Synthesis of a PbTx-2 photoaffinity and fluorescent probe and an alternative synthetic route to photoaffinity probes

A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom. K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence. Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein. Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached. A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds. The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described. An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.

Diving Behaviour of Red-Eared Sliders (Trachemys Scripta Elegans) Under the Risk of Predation

Predation risk influences a variety of behavioral decisions of many organisms and results in animals having to trade-offs safety with other behaviors. The effects of predation, however, have been largely ignored in the study of vertebrates that forage underwater (divers). I tested the predictions of an on optimal diving model that incorporates the risk of predation, using red eared slider turtles (Trachemys scripta elegans). Specifically, I tested the hypothesis that divers will increase their surface time when instantaneous risk decreases with time at the surface. By using a model aerial predator and exposing turtles to both risk and no risk treatments, I tested how turtles perceive risk at the surface and whether they increase or decrease their surface time depending on how they assess risk. The model's predictions for situations in which risk at the surface is decreasing with time spent there-likely to be the case for aerial predation-were supported by the results. I found that surface time and time spent submerged per dive were significantly greater when turtles were at risk and that turtles also spent more time resting at the bottom when exposed to this treatment. Interestingly, turtles under risk engaged in vigilance behaviors while on the bottom just prior to surfacing. This behavior could have implications for model predictions and future experiments are needed to test whether subsurface vigilance may alter diving decisions made under risk.