G-NNACI, a Naja naja atra chymotrypsin inhibitor consists of 57 amino acid residues cross-linked by three disulfide bridges and belongs to the Kunitz/BPTI superfamily, has been successfully cloned and expressed in our laboratory. Since snake venom non-neurotoxic Kunitz/BPTI inhibitors are most conserved in the core and in the N-terminal surface area, Ala-screening mutagenesis, deletion and Domain swapping on the N-terminus were carried out in this study to assess the role of N-terminus in G-NNACI. G-NNACI mutants with single amino acid substitution and deleted mutants were prepared. The secondary structure of all mutated proteins did not significantly alter as evidenced by CD spectra. Although all mutants are found to be functionally active as an inhibitor, their inhibitory potency against chymotrypsin differed. In contrast to G-NNACI and other mutants, R1A¡BP2A and ¡µN3 mutants had a propensity to alter their disulfide linkages under basic conditions. The results of thermal and urea denaturation suggested that amino acid substitution and deletion at the N-terminus lead to a change in the structural stability of G-NNACI. Consequently, the inhibitory potency of G-NNACI mutants along with time was affected. B chain of

PAHs are hydrophobic organic compounds, which have received considerable attention because of their high bioaccumulation, toxicity, carcinogenicity, and mutagenicity. Because of their hydrophobic characteristics, PAHs intent to adsorb on particles, most of PAHs contained in wastewaters are treated biologically, especially in activated sludge systems. But due to the low construction cost and high efficiency, macrophyte-based treatment systems received increasing attention lately. In this work, batch experiments were conducted to study the adsorption behaviors between the aquatic plant (Najas graminea Del.) and PAHs aqueous solutions, including acenaphthene (Acp), fluorene (Flu), phenanthrene (Phe) and pyrene (Pyr). The interactions between different PAHs and if the plant conditions affected the adsorption were also discussed. The kinetic studies showed that the more hydrophobic the PAHs are, the faster the equilibriums achieve, The kinetic constants were found to correlate with Kow. Linear adsorption isotherms were observed in all the adsorption experiments. The adsorption equilibrium constants (K) increase with the Kow indicates that the hydrophobicity of the PAHs dominates the adsorption behaviors. The existence of small mount of Pyr could lower the adsorption of Phe on the plant, while Flu couldn¡¦t. Because the hydrophobicity of Flu is too weak to compete with Phe, so the competition was not observed. The plant after dry process had even better performances in both kinetic and adsorption experiments, which provide more flexibility when put this technology into practice.

The extant medically and socially important cobras have been the subject to several comparative taxonomic studies since the 1940s, but still lack an inclusive and thorough phylogenetic tree. With recent major advancements in phylogenetic analysis, it is now common to use multiple independent loci for studying the phylogenetic relationships within groups. For the first time, 27 from the 29 identified Naja species, alongside 5 putative new or elevated species had 4426 base pairs across 1701 sequences of mitochondrial and nuclear DNA sequence data analysed. The results continue to support the monophyletic core cobra clade encompassing the genera Walterinnesia, Aspidelaps, Hemachatus, Pseudohaje and Naja (1.0 Bayesian posterior probability (BPP)), in addition to the grouping of four monophyletic subgenera within Naja. The group of African spitting cobras, Afronaja, is positioned as the sister group to the rest of the genus. Moderate support (0.8 BPP) is found for the grouping of the Asian cobras, Naja, with the African non-spitting cobras, Ureaus. The closest relative to the genus Naja is Pseudohaje goldii, a genus and species never before included in phylogenetic analysis, followed by the sister taxa Hemachatus haemachatus. The king cobra continues to be positioned outside the core cobra group, sister to Hemibungarus calligaster. The results support the hypothesis of three independent origins of spitting, once in the monotypic Hemachatus haemachatus, once within the subgenus Afronaja, and the final origin within the Asian cobras, subgenus Naja. The relationships found were broadly consistent with previous studies, with the additional inclusion of more species creating the most comprehensive cobra phylogeny to date. Further molecular analysis, specifically species delimitation, must be undertaken to ascertain the position of the 5 putative new species included in this study.

Snake envenomations are often medical emergencies and occur regularly in dogs. Snake venom contains hundreds of enzymes, proteins and peptides that assist in paralysing, killing and digesting prey, or as a defence against predators. Multiple snake venom components affect haemostasis in the victim. Inadvertent activation of coagulation may also result from expression of large amounts of tissue factor (TF) from injured tissues at the envenomation site, especially with potent cytotoxic venoms. The purpose of this study was to investigate the haemostatic functions in dogs envenomed by two South African snakes (Bitis arietans and Naja annulifera) using thromboelastography and traditional plasma-based coagulation assays. This prospective study included 18 client-owned dogs, of which nine dogs were envenomed by African puffadder and nine by snouted cobra. Blood was collected at presentation and at 24 hours post-envenomation. Complete blood count, thromboelastography (TEG), prothrombin time (PT), activated partial thromboplastin time (aPTT), antithrombin (AT) activity and C-reactive protein (CRP) and fibrinogen (Fib) concentrations were measured. Ten healthy client-owned dogs served as controls. These dogs were presented for routine ovariohysterectomy, castration or blood donation. Haematologic and haemostatic assay results at presentation were compared between groups using ANCOVA (analysis of covariance), and results over time between the puffadder and cobra groups were compared using linear mixed models at 5% significance. At presentation, the mean TEG R-time was significantly prolonged in the puffadder group when compared to the cobra and control groups (P=0.01 and 0.05, respectively). Visual appraisal of the thromboelastograms at presentation revealed that 5/9 (56%) of puffadder-envenomed dogs had hypocoagulable thromboelastograms as was demonstrated by prolonged R-time and decreased Angle (á), maximal amplitude (MA) and global clot strength (G). Despite this observation of hypocoagulability, none of the other TEG parameters (á, MA or G) were significantly decreased when compared to the cobra and control groups. This finding of hypocoagulability was surprising, because puffadder venom is cytotoxic, often inducing severe tissue necrosis and potentially leading to limb loss and disability in people. It therefore seems that certain components in puffadder venom affect the thromboelastograph by either interfering with or consuming coagulation factors, resulting in a hypocoagulable tracing. It is also possible that this is a dose-dependent effect, with only dogs with a significant amount of envenoming demonstrating this phenomenon. This effect appears to be transient, as 6/8 dogs (one fatality) envenomed by puffadders reverted to a severely hypercoagulable state at 24 hours post-envenomation. One dog was still hypocoagulable and one dog that was hypocoagulable became normocoagulable but still had a prolonged R-time. In the cobra-envenomed group hypercoagulable thromboelastograms were observed in 5/9 (56%) dogs at presentation as was demonstrated by increased MA and G. At 24 hours post-envenomation all cobra-envenomed dogs demonstrated hypercoagulable thromboelastograms. This hypercoagulability at presentation and 24 hours post-envenoming was not statistically significant between groups. This hypercoagulable state was likely due to tissue factor-activated coagulation promoted by inflammation at the envenomation site. At presentation, marked thrombocytopenia was evident in the puffadder-envenomed dogs when compared to the cobras and controls (P=0.04 and 0.001, respectively). Thrombocytopenia following puffadder envenomation has been reported in dogs and baboons. Components have been identified in puffadder venom that interfere with platelet function either by inhibiting or promoting aggregation. At 24 hours post-envenomation mean platelet count (Plt) was mildly increased compared to its value at presentation in the puffadder-envenomed dogs. There were Plt abnormalities in the cobra-envenomed dogs at presentation or at 24 hours post-envenomation. Marked leucocytosis was detected in the puffadder-envenomed dogs at presentation when compared to the cobras and controls (P=0.003 and 0.001, respectively) and was more severe at 24 hours post-envenomation when compared to the cobra group (P=0.01). Leucocytosis has been reported in different types of snake envenoming including puffadder-envenomed dogs. C-reactive protein (CRP) concentration at presentation was below the lowest detection limit for most dogs (14/18) in this study. At 24 hours post-envenoming all but two dogs (one each in the puffadder and cobra groups) had severely elevated CRP. This increase in CRP was statistically significant in both puffadder and cobra-envenomed dogs when compared to its concentration at presentation (P=0.04 and 0.001, respectively). Fibrinogen (Fib) concentration was not elevated in any envenomed dogs at presentation, but increased 24 hours post-envenoming. Although this increase was not statistically significant, an increase would suggest activation of the inflammatory response, as both Fib and CRP are positive acute phase proteins. Elevated CRP, neutrophilic leucocytosis and increases in cytokine IL-6 and IL-8 has been documented in four human patients bitten by Bothrops and Crotalus snakes (two each) in Brazil. CRP levels were low immediately post-envenoming, peaked at two days post-envenoming and dropped to within normal limits four days post-envenoming. These findings demonstrated a typical acute-phase response and it is likely that a similar acute phase response occurred after puffadder and cobra envenoming in our study. Mean antithrombin (AT) activity was mildly decreased in both the puffadder- and cobra-envenomed dogs compared to the controls (P=0.002 and 0.004, respectively), suggesting that the activation of haemostasis led to some AT consumption. Mean PT and mean aPTT were prolonged in the cobra-envenomed dogs compared to the controls (P=0.03 for both), but were within their reference intervals (RI). At 24 hours post envenomation mean haematocrit (Ht) was significantly decreased in the puffadder group compared to the cobra group (P=0.01), but was within RI. The Ht was significantly lower at 24 hours post envenomation compared to presentation values in both these groups (P<0.001 and 0.02, respectively). At presentation, marked thrombocytopenia, leucocytosis and prolonged clot initiation were common features in puffadder-envenomed dogs and were likely venom-induced. Snouted cobra-envenomed dogs were normo- to hypercoagulable at presentation. Both puffadder- and cobra-envenomed dogs equally showed hypercoagulability at 24 hours post-envenomation and this was more pronounced compared to their coagulability at presentation. TEG proved to be a useful tool to detect abnormal haemostasis in all envenomed dogs in this study. TEG also provided additional insights into certain aspects of snake envenomation (such as hypercoagulability) that has not been reported on previously and cannot be assessed using traditional coagulation assays. TEG may serve as a differentiating tool in early envenomation between these two types of snake envenoming in scenarios where the identity of the snake species involved is not known. / Dissertation (MMedVet)--University of Pretoria, 2012. / Companion Animal Clinical Studies / unrestricted

Im Fokus dieser Dissertation steht die Zerstörung des Lebensraumes von Schlangen durch anthropogene Einflüsse im Zusammenhang mit dem Auftreten von Schlangenbissen bei Menschen. Zum Schutz der Schlangen und dem Problem der Schlangenbisse wurden vier wesentliche Anforderungen erarbeitet. Mit Hilfe eines Artenverteilungsmodells wurden die ökologischen Nischen von 29 Giftschlangenarten aus Bangladesch und von zehn Kobraarten (Naja) aus Asien ermittelt. Auf lokaler Ebene wurde festgestellt, dass Überschwemmungen, Waldtypen, Ökosysteme und klimatische Parameter das Verbreitungsgebiet der Schlangenarten in Bangladesch beeinflussen. Auf regionaler Ebene sind anthropogene Faktoren wie die Zerstörung von Schlangenlebensräumen, Handel, Ausbeutung und Tötung von Schlangen wahrscheinlich die Hauptursachen für den Rückgang der Populationen der asiatischen Naja. Auf beiden Ebenen führt der Klimawandel zu einer Verkleinerung der ökologischen Nischen von Schlangenarten in Bangladesch und Asien. In Bangladesch könnten die untersuchten Arten innerhalb der nächsten 50 Jahre mehr als 90 % ihren derzeitigen Lebensraum verlieren (Asiatische Naja durchschnittlich 56 % (12 bis 100 %)). Wenn günstige Nischen nicht erhalten werden können, könnten viele Schlangenarten in einigen Ländern Asiens, darunter Bangladesch, in wenigen Jahrzehnten aussterben. Darüber hinaus wurde in der vorliegenden Studie geschätzt, dass die Zahl der jährlichen Schlangenbisse in Bangladesch mehr als doppelt so hoch sein könnte wie bisher angenommen. Da der Klimawandel zu einer geografischen Verschiebung günstiger Nischen führen kann, kann sich diese Veränderung auch auf die Häufigkeit von Schlangenbissen auswirken. Bei der Analyse von Giftvariationen und der Wirksamkeit von Antivenomen wurde festgestellt, dass isolierte Populationen deutliche intraspezifischere Giftvariationen zeigen. / Snakes are threatened by habitat degradation and anthropogenic impacts, while accidental encounters between humans and snakes may result in snakebite incidents. Thus, the conservation of snakes snakebite problem, a global public health emergency, are interconnected. I found four primary needs for a combined solution to satisfy both above causes. A species distribution model was employed to identify the ecological niches of 29 venomous snake species from Bangladesh and of ten cobra species (Naja) from Asia. At the local scale, flood events, forest types, ecosystems, and climatic parameters were found to shape the range of snake species in Bangladesh. At the regional scale, anthropogenic factors, like snake habitat destruction, trade, exploitation, and snake killings are likely to be the main causes for the decrease in populations of Asian Naja. At both levels, climate change causes shrinking and fragmenting the ecological niches of snake species in Bangladesh and Asia. In Bangladesh, the studied species may lose more than 90% of their current climatic niche within the next 50 years. In the same timeframe, Asiatic Naja may lose an average of 56% (12- 100%) of the area of suitable climatic niches. If favourable niches cannot be preserved, many snake species might go extinct from several countries in Asia in a few decades, including Bangladesh. In addition, the present study estimated that there might be more than double the amount of annual snakebite incidents in Bangladesh than previously estimated. As climate change may cause geographical shifting of favourable niches, this change may also have consequences for the frequency of snakebites, so snakebite should be considered a climate driven dynamic public health problem. While analysing venom variations and the efficacy of antivenom, I found that venom should be sampled from geographically or ecologically isolated populations of each venomous snake, because isolated populations have shown distinct intraspecific venom variations.