated gene ASAP1 that encodes an Arf GTPase-activating protein (GAP). ASAP1 is known to be involved in regulation of actin and membrane remodeling. My Ph.D. included three projects. In my first project, I used RNAi and CRISPR-Cas9 technologies to study the role of ASAP1 in dendritic cells and macrophages, cells that play critical roles during mycobacterial infection. I demonstrated that in these cells ASAP1 is essential for migration and phagocytosis of mycobacteria. I characterized proteins that ASAP1 interacts with during mycobacterial infection. Finally, I found that the ASAP1-mediated pathway regulates expression of a large number of the immune response genes. These findings emphasize the important role of ASAP1 in mycobacterial infection and explain its involvement in TB pathogenesis. In my second project, I was involved in a large study conducted by our laboratory that characterized transcriptional responses to M. tb infection in macrophages from a cohort of 144 healthy subjects. We used RNA-Seq to study transcriptomes of the infected and non-infected macrophages and identified differentially expressed genes. We also genotyped DNA polymorphisms of these subjects and studied the association between genetic variants and levels of gene expression, which allows us to identify expression quantitative trait loci (eQTLs), i.e., DNA polymorphism that affect gene expression. In particular, we identified an eQTL located in the TLR10-TLR1-TLR6 gene cluster. In non-infected macrophages, a group of polymorphisms in this region was associated in cis with the level of expression of TLR1, but not of the other two TLR genes. In M. tb-infected macrophages the same polymorphisms were associated in trans with levels of expression of 37 genes. This network includes essential immune response proteins, including multiple cytokines and chemokines. The discovery of this TLR1-driven network will help to better understand mechanisms of macrophage responses to mycobacterial infection. Our study also identified a DNA polymorphism located upstream of the ARHGAP27 gene, regulating its expression in infected and non-infected macrophages. In our GWAS this polymorphism was associated with TB risk, which implicated ARHGAP27 in TB pathogenesis. The ARHGAP27 protein is a Rho-GAP involved in the endocytic pathway. In my third project, I used CRISPR technology to establish the ARHGAP27-knockout macrophage cell model and characterized the function of ARHGAP27, showing that it is involved in cell migration and phagocytosis of mycobacteria. Taken together, my studies highlighted functional mechanisms implicating TB-associated GAP proteins ASAP1 and ARHGAP27 in mycobacterial infection and TB pathogenesis.

In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.

This thesis deals with the problematice of measuring membrane potential and monitoring the propagation of electrical activity of cells. For this purpose, fluorescence membrane voltage sensors have been developed to detect changes in the membrane potential by changing their fluorescence intensity. The practical part is focused on the study of the properties of the ASAP1 fluorescence probe, which was transfected into the HEK293 cell line, which are kidney cells from the human embryo. Cell membrane potential was changed using the patch-clamp technique.