An overview on Human Leukocyte Antigen (HLA) region gene expression during Pseudomonas aeruginosa infection

The human leukocyte antigen (HLA) complex is an extensively studied cluster of genes with immunoregulatory function. Pseudomonas aeruginosa is capable of infecting individuals with weakened immune systems, and is associated with a high mortality rate. Previous genetic studies of the HLA region have found correlations between bacterial infection and its effect on regulating HLA gene expressions to establish their infection. This project analyzes the expression of classical HLA loci (A, B, C, DR, DQ, DP) in human B cells and macrophage cells during the infection of virulent strains of P. aeruginosa. Cells were cultured and infected with different virulent live, and heat-killed strains of P. aeruginosa for different time periods. The mRNA was extracted and converted into cDNA followed by real-time quantitative PCR and data analysis. The Western Blot technique was used to identify the targeted protein’s cell surface expression. Infection with P. aeruginosa was found to inhibit the expression of HLA proteins. The PA14 strain inhibited expression of all targeted genes in all experiments. Infections with PA01 and PA103 showed different patterns depending on the incubation time and the targeted gene. These differences suggest that the three strains use various mechanisms to inhibit HLA protein expression.

Real-time biosensor for the assessment of nanotoxicity and cancer electrotherapy

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor's ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell's electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.

Real-time Biosensor for the Assessment of Nanotoxicity and Cancer Electrotherapy

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor’s ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell’s electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.