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Philosophiae Doctor - PhD / Tetrodotoxin (TTX) is a nonpeptidic neurotoxin with a high rate of food poisoning mortality (60%) that has been associated with the consumption of diets from puffer fish and mud snails harbouring TTX-producing bacteria. As this neurotoxin has no known antidote and could not be mitigated by cooking, the only way for safety appears to be the detection of TTX-contaminated fishes at the points of harvest and control. The overall aim of this study was to develop amperometric and impedimetric sensors for TTX based on ionophores and aptamer immobilised on the modified conducting electroactive polyaniline (PANI)/electrode. The undoped polyaniline and poly(4-styrenesulfonic acid) (PSSA) doped electroactive polyanilines were prepared in perchloric acid/acetonitrile and phosphoric acid respectively by electrochemical oxidative polymerisation. Two types of electropolymerisation were applied to prepare the neutral and p-doped PANI−PSSA films composites. The dynamic electroinactivity of TTX was studied which revealed that TTX is not electrochemically active on bare Au, GC, Pt, PG, Ni, Ti and BDD (Boron dopeddiamond) electrodes in acetate buffer pH 4.8. Using ion transfer voltammetry and UV-Vis analysis, the complexation of TTX with two neutral ionophores (sodium ionophore X (NaX) and dibenzo-18-crown6 (B18C6)) was investigated. The cyclic voltammograms (CVs) recorded from ion transfer voltammetry presented no redox peak and no increasing/decreasing current was observed which indicates that no TTX ions transfer from the liquid to the organic phase. In addition, the absorption spectra of the mixture of TTX/NaX and TTX/B18C6 presented the same absorption bands recorded for NaX and B18C6 respectively. Three absorptions bands at 250.4, 278.3, and 370.6 nm for NaX and two at 222.03 and 274.10 nm for B18C6 were observed before and after mixing TTX with NaX and TTX with B18C6 separately. No chemical reaction occurred between the TTX and both ionophores, therefore, sodium ionophore X and dibenzo-18-crown-6 did not form a complex with TTX. Thus, TTX ion sensor cannot be developed based on these two neutral compounds. The electrodynamics of the PANI and PANI−PSSA films electropolymerised on the bare precious metal electrodes were also investigated through various electrochemical techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies in sodium phosphate (SPB) and acetate (OAc) buffer revealed that both neutral and p-doped films synthesized were thin (thickness L < 5 nm in acetate buffer and L < 10 nm in sodium phosphate buffer) film polymers.

Previous reports of the effects of tetrodotoxin (TTX)-induced muscular disuse have demonstrated alterations in muscle force, speed, and fatiguability that might suggest changes in the quality of contractile proteins. These studies were extended to the effects of TTX-induced disuse on the Ca$ sp{2+}$-activation characteristics of myofibrillar ATPase of the rat gastrocnemius. Atrophic responses after TTX treatment were as previously reported with a significant decrease in left gastrocnemius weight (g) compared to the control-pump (C) group (1.25 $ pm$ 0.04 for C vs 0.72 $ pm$ 0.04 for TTX, X $ pm$ SEM, p $ leq$ 0.01). Myofibrillar protein yield (mg$ cdot$g$ sp{-1}$ wet weight) was also depressed (92.8 $ pm$ 4.6 for C vs 70.3 $ pm$ 3.7 for TTX; p $ leq$ 0.01). Maximum ATPase of myofibrils (nmol Pi$ cdot$mg$ sp{-1} cdot$min$ sp{-1}$) was decreased (424 $ pm$ 46 for C vs 199 $ pm$ 27 for TTX, p $ leq$ 0.01). Furthermore, the Hill n which reflects the cooperative aspects of Ca$ sp{2+}$-activation of the myofibrillar ATPase was significantly depressed (1.58 $ pm$ 0.07 for C vs 1.29 $ pm$ 0.09 for TTX; p $ leq$ 0.05) after TTX treatment. The results of the present study suggest that muscle perturbations that result from TTX-induced disuse are at least partially related to changes in the myofibrillar fraction.

In this study, the synthesis of TTX by three species of TTX-producing bacteria (Vibrio alginolyticus, Microbacterium arabinogalactanolyticum and Serratia marcescens) was conducted in a 10-L fermentor under the same controlled fermentation conditions for each of a period of 60 hours. The bacterial growth curves were monitored and the TTX synthesized in the culture medium was determined by HPLC. The TTX biosynthesis was found limited at the microgram level per L of culture medium with toxicities 14.7 MU (mouse unit) and 13.0 MU per mL in the partially purified culture medium of V. alginolyticus and M. arabinogalactanolyticum respectively by mouse bioassay. In the studies on SW480 and SW620 colorectal carcinoma cell lines, the expression, distribution, invasion and proliferation of voltage-gated sodium channels (VGSCs) were investigated by MTT assay (24-48 hours) and wound healing assay (0-120 hours). The different subtypes of VGSCs were expressed by semiquantitative RT-PCR and the locations of Nav1.5 and Nav 1.7 were detected by immunofluorescence microscopy. In the MTT assay, 40μmol/L of TTX showed significant inhibitory effect on both cell lines, with maximum inhibition rate, 33% and 40%, in SW480 and SW620 respectively. In the wound-healing assay, the inhibitory rate of 80μmol/L of TTX on SW480 reached 22% after 120 hours, compared with 30% in the control group. Moreover, VGSCs were highly expressed in both SW480 and SW620, with the main subtypes of Nav1.5 and Nav1.7 located on the cell surface, which might increase the metastatic rate of the cell lines. Keywords: Tetrodotoxin (TTX), Bacterial synthesis, Anticancer, VGSCs

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The temporal nature of ectopic activity is important to elucidating the mechanisms that can lead to arrhythmogenesis. However, challenges remain in distinguishing between ectopic and non-ectopic beats. A new methodology was developed and validated to distinguish between beat types. Rapid pacing was used to induce both ectopic and non-ectopic beats. Using an electrocardiogram, the post-pacing recovery beat cycle length (RCL) and QRS were normalized to pre-paced R-R and QRS intervals and analyzed using a K-means clustering algorithm. Control hearts only produced beats with RCL ratios that increased with rapid pacing, suggestive of non-ectopic activity. Hypercalcemia and digoxin both produced significantly earlier beats with wider QRS durations, suggestive of ectopic activity. Increasing pacing further shortened RCL during digoxin + hypothermia, a mechanistic identifier of ectopic activity. When tested against a previously validated analysis, our algorithm performed well. Therefore, this electrocardiogram based algorithm distinguishes between ectopic and non-ectopic beats. In a prospective study, tetrodotoxin increased RCL ratio without changing the QRS duration of excited beats, suggesting neuronal sodium channels play an important role in ectopic beat timing. The next goal was to create a consistent model of ectopic activity. Both sympathetic and parasympathetic stimulation independently potentiate arrhythmogenesis, and we investigated the effects of independent and simultaneous stimulation on the temporal nature of arrhythmogenesis. Isoproterenol (ISO), a sympathetic agonist, transiently produced ectopic activity and increased heart rate. Acetylcholine (ACh), a parasympathetic agonist, did not significantly produce ectopic activity but did slow heart rate. ACh added after ISO also transiently produced ectopic activity, while heart rate remained slowed. Importantly, ISO following ACh persistently increased ectopic activity and heart rate. Therefore, ISO following ACh is an ideal model for creating sustained ectopic activity. Mature animals exhibited sustained arrhythmogenesis while young animals did not. When ACh was removed and then followed by ISO, ectopic activity and heart rate transiently increased, similar to ISO alone. This suggests that maintained ACh perfusion can sustain ISO sensitivity, in contrast to ISO perfusion alone. The data in this dissertation provide an insight into the mechanisms that affect the ectopic beat timing and arrhythmia propensity. / Ph. D.

Research suggests biological sex differences may affect pain perception, however the difference in analgesic tolerance between male and females not been extensively studied. Therefore, here we studied the response of male and female Sprague-Dawley rats to prolonged duration local anesthetics; including conventional amino-amide anesthetics (bupivacaine), site 1 sodium channel blockers (tetrodotoxin) and a prolonged duration liposomal formulation (Exparel). This study examined the incidence and duration of sensory and motor blockade; systemic side effects, as well as local neurotoxicity and myotoxicity in both male and female rodents in an in vivo model of sciatic nerve blockade. The data collected does not indicate a sexual dimorphism among the agents studied. / 2019-07-03T00:00:00Z

Salvia miltiorrhiza (SM) preconditioning was reported to be helpful in the early recovery of gastrointestinal motility in the intestinal congestion of rats with hepatic ischemia reperfusion. The aim of this study was to determine whether SM stimulates contraction of isolated terminal ileum of Sprague-Dawley rat ex vitro and the mechanisms which regulates that. The roots of SM were extracted by ethanol. One of the indicative marker of SM, Tanshinone IIA, was identified and quantified with high performance liquid chromatography (HPLC), and the results showed that Tanshinone IIA was 1190 £gg/ml in SM extract. The effects of contractile activity of SM extract at various cumulative dosages on the rat isolated terminal ileum were studied in organ bath. The area under curve above the baseline of contractile graphy of SM extract on isolated terminal ileum was recorded. In order to explore the contractile mechanism of SM extract on isolated terminal ileum, the individual pretreatment or use of atropine (a muscarinic receptor antagonist), tetrodotoxin (a sodium channel blocker), nifedipine (a calcium channel blocker), Ca2+ free Kreb¡¦s solution with EGTA, or trifluoperazine (a calmodulin blocker) was given and then cumulative dosages (40 £gL, 100 £gL, 180 £gL, 280 £gL) of SM extract were added. In addition, we used Fura-2 pentakis acetoxymethyl ester to detect the change of intracellular calcium concentration of intestinal epithelial cell-6 (IEC-6) induced in 1/1000-time or 1/10000-time dilution of SM extract. The result indicated SM extract significantly simulated the contraction of isolated terminal ileum in a dose-dependent manner. The individual addition or use of atropine, tetrodotoxin, nifedipine, or Ca2+ free Kreb¡¦s solution with EGTA all could not down-regulate significantly the contraction of SM extract on isolated terminal ileum. Trifluoperazine significantly down-regulated the contraction of SM extract on isolated terminal ileum. In addiation, SM extract was able to increase cytosolic calcium concentration of IEC-6 cells. In conclusion, the mechanisms of contraction of SM extract on isolated terminal ileum of rat were involved in calmodulin/Ca2+ associated contraction pathway.

Many organisms utilize toxic or noxious compounds as a means of deterring predation. Eastern newts (Notopthalmus viridescens), along with other species in the family Salamandridae, possess a potent neurotoxin called tetrodotoxin (TTX). Although TTX can serve as an effective antipredator mechanism in species of newts with high concentrations (e.g., Taricha), eastern newts have relatively low levels of toxicity in comparison to those species, and it may not serve as an effective antipredator mechanism against all threats. In this case, they may benefit rather by utilizing behavioral changes to avoid initial contact with predators. We tested for predator-avoidance behavior in newts by exposing individuals to kairomones from various predators. We recorded activity patterns of newts when they were exposed to cues from potential predators including bullfrogs (Lithobates catesbeiana), water snakes (Nerodia sipedon) and snapping turtles (Chelydra serpentina), as well as a non-predator (bullfrog tadpoles), and a control (deionized water). Newts reduced activity when exposed to snapping turtle stimuli, but did not change activity when exposed to any other chemical cues. We verified that newts interact with this predator by trapping snapping turtles found in ponds from which newts were collected. Finally, we used turtles caught during this sampling to test whether they are an actual predator of newts and whether newts shift microhabitat use when exposed to this predator. In each replicate, turtles consumed newts, and newts spatially avoided the snapping turtle, relative to a control. The results of these experiments indicate newts rely on predator-avoidance behavior to reduce the probability of being consumed by snapping turtles, but do not reduce activity in response to other potential predators that may only consume them rarely.