The design and synthesis of artificial receptors based on molecular imprinting (MI) technology for the development of a new MIP-based biosensor for detection of the stress biomarker α-amylase in human saliva in point-of-care (PoC) applications is described in this work. The portable electrochemical devices for monitoring α-amylase consists of cost-effective and disposable gold screen-printed electrodes (AuSPEs). To build the electrochemical device, the template biomolecule was firstly immobilized directly over the working area of the gold chip previously activated with a self-assembled monolayer (SAM) of cysteamine (CA). Then, pyrrole (Py) monomer was selected as building block of a polymeric network prepared by CV electropolymerization. After the electropolymerization process, the enzyme was removed from the polymer film in order to build the specific recognition sites for the target enzyme. The MIP biosensor showed a very wide linear concentration range (between 3.0 × 10⁻⁴ to 0.60 mg mL⁻¹ in buffer solution and between 3.0 × 10⁻⁴ to 3.0 × 10⁻² mg mL⁻¹ in human saliva) and low detection levels were achieved (LOD < 3.0 × 10⁻⁴ mg mL⁻¹) using square wave voltammetry (SWV) as the electroanalytical technique.

Within the thymus, thymic epithelial cells (TECs) create dedicated microenvironments for T cell development and selection. Considering that TECs are sensitive to distinct pathophysiological conditions, uncovering the molecular elements that coordinate their thymopoietic role has important fundamental and clinical implications. Particularly, medullary thymic epithelial cells (mTECs) play a crucial role in central tolerance. Our previous studies, along with others, suggest that mTECs depend on molecular factors linked to genome-protecting pathways, but the precise mechanisms underlying their function remain unknown. These observations led us to examine the role of Foxo3, as it is expressed in TECs and involved in DNA damage response. Our findings show that mice with TEC-specific deletion of Foxo3 (Foxo3^cKO) displayed a disrupted mTEC compartment, with a more profound impact on the numbers of CCL21⁺ and thymic tuft mTEC^lo subsets. At the molecular level, Foxo3 controls distinct functional modules in the transcriptome of cTECs and mTECs under normal conditions, which includes the regulation of ribosomal biogenesis and DNA damage response, respectively. These changes in the TEC compartment resulted in a reduced total thymocyte cellularity and specific changes in regulatory T cell and iNKT cell development in the Foxo3^cKO thymus. Lastly, the thymic defects observed in adulthood correlated with mild signs of altered peripheral immunotolerance in aged Foxo3^cKO mice. Moreover, the deficiency in Foxo3 moderately aggravated the autoimmune predisposition observed in Aire-deficient mice. Our findings highlight the importance of Foxo3 in preserving the homeostasis of TECs and in supporting their role in T cell development and tolerance.

Within the thymus, thymic epithelial cells (TECs) create dedicated microenvironments for T-cell development and selection. Considering that TECs are sensitive to distinct pathophysiological conditions, uncovering the molecular elements that coordinate their thymopoietic role has important fundamental and clinical implications. Particularly, medullary thymic epithelial cells (mTECs) play a crucial role in central tolerance. Our previous studies, along with others, suggest that mTECs depend on molecular factors linked to genome-protecting pathways, but the precise mechanisms underlying their function remain unknown. These observations led us to examine the role of Foxo3, as it is expressed in TECs and involved in DNA damage response. Our findings show that mice with TEC-specific deletion of Foxo3 (Foxo3^cKO) displayed a disrupted mTEC compartment, with a more profound impact on the numbers of CCL21⁺ and thymic tuft mTEC^lo subsets. Notably, the combined deficiency in Foxo3 and Aire revealed a central role of Foxo3 in regulating Aire-dependent and Aire-independent mTEC lineages. At the molecular level, Foxo3 controls distinct functional modules in the transcriptome of cTEC and mTEC under normal conditions, which includes the regulation of ribosomal biogenesis and DNA damage response, respectively. These changes in the TEC compartment resulted in a reduced total thymocyte cellularity and specific changes in regulatory T cell and iNKT cell development in the Foxo3^cKO thymus. Lastly, the thymic defects observed in adulthood extended to the periphery, as aged Foxo3^cKO mice presented moderate signs of altered peripheral immunotolerance, which were aggravated in the Aire-deficient autoimmune background. Our findings highlight the importance of Foxo3 in preserving the homeostasis of TECs and in supporting their role in T-cell development and tolerance.