Pharmacology and Therapeutics - Theses

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    Transcriptomic, proteomic and biological analyses of venom proteins from two Chrysaora jellyfish
    Ponce-Garcia, Dalia Patricia ( 2017)
    Chrysaora jellyfish (Class Scyphozoa) are considered mild to severe stingers. In particular, Chrysaora fuscescens and Chrysaora quinquecirrha are known for their ability to inflict painful stings that cause both localised and systemic injury in humans. The bioactivities in jellyfish venom are associated with a payload of toxic proteins but the molecular identities and mechanism(s) of action for those toxins require elucidation. Because of the complexity of venom composition and the concomitant difficulties related with the isolation and characterisation of individual toxins, high-throughput sequencing methods were used in this thesis to identify the major toxin groups in an effort to provide clues about their functional roles. Concurrently, the venom of C. quinquecirrha was assessed and compared against the venom of cubozoan jellyfish, C. fleckeri, to provide a quantitative and standardised measurement of the toxic properties and potency of venoms. The potential toxin repertoires of C. fuscescens and C. quinquecirrha jellyfish were determined from sequence data generated from the tentacle transcriptomes and the venom proteomes of each species. The strategy followed included the construction of the tentacle transcriptomes using a combination of mRNA high-throughput sequencing (Illumina), de novo assembly, functional annotation based on protein homology and sequence analysis assisted by bioinformatics tools. In parallel, the venom proteomes were characterised using SDS-PAGE and tandem mass spectrometry. From this combined approach, 158 and 75 potential toxins were identified in both transcriptomes and proteomes of C. fuscescens and C. quinquecirrha, respectively. Recognised toxin families included C-type lectins, CAP domain-containing proteins, deoxyribonucleases, esterases, glycosidases, lipases, nucleotidases, phosphatases, pore-forming toxins, proteases, protease inhibitors, transpeptidases and venom allergens. In both species, toxin-encoding transcripts and venom proteomes were dominated by hydrolytic enzymes, in particular metalloproteases. Using in vitro assays, C. quinquecirrha venom proved to be cytotoxic to fish gill cells and rat cardiomyocytes, and cytolytic to sheep erythrocytes in a concentration-dependent manner. Comparative analysis showed the venom of C. fleckeri to be substantially more potently cytotoxic to fish gill cells and cytolytic than C. quinquecirrha venom. The toxicity and potency of these venoms appeared to be associated with cytolysins of the cnidarian pore-forming toxin group present in their venoms. Potential cytolysins identified in C. quinquecirrha were homologues to CfTX-1 and CfTX-2 toxins from C. fleckeri and CqTX-A toxin from Chironex yamaguchii representing the first pore-forming toxins identified in Chrysaora venoms. Further biological assays targeted the protease and phospholipases A2 (PLA2) activities in C. quinquecirrha venom based on the identification of these enzymes in proteome and transcriptome datasets and their potential association with some of the toxic effects of C. quinquecirrha venom. However, no protease activity was observed using in gel zymography and the PLA2 activity was low, as determined using a colourimetric assay, suggesting that further experiments are needed to properly address the function of these enzymes and other toxin enzymes that may participate in the major toxic effects of C. quinquecirrha venom. This thesis reports for the first time a comprehensive study of the molecular diversity of toxins and the potential range of bioactivities that underlie the toxic effects of venoms from two Chrysaora species. The identification, description and sequence data of more than 200 potential toxins presented herein adds substantially to the field of jellyfish toxinology by making a strong contribution to the growing number of toxins discovered in cnidarian venoms. Moreover, this thesis reports a comparative analysis of the in vitro toxic effects of C. quinquecirrha and C. fleckeri venoms, which uncovered toxins that may explain variations in the toxicity and potency of venoms from different jellyfish Classes. Accordingly, this work also provides an enhanced understanding of jellyfish venom that could inform treatments and guidelines for human envenomation procedures. Finally, this study provides a catalogue of putative bioactive proteins that can be used for future comparative studies, toxin evolution analysis and drug discovery.
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    Urodacus yaschenkoi, Australian scorpion: phylogeny, venom characterization and pharmacology
    Luna Ramirez, Karen Sofia ( 2014)
    Australia has over 40 species of scorpions organised in four families: Buthidae, Bothriuridae, Urodacidae and Liochelidae (old name Ischnuridae). Unlike their overseas counterparts, Australian scorpions have received little attention and there is a lack of knowledge about their venoms and phylogeny. Urodacus yaschenkoi belongs to the family Urodacidae of scorpions, the most widely distributed in Australia. In a multidisciplinary approach, the aims of this study were to: (i) verify if the Urodacus yaschenkoi scorpion is in fact one species via a phylogenetic study with molecular markers; (ii) fully understand and characterise the venom (using proteomic and genomic approaches); and (iii) determine if there are specific toxins in the venom of Urodacus yaschenkoi that may be useful as pharmacological tools or as drug leads. The phylogenetic review of this species was conducted with 3 loci (2 mitochondrial and 1 nuclear genes) and suggested that the U. yaschenkoi population is in expansion and has given rise to at least two different clades where the Western Australian clade shows strong molecular divergence and some morphological difference. These results indicate that samples from WA need to be treated as a distinct group when performing toxinological characterisation. This study represents the first phylogenetic review of this species since Koch (1977) and the first one made with genetic analysis and not only with morphological characters. The venom characterisation showed that U. yaschenkoi venom is as complex as other scorpion venoms; several bioactive molecules were identified and assayed. The venom profile rendered 74 fractions where approximately 250 different molecular masses were identified with molecular weights varying from 291 to 16,290 Da. The most abundant peptides were those from 1 and 4-5 KDa representing antimicrobial peptides and putative potassium channel toxins, respectively. Also, the construction of a cDNA library from a replete venom gland was performed. The average titre of the cDNA library obtained was 1 X 105 cfu/ml with 70% recombinant clones, but only 172 expressed sequence tags were analysed (based on a size >400 bp). These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins could be assorted in several groups such as precursors similar to gene products implicated in common cellular processes, others representing putative neurotoxins and antimicrobial peptides. Further, characterisation of four antimicrobial peptides named UyCT peptides – was carried out. The activity of these peptides against Gram-positive, Gram-negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release of the fluorophore calcein from liposomes and isothermal titration calorimetry; their secondary structure was determined by circular dichroism. Three different lipid systems were used to mimic red blood cells, E. coli and S. aureus membranes. UyCT peptides exhibited broad-spectrum antimicrobial activity with low Minimal Inhibitory Concentrations for S. aureus and multi-drug resistant Gram-negative strains. Peptide combinations showed synergy enhancing their potency, but not haemolytic activity. These results suggest that UyCT peptides may be candidates for peptidomimetics to maximise their antibacterial activity. Additionally, the first K+ channel toxin, called urotoxin, from an Australian scorpion was identified and studied. Urotoxin was tested against eight different potassium channels and proved to be a potent blocker of human Kv1.2 channels with an IC50 of 160.5 pM. Its affinity for hKv1.1 channels is in the order of 253.5 nM, while for hKv1.3 is 90.9 nM and KCa3.1 is 69.9 nM. Urotoxin has no effect on Kv1.4, Kv1.5, hERG1 and ELK2. By constructing the whole transcriptome by means of Next Generation Sequencing, six new members – isoforms – similar to urotoxin were also identified. The computer modelling of urotoxin’s 3D structure suggested the presence of the characteristic α/β-scaffold of other scorpion toxins, very likely forming a disulfide pairing similar to maurotoxin. Using molecular dynamics, a model for the binding of this peptide to human Kv1.2 and hKv1.1 channel is included. Urotoxin may still be considered a selective hKv1.2 blocker as a minimum of 100-fold difference in IC50 makes a toxin selective for a given channel. Urotoxin may be considered a leading pharmacological tool to study K+ channel subtypes.
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    Cardiovascular, coagulation and haematological effects of brown snake and tiger snake venoms
    TIBBALLS, JAMES ( 1998)
    Transient hypotension has been observed in human victims of Australian elapid snake bite. Its aetiology and mechanism are unknown. This thesis concerns the study of the cardiovascular, coagulation and haematological effects of Brown snake (Pseudonaja spp) and Tiger snake (Notechis scutatus) venom and procoagulants derived from both. Whole venom from Pseudonaja textilis, Pseudonaja affinis and Pseudonaja nuchalis induced severe depression of cardiovascular function after intravenous and subcutaneous injection in anaesthetised mechanically ventilated dogs. The hypotension occurred within several minutes of intravenous injection and within fifteen minutes, sometimes sooner after subcutaneous injection, and persisted for approximately 30 minutes. It was accompanied by diminution in cardiac output and stroke volume and by increases in heart rate, pulmonary artery pressure, pulmonary artery occlusion pressure, central venous pressure and by increases in peripheral and pulmonary vascular resistances. Electrocardiographic signs of myocardial ischaemia occurred during hypotension. These observations are consistent with acute biventricular cardiac failure. Coagulation and haematological studies during cardiovascular depression revealed evidence of disseminated intravascular coagulation which consisted of prolongation of prothrombin and activated partial thromboplastin times and depletion of serum fibrinogen. Fibrinolysis was not detected up to two hours after envenomation. Thrombocytopenia and leucopenia were observed. Limited post-mortem examinations revealed fluidic blood in the major thoracic and abdominal vessels. Small haemorrhages were observed within or on the surfaces of the lungs, kidneys, liver and spleen. However, the magnitude and extent of the haemorrhages were not considered to be the cause of hypotension. Macroscopic thrombi were observed in the heart chambers including the left ventricle. The cardiovascular and haematological effects of the whole venoms were duplicated by intravenous infusion of procoagulants (prothrombin activators) purified from Pseudonaja textilis venom and from Notechis scutatus venom. The effect of heparin on the activities of Pseudonaja textilis prothrombin activator and to a lesser extent on Notechis scutatus prothrombin activator was also studied. Prior administration of heparin prevented the systemic and pulmonary haemodynamic effects of both prothrombin activators. These observations support the concept that cardiovascular depression is a consequence of coagulopathy, but do not exclude other causes including direct depression of cardiac function by the venom or by vasoactive substances released by the venom. Heparin therapy was ineffective in treatment of established Pseudonaja textilis envenomation. The cardiovascular and haematological effects of whole venom were neutralized by Brown snake antivenom but in doses well in excess of those currently recommended for clinical treatment of human snake bite victims. Echocardiographic studies revealed formation of multiple thrombi within the cardiac chambers during intravenous administration of Notechis scutatus prothrombin activator. This was associated with pulmonary hypertension, right heart dilatation, impeded filling of the left heart and by systemic hypotension and low cardiac output. Histological sections of lung revealed thrombi within the pulmonary circulation. Although the coagulation process is known to release vasoactive substances from platelets which may cause pulmonary hypertension and systemic hypotension, no evidence of involvement of platelet activating factor, thromboxane A2 or other eicosanoids, serotonin or histamine could be demonstrated. Pulmonary hypertension was not responsive to inhaled nitric oxide. It was concluded that systemic hypotension was a consequence of thrombo-embolic obstruction of the pulmonary circulation and may explain transient hypotension sometimes observed in humans after Pseudonaja or after Notechis envenomation. This phenomenon may be exacerbated by depression of myocardial contractility as a consequence of coagulation in coronary vessels with subsequent ischaemia.
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    Studies in animal venoms: the venoms of the Stone fish (Synanceja trachynis), the Red-back spider (Latrodectus hasseltii) and the funnel-web spider (Atrax robustus), with particular reference to the production of antivenenes
    Wiener, Saul ( 1959)
    Although severe and occasionally fatal results have followed the bite of spiders and the sting of fish in Australia, relatively few investigations have been carried out in the past on the venom the species of animals responsible for them. Antivenens have been produce against the venom of various species of spiders belonging to the genus Latrodectus in other parts of the world, but no attempt has been made in the past to produce an antivenene against the venom of the red-back spider which is the Australian representative of this genus. With the venom of the Sydney funnel-web spider, previous investigators have not been able to demonstrate any toxic effects in laboratory animals. Only a few studies have been carried out with the venom of the stone-fish but no attempt to produce an antivenene has been made. Treatment of envenomation following an effective bite or sting by these animals has therefore remained empirical and in the absence of a specific antivenene was not very successful. The present investigation was undertaken with the view to producing a specific antivenene against the venom of the red-back spider (Latrodectus hasseltii), the venom of the funnel-web spider (Atrax robustus) and the venom of the stone-fish (Synanceja trachynis) respectively. The studies presented in this thesis were therefore primarily concerned with the accomplishment of a practical task for which no efforts were spared. (From Introduction)