No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

The synthesis of peptides can be considered as the rate-limiting step in the development of peptide-based medicines. Synthetic methods currently available are limited by several aspects, including the high cost of production or excessive waste when applied to large-scale synthesis. The development of a new solution-phase synthesis of peptides is described herein. Initial studies focus on the synthesis of α-peptides in the C-terminal direction, though the occurrence of epimerisation during chain elongation shows the limitation of this approach. Attempts to reduce this problem and to gain a better insight into the epimerisation processes involved are described. The unsuccessful application of this initial strategy to the synthesis of β-peptides is also discussed. A new procedure involving the coupling of amino acids or peptide acids with slight excesses of pentafluorophenyl esters in a THF/water solvent mixture in the N-terminal direction is developed and discussed. Contrary to modern repetitive solution-phase peptide synthesis procedures, this approach does not require time-consuming neutralisation reactions and reduces significantly the number of operation units that are necessary to obtain peptide intermediates. The efficiency of the new method is demonstrated by the rapid synthesis of short hydrophobic and hydrophilic peptides, the antimalarial cyclopeptide mahafacyclin B and a protected form of the hydrophilic pentapeptide Gly-Arg-Gly-Asp-Ser. Binding studies of the complex between 1-(3-Dimethylaminopropyl)-3-ethylurea hydrochloride (EDU.HCl) and triethylammonium trifluoroacetate are described and the potential application of EDU.HCl as an artificial carboxylate receptor to increase the acidity of trifluoroacetic acid is discussed.

Regulated survival, proliferation and differentiation of cells in the nervous system is crucial for development. Much of regulation is controlled by hormones. There is abundant evidence that a member of the glucagon superfamily, pituitary adenylate cyclase-activating polypeptide (PACAP), is important in this process. PACAP functions have been described in the peripheral and central nervous systems of many species. Although the primary function of PACAP is not known, its high conservation and presence in all species examined to date suggest it is vital to normal development. My thesis objective was to determine the response of early CNS neuroblasts to PACAP, in conjunction with another glucagon superfamily member, growth hormone releasing hormone (GHRH). GHRH is best known for causing release of growth hormone from the pituitary, but it also has functions in nervous system development. Because PACAP and GHRH are encoded on the same gene in non-mammalian vertebrates, it is possible that they have similar or coordinated functions. PACAP affects development by altering levels of proliferation and differentiation and decreasing apoptosis. For these reasons, I focused my research in these areas. Using neuroblast-enriched cultures from embryonic day 3.5 chick, my first goal was to show that PACAP and GHRH affected these cells. Radioimmunoassays for cAMP revealed that all but one form of PACAP, and only one form of GHRH, caused an increase in cAMP relative to controls. As to the former, comparison of differing PACAP structures suggested that conservation at the amino terminus was important in binding the hormone to the receptor. The fact that PACAP, but not GHRH, increased cAMP, indicated that evolution of PACAP and GHRH had altered their functions. Chick neuroblasts were also shown to produce PACAP and its primary receptor, suggesting an autocrine/paracrine role for PACAP. My next goal was to examine the nature of the downstream effects of increased cAMP. To study cell cycle, I developed a protocol using proliferating cell nuclear antigen (PCNA) and propidium iodide (PI), in fixed cell populations. PCNA is present in low amounts in non-cycling cells, but rises sharply in actively proliferating cells. The PI helped delineate cell cycle compartments, because in permeabilized cells it binds to and quantifies DNA. Changes in G0, G1, S and G2/M were recorded using flow cytometry. Because the cells were producing PACAP and most were cycling, rather than add more PACAP I chose to block the PACAP receptor. This caused cell cycle exit. I also blocked the cell cycle at two points, and showed that exogenous PACAP could release some cells from the block, and return them to cycling. PACAP affected apoptosis also, but because the protocol was not designed to measure this, I adopted another protocol using flow cytometry. With live cells, and fluorescein diacetate, which is retained and fluoresces in healthy cells, and PI, which enters only cells with damaged membranes, I used the characteristic of apoptotic cells to die with membranes intact to confirm increased apoptosis when the PACAP receptor was blocked. This left the question of whether PACAP affected differentiation. The cell cycle protocol had shown some cells were still quiescent, not dying, at 24 h, so I hypothesized that they might be differentiating. I used proteomics to test this. With isotope-coded affinity tagged (ICAT) analysis, I measured changes in protein content in cells that had been treated with the receptor blocker, compared to control. This confirmed previous work and my hypothesis that some cells were differentiating. Because this technique is not commonly used in molecular biology, I also evaluated the effectiveness of the technique. My work showed that endogenous PACAP keeps chick neuroblasts alive and cycling, but will allow some to differentiate rather than die, when the hormone is withdrawn. Obviously, PACAP plays a crucial role in early chick brain development. / Graduate

University of Macau / Faculty of Science and Technology. / Department of Computer and Information Science

The use of peptide drugs has gained popularity recently. Peptides are attractive drug targets due to their high specificity and potency towards their biological targets. A drawback for peptide drugs is a lack of stability for oral delivery. Two classes of disulfide-rich peptides, conotoxins and cyclotides, have been shown to have higher stability than linear peptides thanks to their disulfide connectivity. Conotoxins are present in the venom of cone snails, a carnivorous marine mollusk that preys upon fish, worms, or other mollusks. Conotoxins are promising drugs leads with great prospects in the treatment of diseases and disorders such as chronic pain, multiple sclerosis and Parkinson’s and Alzheimer’s diseases. Cyclotides, which are cyclic cysteine knot containing peptides, isolated from the Violaceae (violet), Rubiaceae (coffee), and Cucurbitaceae (cucurbit) families and they have a wide range of biological activities, such as anti-HIV, uterotonic, and antimicrobial. P-superfamily framework IX conotoxins (C-C- C-CXC- C) contain the same cysteine framework, homologous sequences, and similar 3D structures to cyclotides. The knot containing conotoxins have been identified in several Conus species, but this work focuses on those from Conus brunneus, Conus purpurascens, and Conus gloriamaris. The cysteine knot motif of cyclotides and P-superfamily conotoxins is characterized by a cyclic backbone and six-conserved cysteine residues that form the three-disulfide bridges of the “knot”. This motif provides cyclotides and conotoxins with superior stability against thermal, chemical, and enzymatic degradation; marking them as potential frameworks for peptide drug delivery. Presented are details on the isolation of conotoxins and cyclotides, from Viola tricolor, and the characterization of their activity in the well-characterized Drosophila melanogaster giant fiber system (GFS) neuronal circuit, which contains GAP, acetylcholine, and glutamate synapses. The transcriptomes of two Conus brunneus specimens were assembled and mined for P-superfamily framework IX conotoxins. Eleven mature P-superfamily framework IX conotoxins were identified in the crude venom. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection