Characterization of the PGE2 pathway in arthritis and inflammation : mPGES-1 as a therapeutic target

The inducible prostaglandin (PG) E2 pathway is defined by the concerted activities of the enzymes cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 in producing PGE2. PGE2 has pro-inflammatory and immunomodulatory functions and is involved in an array of diseases with an autoimmune and chronic inflammatory component, including rheumatoid arthritis (RA). In RA, mPGES-1 and COX-2 are up-regulated in the inflamed synovium of patients. COX inhibitors like non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors (COXibs) relieve inflammation and pain in RA, but their respective GI tract and cardiovascular side effects preclude long-term use. Moreover, other effective RA therapies like TNF blockade and B-cell depletion therapy leave the inducible PGE2 pathway unaffected, suggesting that targeting this pathway could have additional benefits in a combinatorial approach. mPGES-1 is currently investigated as a target that could dissociate the anti-inflammatory benefits of COX inhibitors from their detrimental side effects. However, most inhibitors of human mPGES-1 activity generated so far failed to inhibit the murine enzyme ortholog, which complicates their characterization in vivo in relevant disease models. The first objective of this thesis was to extend the characterization of the PGE2 pathway in arthritis. The second objective was to investigate mPGES-1 as a therapeutic target. The latter was accomplished in part through the development and evaluation of pharmacological inhibitors active on both human and murine mPGES-1. To fulfill the first objective, we characterized a new mechanism for the induction of PGE2 production in RA synovial fibroblasts (RASFs), whereby complexes of the alarmin high mobility group box protein-1 (HMGB1) and the cytokine IL-1β cause an up-regulation of COX-2 and mPGES-1 expression. We also characterized 15- prostaglandin dehydrogenase (15-PGDH) to co-localize with mPGES-1 and the COX enzymes in the synovium of RA patients, suggesting a concerted activity of the anabolic and catabolic parts of the PGE2 cascade in the joint. In the same patients, we determined that methotrexate therapy did not interfere with the expression of COXs, mPGES-1 or 15-PGDH, adding that therapy to the list of approaches leaving the PGE2 pathway unaffected. Lastly, we could not confirm an association between the expression of COXs, mPGES-1 and 15-PGDH and quantitative pain assessment or arthritis development in arthralgic individuals at risk of developing arthritis or in early arthritis patients. In line with the second objective, we developed compounds II and III, which inhibit PGE2 synthesis in vitro in different human and murine cell assays and in vivo in the air pouch model of acute inflammation. Compound II also reduced edema in the rat adjuvant-induced arthritis (AIA) model. When the PG profile elicited by mPGES-1 inhibition with compound III was compared to that resulting from mPGES- 1 gene deletion in the air pouch model, different results were observed suggesting the two modes of inhibition might not have the exact same outcome. While inhibition of PGE2 synthesis with both compound II and III did not result in the shunting of PGH2 to other prostanoids, a shunt to thromboxane (TX) B2 was observed in the mPGES-1 knockout mouse. We also used the mPGES-1 knockout mouse to investigate the impact of mPGES-1 gene deletion on the eicosanoid and fatty acid profiles in inflammation. We discovered that it resulted in macrophages producing more 15- deoxy-Δ 12,14 PGJ2 and the spleen containing more eicosadienoic acid (EDA). This suggests mPGES-1 inhibition could not only inhibit the synthesis of the pro- inflammatory PGE2, but also cause the up-regulation of anti-inflammatory pathways. In conclusion, this thesis further advances the knowledge about the PGE2 synthesis cascade in arthritis and describes two new mPGES-1 inhibitors with an in vivo activity in native rodent models of disease. The latter constitute new valuable tools for the study of mPGES-1 in whichever pathology it has an involvement

Epistemic Logic Programs with World View Constraints

An epistemic logic program is a set of rules written in the language of Epistemic Specifications, an extension of the language of answer set programming that provides for more powerful introspective reasoning through the use of modal operators K and M. We propose adding a new construct to Epistemic Specifications called a world view constraint that provides a universal device for expressing global constraints in the various versions of the language. We further propose the use of subjective literals (literals preceded by K or M) in rule heads as syntactic sugar for world view constraints. Additionally, we provide an algorithm for finding the world views of such programs

Impact of Backbone fluorination on -conjugated polymers in organic photovoltaic devices: a review

Solution-processed bulk heterojunction solar cells have experienced a remarkable acceleration in performances in the last two decades, reaching power conversion efficiencies above 10%. This impressive progress is the outcome of a simultaneous development of more advanced device architectures and of optimized semiconducting polymers. Several chemical approaches have been developed to fine-tune the optoelectronics and structural polymer parameters required to reach high efficiencies. Fluorination of the conjugated polymer backbone has appeared recently to be an especially promising approach for the development of efficient semiconducting polymers. As a matter of fact, most currently best-performing semiconducting polymers are using fluorine atoms in their conjugated backbone. In this review, we attempt to give an up-to-date overview of the latest results achieved on fluorinated polymers for solar cells and to highlight general polymer properties’ evolution trends related to the fluorination of their conjugated backbone

Impact of Backbone fluorination on -conjugated polymers in organic photovoltaic devices: a review

Solution-processed bulk heterojunction solar cells have experienced a remarkable acceleration in performances in the last two decades, reaching power conversion efficiencies above 10%. This impressive progress is the outcome of a simultaneous development of more advanced device architectures and of optimized semiconducting polymers. Several chemical approaches have been developed to fine-tune the optoelectronics and structural polymer parameters required to reach high efficiencies. Fluorination of the conjugated polymer backbone has appeared recently to be an especially promising approach for the development of efficient semiconducting polymers. As a matter of fact, most currently best-performing semiconducting polymers are using fluorine atoms in their conjugated backbone. In this review, we attempt to give an up-to-date overview of the latest results achieved on fluorinated polymers for solar cells and to highlight general polymer properties’ evolution trends related to the fluorination of their conjugated backbone

Influence of Sp1/Sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues

PURPOSE : Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS : HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS : Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS : Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes

Transcriptional regulation of the human α6 integrin gene by the transcription factor NFI during corneal wound healing

Purpose. Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human α6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human α6 gene, and its regulatory influence was characterized in corneal epithelial cells. Methods. Plasmids bearing the α6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the α6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. Results. All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of α6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of α6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. Conclusions. Repression of α6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane