Using 2 - D NMR spectroscopy, the structure of the sex pheromone from Litoria splendida has been determined, in order to elucidate its mode of transport through the aquatic environment. The peptide was found form an α - helical structure, with a central flexible hinge region. The mode of transport through the aquatic environment has been discussed in relation to the structure. Previous work indicated that the Australian amphibian host defence skin peptides that inhibit neuronal nitric oxide synthase ( nNOS ) were likely to act indirectly on the enzyme, by binding to the co - enzyme of nNOS, calmodulin. [superscript 15] N labelled calmodulin was expressed and purified via a bacterial protein expression system and a series of 2 - D NMR [superscript 15] N - HSQC titrations was performed with Australian amphibian host defence skin peptides. in order to determine whether these peptides bind to calmodulin. The three peptides tested were found to bind, and with differing strengths of interaction. One of these was selected for further study. [superscript 15] N and [superscript 13] C doubly labelled calmodulin was then prepared in order to study the complex between this protein and the selected peptide, caerin 1.8, an Australian amphibian skin peptide isolated from Litoria chloris. A series of 3 - D NMR spectra has been recorded on this complex. The backbone atom resonances have been assigned for free calmodulin and for the calmodulin - peptide complex, using a combination of main chain directed and sequential assignment strategies. By analysing the changes in chemical shift that occur upon binding the peptide, it was determined that the mode of binding involves a stronger interaction with the C - terminal domain than the N - terminal domain. / Thesis (Ph.D.)--School of Chemistry and Physics, 2006.

Antibiotics have been used widely for the treatment of bacterial infections for over half a century. However, the emergence of resistance to antibiotics has aroused public health concern, leading to the development of antimicrobial peptides (AMPs) as potential alternative therapeutic agents against bacterial infections. AMPs are naturally found in many species and have important roles in our innate immune defense systems. AMPs are usually cationic amphipathic peptides with membrane destabilizing property. They have a relatively broad spectrum of antimicrobial activity and pathogens are less likely to develop resistance against AMPs. The major challenge of using AMPs as therapeutic agents is their toxicity towards mammalian cells. The biological stability of AMPs to protease in human body is another concern. To address the latter problem, instead of the naturally occur L-enantiomers, Denantiomeric AMPs were introduced to enhance their stability. This study aimed to test the hypothesis that the D-enantiomeric AMPs are more resistant than the Lenantiomeric AMPs against proteolytic degradation. Three pairs of synthetic D-/LAMPs (D-LAO160-P13/LAO160-P12; D-LAO160-H/LAO160-H; and D-LAK-120-HP13/LAK-120-HP13) were employed to test for their stability when treated with trypsin, serum and gastric fluid, and the samples were analyzed by high performance liquid chromatography (HPLC). Generally, all the D-enantiomeric AMPs were found to be resistant towards proteolysis. Besides, to compare the cytotoxicity of D-/LAMPs, MTT and LDH assays of the D/L-LAK120-HP13 pair were carried out on two different cell lines, A549 cells (human lung adenocarcinoma epithelial cells) and RAW264.7 cells (mouse macrophage cells). Significant difference in cytotoxicity of D-LAK120-HP13 and LAK120-HP13 on RAW264.7 cells were obtained from MTT assay, but not in LDH assays or on A549 cells. Further analysis has to be done to validate the findings obtained from this research. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Copy of author's previously published works inserted. / Bibliography: leaves 183-220. / xiii, 226 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Three antimicrobial peptides, maculatin 1.1, uperin 3.6 and caerin 4.1 have been isolated from the respective skin glands of the Australian amphibians Litoria genimaculata, Uperoleia mjobergii and Litoria caerulea. To gain a deeper insight into their mechanisms of action, three dimensional structural studies have been conducted using circular dichroism, two-dimensional nuclear resonance and computer modelling techniques. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is also discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 2000

Copy of author's previously published works inserted. Bibliography: leaves 183-220. Three antimicrobial peptides, maculatin 1.1, uperin 3.6 and caerin 4.1 have been isolated from the respective skin glands of the Australian amphibians Litoria genimaculata, Uperoleia mjobergii and Litoria caerulea. To gain a deeper insight into their mechanisms of action, three dimensional structural studies have been conducted using circular dichroism, two-dimensional nuclear resonance and computer modelling techniques. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is also discussed.

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.

Choy Wai Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 98-122). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.x / Table of contents --- p.xi / List of figures --- p.xv / List of tables --- p.xviii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Severe acute respiratory syndrome (SARS) - An overview --- p.1 / Chapter 1.1.1 --- Epidemiology of SARS --- p.1 / Chapter 1.1.2 --- Clinical presentation of SARS --- p.2 / Chapter 1.1.3 --- Diagnostic tests of SARS --- p.5 / Chapter 1.1.4 --- Treatment of SARS --- p.7 / Chapter 1.2 --- Severe acute respiratory syndrome coronavirus (SARS- CoV) --- p.8 / Chapter 1.2.1 --- The etiological agent of SARS --- p.8 / Chapter 1.2.2 --- The coronaviruses --- p.9 / Chapter 1.2.3 --- Genome of SARS-CoV --- p.11 / Chapter 1.3 --- Spike (S) glycoprotein of SARS-CoV --- p.14 / Chapter 1.3.1 --- Functions of SARS-CoV S glycoprotein --- p.15 / Chapter 1.3.2 --- Receptors for S glycoprotein of SARS-CoV --- p.17 / Chapter 1.4 --- 3C-like protease (3CLPro) of SARS-CoV --- p.20 / Chapter 1.4.1 --- Extensive proteolytic processing of SARS-CoV replicase polyproteins --- p.20 / Chapter 1.4.2 --- SARS-CoV 3CLPro --- p.21 / Chapter 1.4.3 --- Substrate specificity of SARS-CoV 3CLPro --- p.22 / Chapter 1.5 --- Combating SARS - Vaccine and drug development --- p.24 / Chapter 1.5.1 --- Vaccine development against SARS --- p.24 / Chapter 1.5.2 --- Drug development against SARS --- p.25 / Chapter 1.6 --- Project objectives of this thesis --- p.27 / Chapter 1.6.1 --- Synthetic Peptide Studies on SARS-CoV S glycoprotein --- p.27 / Chapter 1.6.2 --- Synthetic Peptide Studies on SARS-CoV 3CLPro --- p.28 / Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.30 / Chapter 2.1.1 --- Bioinformatics analyses of SARS-CoV S gly- coprotein --- p.30 / Chapter 2.1.2 --- Peptide design and molecular modeling --- p.32 / Chapter 2.1.3 --- Solid phase peptide synthesis (SPPS) --- p.33 / Chapter 2.1.4 --- Peptide conjugation --- p.35 / Chapter 2.1.5 --- Immunization in rabbits and monkeys --- p.36 / Chapter 2.1.6 --- ELISA analysis --- p.37 / Chapter 2.1.7 --- Immunofluorescent confocal microscopy --- p.39 / Chapter 2.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.40 / Chapter 2.2.1 --- Protein expression and purification --- p.40 / Chapter 2.2.2 --- Solid phase peptide synthesis (SPPS) --- p.41 / Chapter 2.2.3 --- Peptide cleavage assay --- p.44 / Chapter 2.2.4 --- Molecular docking --- p.46 / Chapter 3 --- Results --- p.48 / Chapter 3.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.48 / Chapter 3.1.1 --- General features and structural analyses of the S glycoprotein --- p.48 / Chapter 3.1.2 --- Peptides design and synthesis --- p.53 / Chapter 3.1.3 --- ELISA analysis and immunofluorescent con- focal microscopy --- p.55 / Chapter 3.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.62 / Chapter 3.2.1 --- Substrate specificity of SARS-CoV 3CLPro . . --- p.62 / Chapter 3.2.2 --- Molecular docking of SARS-CoV 3CLPro and peptide substrates --- p.74 / Chapter 4 --- Discussion --- p.78 / Chapter 4.1 --- Synthetic peptide studies on SARS-CoV S glycoprotein --- p.78 / Chapter 4.1.1 --- Synthetic peptides elicited SARS-CoV specific antibodies --- p.78 / Chapter 4.1.2 --- Factors affecting the specificity and antigenic- ity of synthetic peptides --- p.80 / Chapter 4.1.3 --- Next step towards vaccine development --- p.83 / Chapter 4.1.4 --- A synthetic peptide-based approach --- p.84 / Chapter 4.2 --- Synthetic peptide studies on SARS-CoV 3CLpro --- p.86 / Chapter 4.2.1 --- A comprehensive overview of the substrate specificity of SARS-CoV 3CLpro --- p.87 / Chapter 4.2.2 --- Sequence comparison between SARS-CoV 3CLpro cleavage sites --- p.90 / Chapter 4.2.3 --- A rapid and high throughput approach to screen protease substrate specificity --- p.94 / Bibliography --- p.98

Colorectal cancer (CRC) is one of the most common malignancies worldwide. However, adjuvant chemotherapeutic agents exhibit poor accumulation in the tumor mass and frequently result in serious side effects due to nonspecific damage to normal organs. Therefore, the development of more selective anticancer drugs with targeted delivery to tumor sites is the current trend in cancer therapies. Among these sites, tumor neovasculature is an attractive target for anticancer agents. It is because tumor growth is largely limited by blood supply which is dependent on the extent of angiogenesis in the tumor. / Experimental analysis suggested that TCP-1 phage and synthetic TCP-1 peptide specifically homed to colorectal cancer tissues and co-localized with the tumor vasculature. Moreover, TCP-1 peptide also recognized the vasculature of human colorectal cancer specimens. Subsequently, the homing abilities of TCP-1 phage were extensively tested in other cancer models. Results showed that TCP-1 peptide could also target the vasculature of orthotopic gastric cancer induced by human colon cancer cell line (MKN45) in BALB/c nude mice. Meanwhile, TCP-1 phage exhibited binding activity to colorectal cancer cells such as colon 26 and SW1116. TCP-1 peptide could carry a pro-apoptotic peptide into these cells and markedly enhanced its pro-apoptotic action. / In summary, we have used the phage display technology to isolate two unique peptides TCP-1 and TCP-2, which targeted the vasculature of orthotopic colorectal cancer and also recognized the vasculature of human colorectal cancer. Moreover, they could deliver fluorescein or pro-apoptotic peptide only to the tumor vasculature but not to other normal tissues, for imaging detection and targeted therapy. In conclusion, both TCP-1 and TCP-2 may have significant clinical applications as carriers in diagnostic imaging and ligand-mediated targeted therapy for human colorectal cancer. / Similarly, TCP-2 phage or its peptide also targeted specifically the orthotopic colorectal cancer, and co-localized with the tumor vasculature in mice. Meanwhile, TCP-2 peptide recognized the vasculature of human colorectal cancer specimens. FITC-labeled TCP-2 peptide could also be used to detect cancer tissues in tumor-bearing mice. / To identify specific ligands targeting the tumor neovasculature, in vivo phage display technology has been extensively used. Several dozens of peptides homing to normal or diseased vasculature have been identified through this technology. However, these peptides target mainly the tumors growing at distant sites but not at the primary organ, thus limiting their clinical application. To obtain specific peptides targeting the neovasculature of colorectal cancer growing in situ, we established an orthotopic colorectal cancer model in normal BALB/c mice by using syngeneic colon cancer cells (colon 26). Subsequently, in vivo phage display technology was utilized to isolate peptides which specifically recognized the vasculature of the cancer. Four peptides (termed TCP-1, 2, 3, 4) were enriched more than once after four-round selections. Further investigation disclosed that TCP-1 and TCP-2 phages had relatively stronger binding abilities to cancer tissues among the four phage clones. They were chosen for further study. / We further demonstrated that TCP-1 could serve as a carrier for image detection and drug delivery. FITC-labeled TCP-1 could specifically produce a strong fluorescence signal in the tumors after intravenous injection into the orthotopic tumor-bearing mice. Moreover TCP-1, when conjugated with a pro-apoptotic peptide, could also specifically induce apoptosis of tumor vasculature in vivo. / Li, Zhijie. / Adviser: Cho Chiltin. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 194-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

ESF1 is a 2.052 kDa antimicrobial peptide, mimicking the charge distribution and amphipathic alpha-helical structure of magainin, pGLa, a naturally occurring antimicrobial peptide. ESF1 has been reported to display high activity against Fusarium oxysporum f. sp lycopersici and F. oxysporum f. sp cubense race 4, the tomato and banana crop plant, wilt-causing pathogens, respectively. To assess whether this synthetic peptide can be heterologously expressed in yeast in significant quantities, and still retain full bioactivity, within a eukaryotic system, the ESF1 gene was designed and synthesized from five oligonucleotides, and cloned into pUC18. From the pUC18/ESF1 recombinant plasmid, the ESF1 gene sequence was amplified and cloned into the pBluescript-based vector, pVD4, downstream of the yeast pheromone mating factor alpha (MFa1) promoter, and in frame with the MFa1 signal peptide sequence for expression and secretion in yeast. The expression cassette comprising the MFa1 promoter and signal peptide sequence, and ESF1 gene was subsequently cloned into the yeast/ E. coli shuttle vector, pTG3828 and transformed into Saccharomyces cerevisiae. Chicken IgY antibodies against ESF1 peptide were raised and immunoaffinity purified. Following this, western dot blot analysis and mass spectrometry confirmed the presence of ESF1 in partial HPLC purified fractions of the recombinant yeast culture supernatant. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Venoms, toxins and host-defence systems constitute rich sources of biologically active molecules, many of which have enormous therapeutic and biotechnological potential. In particular, peptides are often a significant component of these chemical arsenals, and are fundamentally important as biological effector molecules. The research presented in this thesis is centred on the isolation and investigation of peptides from both frogs and spiders, and endeavours to probe the important structural and mechanistic features of these bioactive compounds. The skin peptide profiles of interspecific hybrids between the green tree frog Litoria caerulea and the magnificent tree frog Litoria splendida have been investigated in a ninemonth survey. Fourteen peptides were characterised primarily using mass spectrometry, of which three had not been identified previously in the skin secretions of either parent. A number of these peptides are antibacterial agents, while others effectively inhibit the formation of nitric oxide by neuronal nitric oxide synthase. Implications for the genetics and expression of amphibian dermal peptides are also discussed. The majority of frogs of the genus Litoria contain at least one peptide in their glandular secretion capable of inhibiting the formation of nitric oxide by the enzyme neuronal nitric oxide synthase. This was proposed to occur by preventing the association of the regulatory cofactor, Ca²⁺ -calmodulin, with its binding site on the enzyme. Non-covalent binding of the amphibian peptides to calmodulin in the presence of Ca²⁺ has been confirmed using electrospray ionisation mass spectrometry, by the observation of complexes in the gas phase with a 1 : 1 : 4 calmodulin / peptide / Ca²⁺ stoichiometry. In addition, the structure and binding interactions of caerin 1.8, a potent nitric oxide synthase inhibitor, have been further probed using mass spectrometry and nuclear magnetic resonance spectroscopy techniques. Recently a number of small, disulfide - containing neuropeptides of the signiferin and riparin families have been characterised from the skin secretion of frogs of the Crinia genus. Of these, signiferin 1 and riparin 1.1 are both ten residue peptides with similar primary sequences, however appear to have a significantly different spectrum of bioactivity. Although both act at cholecystokinin-2 receptors, signiferin 1 is smooth muscle active while riparin 1.1 is not, and instead causes proliferation of lymphocytes. The three-dimensional structures of these peptides were determined using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Both signiferin 1 and riparin 1.1 adopt β - turn type conformations, however differences in these structures may be responsible for the variation in biological activity noted for these peptides. The dermal secretions of most Australian frogs contain at least one broad-spectrum peptide antibiotic, and often a series of peptides with differing activity to afford greater protection against microbial pathogens. Solid state nuclear magnetic resonance spectroscopy studies were carried out to investigate the interaction of a number of these antibacterial peptides with anionic model membranes, and the results are compared with work previously reported using neutral lipids. It appears the peptides may have a different mode of interaction with the membranes depending upon the charge of the lipid head group. The cupiennin 1 peptides have been identified in the venom of the neotropical wandering spider, Cupiennius salei, and demonstrate potent wide-spectrum antibacterial activity. Primary sequence analysis of these peptides suggests a unique amphipathic structure distinctly different from that of other potentially helical cationic antimicrobial peptides isolated thus far. Using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations, cupiennin 1a was found to adopt an α- helical structure with a flexible central hinge region in membrane mimicking solvents. Following this, nuclear magnetic resonance spectroscopy methods were used to further probe the antibacterial and the newly identified neuronal nitric oxide synthase inhibitory activity of this peptide. / Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, Discipline of Chemistry, 2006

The skin extracts from amphibians have been investigated for over fifty years and have been found to contain numerous components with therapeutic and medicinal uses. Host - defence compounds are secreted onto the dorsal surface of the animal from specialised granular glands in response to a variety of stimuli, such as stress induced by a predator. Isolated peptides can exhibit either pharmacological properties or antibiotic activity. Previous studies isolated a potent hypotensive neuropeptide, crinia angiotensin II, within skin secretions of the Australian frog Crinia georgiana. This prompted further investigations into the isolation and sequence determination of host - defence compounds from other species in this genus - C. signifera, C. riparia and C. deserticola. Fifteen novel peptides were identified. The major peptide components were potent disulfide containing neuropeptides of a type not observed in other Australian anurans that have been previously investigated. The remaining peptides demonstrate either antibiotic activity or inhibit the enzyme neuronal nitric oxide synthase. The skin components from anurans of the Litoria genus have been extensively studied, with a number of peptides exhibiting both antibacterial and pharmacological activity. The skin secretion of Litoria dentata has been investigated, with five novel peptides identified. The neuropeptide tryptophyllin L 1.3 was previously isolated from the related frog L. rubella. Other components that are unique in structure have not yet been tested for biological activity. The parasitic disease malaria is responsible for over one million deaths per year. The increase in resistance of current antimalarial compounds has led to the development of new treatments from various animal - derived peptide antimicrobials. A number of amphibian peptides and their derivatives were investigated as potential antiplasmodial agents against the malaria parasite Plasmodium falciparum. Results indicate that these compounds inhibit parasite growth with minimal haemolytic activity, making them promising tools for malaria research.The defence chemistry of amphibian neuropeptides has been extensively studied and is important in understanding both the ecology and physiology of the vertebrate. Neuropeptides are classified into groups with similar structural characteristics. Biological activity occurs via interaction with a G protein - coupled receptor. The most studied of all amphibian neuropeptides is caerulein, which has a similar spectrum of activity to the mammalian peptide cholecystokinin. This includes smooth muscle contraction that occurs via interaction with cholecystokinin receptors. The pharmacological activity of Australian anuran neuropeptides from various genera was investigated. Two biological assays were conducted - a smooth muscle contraction test and a lymphocyte proliferation assay. A range of neuropeptides contracted smooth muscle at nanomolar concentrations, while others only proliferated lymphocytes. Some peptides were inactive in both assays. Young marsupials are born at an immature stage of development and rely on immune protection provided by the mother. Eugenin is a host - defence compound isolated from pouch secretions of the Tammar wallaby. The immunomodulator activates CCK2 receptors, resulting in lymphocyte proliferation. Therefore, eugenin stimulates immune cells in the pouch providing vital immune protection for pouch young. / Thesis (Ph.D.)--School of Chemistry and Physics, 2006.