Our laboratory has demonstrated previously that the proglucagon gene (gcg), which encodes the incretin hormone GLP-1, is among the downstream targets of the Wnt signaling pathway; and that Pak1 mediates the stimulatory effect of insulin on Wnt target gene expression in mouse gut non- endocrine cells. Here, I asked whether Pak1 controls gut gcg expression and GLP-1 production, and whether Pak1 deletion leads to impaired metabolic homeostasis in mice. I detected the expression of Pak1 and two other group I Paks in the gut endocrine L cell line GLUTag, and co-localized Pak1 and GLP-1 in the mouse gut. Insulin was shown to stimulate Pak1 Thr423 and β-cat Ser675 phosphorylation. The stimulation of insulin on β-cat Ser675 phosphorylation, gcg promoter activity and gcg mRNA expression could be attenuated by the Pak inhibitor IPA3. Male Pak1-/- mice showed significant reduction in both gut and brain gcg expression levels, and attenuated elevation of plasma GLP-1 levels in response to oral glucose challenge. Notably, the Pak1-/- mice were intolerant to both intraperitoneal and oral glucose administration. Aged Pak1-/- mice showed a severe defect in response to intraperitoneal pyruvate challenge (IPPTT). In primary hepatocytes, however, IPA3 reduced basal glucose production, attenuated glucagon-stimulated glucose production, and inhibited the expression of Pck1 and G6pc. This implicates that the direct effect of group I Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in IPPTT in aged Pak1-/- mice is due to the lack of Pak1 elsewhere. The defect in IPPTT in aged Pak1-/- mice could be rescued by stimulating gcg expression with forskolin injection or by enhancing the incretin effect via sitagliptin administration. In summary, my study demonstrates that: 1) Pak1 positively regulates GLP-1 production, 2) Pak1/β-cat signaling plays a role in gut/liver axis or gut/pancreas/liver axis governing glucose homeostasis, and 3) Pak1-/- mice can be utilized as a novel model for metabolic research.

Our laboratory has demonstrated previously that the proglucagon gene (gcg), which encodes the incretin hormone GLP-1, is among the downstream targets of the Wnt signaling pathway; and that Pak1 mediates the stimulatory effect of insulin on Wnt target gene expression in mouse gut non- endocrine cells. Here, I asked whether Pak1 controls gut gcg expression and GLP-1 production, and whether Pak1 deletion leads to impaired metabolic homeostasis in mice. I detected the expression of Pak1 and two other group I Paks in the gut endocrine L cell line GLUTag, and co-localized Pak1 and GLP-1 in the mouse gut. Insulin was shown to stimulate Pak1 Thr423 and β-cat Ser675 phosphorylation. The stimulation of insulin on β-cat Ser675 phosphorylation, gcg promoter activity and gcg mRNA expression could be attenuated by the Pak inhibitor IPA3. Male Pak1-/- mice showed significant reduction in both gut and brain gcg expression levels, and attenuated elevation of plasma GLP-1 levels in response to oral glucose challenge. Notably, the Pak1-/- mice were intolerant to both intraperitoneal and oral glucose administration. Aged Pak1-/- mice showed a severe defect in response to intraperitoneal pyruvate challenge (IPPTT). In primary hepatocytes, however, IPA3 reduced basal glucose production, attenuated glucagon-stimulated glucose production, and inhibited the expression of Pck1 and G6pc. This implicates that the direct effect of group I Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in IPPTT in aged Pak1-/- mice is due to the lack of Pak1 elsewhere. The defect in IPPTT in aged Pak1-/- mice could be rescued by stimulating gcg expression with forskolin injection or by enhancing the incretin effect via sitagliptin administration. In summary, my study demonstrates that: 1) Pak1 positively regulates GLP-1 production, 2) Pak1/β-cat signaling plays a role in gut/liver axis or gut/pancreas/liver axis governing glucose homeostasis, and 3) Pak1-/- mice can be utilized as a novel model for metabolic research.

This thesis aims to implement a combination of Linear Programming Column Generation and a Large Neighbourhood Search heuristic to solve scheduling problems. The resulting method is named Integer Programming Column Search (IPCS). For computational evaluation, the IPCS method is applied to the problem Prize-Collecting Job Sequencing with One Common and Multiple Secondary Resources generalised to parallel identical machines. The interest of combining exact procedures with heuristic approaches is quickly growing since scheduling problems have many and complex real-world applications. Most of these problems are NP-hard and therefore very challenging to solve. By using a combination of heuristic strategies and exact procedures, it can be possible to find high-quality solutions to such problems within an acceptable time horizon. The IPCS method uses a greedy integer programming column generating problem introduced in a previous work. This problem is designed to generate columns that are useful in near-optimal integer solutions. A difference to previously introduced method is that we here build a master problem, an Integer Programming Column Search Master (IPCS-Master). This is used to update the dual solution that is provided to the greedy integer programming column generating problem. The computational performance of the IPCS method is evaluated on instances with 60, 70, 80, 90 and 100 jobs. The result shows that the combined design encourage the generation of columns that benefit the search of near-optimal integer solutions. The introduction of an IPCS-Master, which is used to update the dual variable values, generally leads to fewer pricing problem iterations than when no master problem is used.

Cysteinyl peptides likely played an important role in the prebiotic synthesis of cofactors, such as iron-sulfur clusters. However, cysteinyl peptides must be reduced in order to coordinate iron-sulfur clusters. Mixtures of ferric ions and cysteinyl peptides leads to the reduction of ferric to ferrous ions and the concomitant formation of disulfide bridged, oxidized cysteinyl peptides that are incapable of coordinating an iron-sulfur cluster. Here, we develop a photochemically driven solution to this problem. Lipoic acid (( R )-5-(1,2-dithiolane-3-yl)pentanoic acid), a molecule structurally similar to fatty acids, can be photochemically reduced and can subsequently reduce the oxidized cysteinyl peptides needed for the coordination of an iron-sulfur cluster. Other dithilane ring containing molecules possess similar activity to lipoic acid. The synthesis of small peptides containing cysteines, such as glutathione and GCG (Gly-Cys-Gly) is easy by both solid phase and solution phase methodologies. However, as the length of the peptide increases, the yield begins to decrease, especially for peptides containing cysteines due to oxidation. One solution could be to exploit a previously uncovered mechanism for the joining of peptides into longer peptides. Such mechanisms, referred to as CPL for catalytic peptide ligation, rely on either thiols or metals as catalysts and peptide nitriles as substrates. Thus far, CPL has only been exploited with non-cysteinyl peptides. In this thesis, we extend CPL to cysteine containing peptides by taking advantage of the templating effects of Zn2+. Longer peptides with properly spaced cysteines are frequently better able to stabilize iron-sulfur clusters in aqueous solution than shorter peptides. Coordination can either be complete or an open coordination position, filled by solvent, can be used to bind substrate. Two well-known examples of such an arrangement are the radical SAM (S-adenosylmethionine) enzyme and aconitase being an enzyme of the citric acid cycle. We designed and synthesized peptide sequences that could coordinate a [4Fe-4S]2+ cluster with three cysteinyl ligands, leaving an open coordination position. The stability of the [4Fe-4S] cluster was affected by the intermediates of the citric acid cycle. The iron-sulfur can be reconstituted with the long peptidyl sequences from proteins such as SLC25A39 which contains four cysteine ligands to form [2Fe-2S] cluster, which is necessary for glutathione transport from cytosol to mitochondria.