•NF-IL6: Regulation of the promoter region during macrophage differentiation.

Regulation of eukaryotic gene expression occurs when nuclear proteins called transcription factors, recognize short and double stranded nucleotide sequences located further upstream from the transcription start site. These factors can remodel chromatin structure and communicate with the basal complex through a series of strong DNA-protein and protein-protein interactions. During monocyte differentiation, several transcription factors are activated, but only one member of the C/EBP family, called as Nuclear Factor for Interleukin-6 (NF-IL6), allows macrophages to become fully matured. Several molecular biology techniques included cell and bacterial culture, electrophoresis, band shift and transient expression assays are employed in this study for a detailed mechanism on the control of this promoter during gene expression. Three well-related cell lines (U937, HL-60 and THP-1) will be also considered here for a better understanding on how this promoter in controlled when specific conditions are used for cellular differentiation.

•FAP1: Determination of protein function and structure.

In Saccharomyces cerevisiae, the small molecule rapamycin causes a G1 cell cycle arrest mediated through the formation of a ternary complex between the primary cytoplasmic receptor FKBP12 and one of the two TOR gene products.  The interaction involves a conserved serine residue located at the C-terminal of the phosphatidylinositol kinase-related domain of the TOR1, essential for the signaling pathway that regulates growth in response to nutrient availability.  Depletion of TOR function has been shown to display a phenotype similar to that of cells entering G0 during the starvation response.

Rapamycin toxicity also causes overexpression of a novel yeast gene termed FAP1 and the presence of FAP1 protein provides to the yeast cells enough resistance by competing with rapamycin for binding to FKBP12, in a concentration dependence manner.  FKBP12:FAP1 complex was readily detectable but drastically reduced in the presence of increasing amounts of rapamycin.  Therefore, the goal of this proposal is to address the role of FAP1 as a protein responsible for supporting of cell cycling process and as a putative transcription factor responsible for gene expression in yeast.  If successful, the proposed research would have a significant impact on how the yeast cells can manage the cell cycling process in response to rapamycin and nitrogen starvation, through the biophysical interactions between FAP1, FKBP12 and/or TOR-related complexes.

PDGF/VEGF is released into the plasma blood through cellular mechanisms related to protein secretion in order to activate certain specialized cells.  An increase in PDGF/VEGF concentration has been implicated with abnormal cellular growth.  One possible way to detect submolar concentrations of these proteins is by the use of biosensors, constructed with one kind of small molecules called aptamers.  An aptamer is a segment of single-stranded DNA or RNA, which its secondary structure can be recognized by a specific molecule, in our case PDGF/VEGF.  This research focuses on a method suitable for the detection of PDGF/VEGF directly from human plasma blood.  Aptamers are synthesized and studied through several analytical techniques included UV, CD and proton NMR.  Different experimental conditions will also be considered in order to obtain the most stable interaction between an aptamer and PDGF/VEGF.

SAA: Characterization of targeted proteins obtained after co-immunoprecipitation.

Serum amyloid A (SAA) proteins are a family of apoliproteins associated with high-density lipoproteins (HDL) in plasma. Different isoforms known of SAA (1-4) are expressed constitutively at different levels or in response to inflammatory stimuli. During an acute-phase response, the levels of SAA (1-3) expression could reach a 100 to 1,000 fold increase. High expression of SAA in somatic cells causes many metabolic changes such as platelet formation, stimulation of tissue factors, and its accumulation causes amyloidosis (brain, liver, lungs, etc.) which is related to several well known diseases: cancer, thrombosis, and rheumatoid arthritis. Due to their association with acute phase response, we hypothesize that SAA proteins may have a regulatory role mediated through protein:protein interactions with their C-terminal domain, where it may recruit proteins involved in membrane signaling and transcription regulation.  This research aims to isolate those SAA-bound proteins by co-immunoprecipitation, SDS-PAGE and to determine their peptide sequence by mass spectrometry.