Thesis (Ph. D.)--Mississippi State University. Department of Biochemistry and Molecular Biology. / Title from title screen. Includes bibliographical references.

<p>Several lines of cellular and physiological evidence have suggested the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies generated against vertebrate atrial natriuretic peptides (ANPs). Functional characterization of these immunoanalogues, referred to as immunoreactive plant natriuretic peptides (irPNPs) or plant natriuretic peptides (PNPs), has shown that they play important roles in a number of cellular processes crucial for plant growth and maintenance of cellular homeostasis. Although the various biological roles of PNPs in plants are known, their exact mode of action remains elusive. To elucidate the mechanisms of action for these immunoanalogues, we have prepared a biologically active recombinant PNP from Arabidopsis thaliana (AtPNP-A) and the biological activity was demonstrated by showing its ability to induce water uptake into Arabidopsis thaliana protoplasts. In addition, the molecule was shown to downregulate photosynthesis while at the same time up-regulating respiration, transpiration as well as net water uptake and retention capacities in the sage Plectranthus ecklonii. Further analysis of the recombinant AtPNP-A indicated that the peptide can induce systemic response signalling though the phloem. A recombinant Arabidopsis wall associated kinase-like protein (AtWAKL10) that has a domain organization resembling that of vertebrate natriuretic peptide (NP) receptors was also partially characterized as a possible receptor for the recombinant AtPNP-A. Vertebrate NP receptors contain an extracellular ligand-binding domain and an intracellular guanylate cyclase (GC)/kinase domain and signal through the activity of their GC domain that is capable of generating intracellular cGMP from GTP. The structural resemblance of AtWAKL10 to vertebrate NP receptors could suggest a functional homology with receptor molecules and it is conceivable that such a receptor may recognize PNPs as ligands. The characterization of the recombinant AtWAKL10 showed that the molecule functions as both a GC and a kinase in vitro. This strengthened the suggestion that AtWAKL10 could be a possible AtPNP-A receptor especially considering the fact that AtPNP-A applications to plant cells also<br /> trigger cGMP transients. Furthermore, a bioinformatic analysis of the functions of AtPNP-A and AtWAKL10 has inferred both molecules in plant pathogen responses and defense mechanisms, thus indirectly functionally linking the two proteins.</p>

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1989. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

M.Sc. (Biochemistry) / Peptides have become useful therapeutic and targeting molecules in the treatment of various diseases. In nano-medicine, gold nanoparticles (AuNPs) are potential carrier systems of various targeting and therapeutic molecules including peptides. This study investigated the ability of 14 nm AuNPs as intracellular carriers of therapeutic and targeting peptides by assessing the toxic effects of these peptides when conjugated to AuNPs, on U937 monocyte-derived macrophages. These peptides include the proapoptotic peptide (klaklak)2, the targeting Glucose-Regulated Protein 78 (GRP-78) binding peptide and the carrier Trans-Activating Transcriptional (TAT) cell penetrating peptide (CPP). These peptides were conjugated to the AuNPs via poly(ethylene glycol) (PEG) polymers. The size, morphology, aggregation state and surface charge of citrate-stabilized, PEGylated, as well as peptide-conjugated PEG-AuNPs were determined using Transmission Electron Microscopy (TEM), Ultraviolet-Visible Spectroscopy (UV-vis), Dynamic Light Scattering (DLS) and Zeta Potential (ζ-Potential). Intracellular uptake of the tested AuNPs was investigated using the Cytoviva® dark-field hyperspectral imaging system. The toxicity was assessed using the conventional toxicity assay systems including adenosine triphosphate (ATP) and lactate dehydrogenase (LDH) assays, as well as the impedance based technology, xCELLigence real time cell analysis (RTCA) single plate (SP) system. The genotoxicity was investigated with the alkaline comet assay and the mechanisms of toxicity were investigated using western blotting through the ability of the AuNPs to activate the oxidative stress pathways, namely, the nuclear factor erythroid 2-related factor 2 (Nrf2) and factor kappa B (Nf-κB) pathways. Characterisation of the AuNPs revealed that the physicochemical properties of the particles were altered when suspended in culture medium. All the AuNPs tested have shown increase in size through aggregation. Although they all kept their negative charge, this charge was increased, with the greatest increase in charge shown for PEGylated AuNPs (OHPEG-AuNPs). Intracellular uptake was confirmed with 14 nm citrate stabilized AuNPs, TAT and GRP PEG-AuNPs. Some degree of uptake was also observed with (klaklak)2 PEG-AuNPs but not with OHPEG-AuNPs which was generally inaccessible to cells.

Philosophiae Doctor - PhD / Several lines of cellular and physiological evidence have suggested the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies generated against vertebrate atrial natriuretic peptides (ANPs). Functional characterization of these immunoanalogues, referred to as immunoreactive plant natriuretic peptides (irPNPs) or plant natriuretic peptides (PNPs), has shown that they play important roles in a number of cellular processes crucial for plant growth and maintenance of cellular homeostasis. Although the various biological roles of PNPs in plants are known, their exact mode of action remains elusive. To elucidate the mechanisms of action for these immunoanalogues, we have prepared a biologically active recombinant PNP from Arabidopsis thaliana (AtPNP-A) and the biological activity was demonstrated by showing its ability to induce water uptake into Arabidopsis thaliana protoplasts. In addition, the molecule was shown to downregulate photosynthesis while at the same time up-regulating respiration, transpiration as well as net water uptake and retention capacities in the sage Plectranthus ecklonii. Further analysis of the recombinant AtPNP-A indicated that the peptide can induce systemic response signalling though the phloem. A recombinant Arabidopsis wall associated kinase-like protein (AtWAKL10) that has a domain organization resembling that of vertebrate natriuretic peptide (NP) receptors was also partially characterized as a possible receptor for the recombinant AtPNP-A. Vertebrate NP receptors contain an extracellular ligand-binding domain and an intracellular guanylate cyclase (GC)/kinase domain and signal through the activity of their GC domain that is capable of generating intracellular cGMP from GTP. The structural resemblance of AtWAKL10 to vertebrate NP receptors could suggest a functional homology with receptor molecules and it is conceivable that such a receptor may recognize PNPs as ligands. The characterization of the recombinant AtWAKL10 showed that the molecule functions as both a GC and a kinase in vitro. This strengthened the suggestion that AtWAKL10 could be a possible AtPNP-A receptor especially considering the fact that AtPNP-A applications to plant cells also trigger cGMP transients. Furthermore, a bioinformatic analysis of the functions of AtPNP-A and AtWAKL10 has inferred both molecules in plant pathogen responses and defense mechanisms, thus indirectly functionally linking the two proteins. / South Africa

Two highly homologous synthetic peptides MLC(3-13) (K-R-A-K-A-K-T-TK-K-R-G) and MLC(5-13) (A-K-A-K-T-T-K-K-R-G) corresponding to the amino terminal amino acid sequence of smooth muscle myosin light chain were utilized as substrates for protein kinase C purified from murine lymphosarcoma tumors to determine the role of the primary amino acid sequence of protein kinase C substrates in defining the lipid (phosphatidyl serine and diacylglycerol) requirements for the activation of the enzyme. Removal of the basic residues lysine and arginine from the amino terminus of MLC(3-13) did not have a significant effect on the Ka value of diacylglycerol. The binding of effector to calcium-protein kinase C appears to be random since binding of one effector did not block the binding of the other.

"The photophysics of several bichromophoric dipeptide model compounds and two trichromophoric 15-residue peptides have been studied by a combination of absorption, fluorescence, phosphorescence and laser flash photolysis. Intramolecular singlet-singlet energy transfer (SSET) occurs efficiently within these systems. Trichromophore 14 undergoes intramolecular SSET from the central chromophore to the termini, kSSET = 5.8 x109 s-1 , with a five fold increase over 13, kSSET = 1.1 x 109 s-1 . Evaluation of SSET mechanisms via the Förster treatment and molecular modeling indicates that the dipole-induced dipole mechanism is sufficient to account for the observed SSET. However, given the close distances of the chromophores (~10 Å), an electron exchange mechanism can not be ruled out. Low-temperature phosphorescence in 1:1 methanol/ethanol and room-temperature laser flash photolysis in acetonitrile results indicate that intramolecular triplet-triplet energy transfer (TTET) is efficient in dipeptides 7,9-12 and proceeds with a rate constant of kTTET > 5 x 10 8 s-1. The occurrence of TTET in dipeptide 8, (biphenyl-naphthalene), could not be confirmed due to the fact that SSET from biphenyl to the naphthalene moiety was 26 times greater than kISC. Thus nearly all absorbed light was funneled directly the to the singlet manifold of the naphthalene moiety. TTET in the trichromophores could not be fully evaluated due to their low solubility. However, it is shown from 77°K experiments that kTTET is at least 2.2 x 102 and 2.6 x 102 s-1 for 13 and 14 respectively."

No description available.

Current cancer therapies have low specificity for tumor cells and have serious toxic side effects. Targeting drugs to the cancer cells can help improve the outcome of existing cancer therapies. In recent years, a number of peptides have been identified by peptide phage display for targeting different tumor types. Peptides identified from the phage display for targeting cancer cells can be further improved for specific binding and metabolic stability by chemical manipulation of their structures. The aims of this work were: (i) to develop a peptide array-whole cell binding assay for screening peptides with specific binding affinity for cancer cells (ii) design of novel peptidomimetics to improve their properties as drug candidates. First, peptide arrays based on the lead peptide sequences NGR and p160 were designed and synthesized. A direct peptide-cell binding assay using CyQUANT dye allowed identification of several new peptides with higher affinity for MDA-MB-435 and MCF-7 cancer cells compared to the wild type p160. These peptides did not recognize the normal endothelial HUVEC cells. Three p160 peptide analogues, namely, 11 (RGDPAYQGRFL), 18 (WXEAAYQRFL), and 40 (WXEPAYQRKL), that displayed highest affinity for the cancer cells were manually synthesized and labelled with FITC. The binding ability of these peptides was confirmed using fluorescence imaging and flow cytometry. The results confirmed the high and specific affinity of peptides 11 and 18 for the cancer cells. The peptide array-cell binding assay established in this study is not only useful for the identification of cancer targeting peptides. It can also be used for the generation of diagnostic tools for cancer. Secondly, two new classes of -peptides, 3- and 2-peptides derived from L-Asp and L-Dap monomers, respectively, were synthesized. The methodology allowed independent buildup of the -peptide backbone and the introduction of sequential side chain substitutions. It is shown that / mixed peptide increases target recognition and retains the proteolytic stability. Moreover, -peptides impart no cytotoxicity, which will expand their potential application in the design of biologically active peptides. As a result, these compounds represent good candidates for new drugs and as tools to gain further insight into protein folding and molecular recognition processes. / Pharmaceutical Sciences