148 Chapter 5 pro-inflammatory cytokine that plays a central role in initiating and amplifying inflammatory responses by inducing the production of other pro-inflammatory mediators [28]. For instance, TNF-α activates endothelial cells and, through the production of chemokines by macrophages, other immune cells, such as neutrophils are recruited to the site of infection or inflammation. TNF-α can also enhance the phagocytic capacity of macrophages and their ability to eliminate intracellular pathogens [29]. IL-6 is a pleiotropic cytokine that has pro-inflammatory functions when it signals in trans via soluble IL-6 receptors binding to gp130 [30]. IL-6 can be induced by other cytokines, including TNF-α, and controls the development of chronic inflammatory diseases [31]. Protein Analysis: Techniques such as western blotting, immunoprecipitation, and mass spectrometry can be used to study changes in protein expression and post-translational modifications within macrophages. This helps to understand changes in signaling pathways and the production of specific proteins involved in the immune response. In Chapters 3 and 4 I investigated the intracellular proteome of tissue-resident alveolar-like macrophages with the goal of gaining insights into the molecular mechanisms leading macrophages behavior and their roles in immune responses, in combination with KDACi treatment. I have made use of both targeted and untargeted approaches. Untargeted proteomics involves analyzing all detectable proteins in a sample. I started by employing this approach as it provides a more comprehensive view of the proteome and it provided a direction into which to further focus our research. In targeted proteomics, on the other hand, I selected specific proteins of interest for analysis. This approach is useful as I wanted to focus on a particular subset of proteins relevant to our study, the proteins involved in macrophage metabolism. Using a targeted approach also offers an increased detection sensitivity and improved protein quantification over an untargeted approach. Gene analysis: Gene expression profiling, also known as transcriptomics, is a technique that simultaneously assesses the mRNA expression levels for thousands of genes. There are several approaches to studying mRNA: techniques such as microarrays that can offer a snapshot of the transcriptome compared to a control condition, and RNA sequencing (RNA-Seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) can quantify the expression levels of (particular) genes. The identification of genes whose expression is increased or downregulated during macrophage activation can provide insight into the pathways and processes involved in macrophage activation. In Chapter 3, in order to validate the effects of KDAC inhibition, we performed reverse transcription-quantitative
RkJQdWJsaXNoZXIy MTk4NDMw