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Title: Mechanisms of tetrodotoxin production and resistance in the poisonous rough-skinned newt (Taricha granulosa
Creator: Vaelli, Patric M.
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Rough-skinned newts (Taricha granulosa) are poisonous salamanders that possess high concentrations of tetrodotoxin (TTX), a potent neurotoxin that blocks voltage-gated sodium channel (Nav) conductance in neurons and muscle cells. TTX is present in all species of the genus Taricha, but some populations of T. granulosa (hereafter “newts”) possess extreme quantities not seen in any other TTX-bearing species, including puffer fishes, blue-ringed octopuses, and many diverse marine invertebrates....
Show moreRough-skinned newts (Taricha granulosa) are poisonous salamanders that possess high concentrations of tetrodotoxin (TTX), a potent neurotoxin that blocks voltage-gated sodium channel (Nav) conductance in neurons and muscle cells. TTX is present in all species of the genus Taricha, but some populations of T. granulosa (hereafter “newts”) possess extreme quantities not seen in any other TTX-bearing species, including puffer fishes, blue-ringed octopuses, and many diverse marine invertebrates. Geographic variation in TTX toxicity across different newt populations is thought to be driven by ecological interactions with predators. Despite the central role of TTX in the physiology and evolution of newts, the mechanisms of TTX production and neurophysiological resistance are unknown. Because of the polyphyletic distribution of TTX toxicity among animals, we explored the hypothesis that TTX is produced by symbiotic skin bacteria in newts. We conducted 16S rRNA gene-based sequencing surveys to characterize skin- associated bacterial communities of newts from toxic and non-toxic populations. From here, we employed ecologically-guided cultivation strategies to target skin-associated symbionts and produce pure cultures. We screened cultures for TTX production using a customized HILIC-MS/MS method and confirmed TTX production in multiple isolated bacterial strains. Furthermore, we investigated the molecular adaptations underlying apparent TTX resistance in the Navs of newts. We cloned and sequenced the TTX binding site, the S5-S6 pore loop regions, of all six Nav genes present in this species and compared sequences from toxic and non-toxic populations, as well as from other vertebrates. As a result, we identified several mutations present in the S5-S6 pore loops of all six genes, indicating a remarkable parallel evolution of TTX resistance across the Nav gene family. To determine whether these mutations impact TTX resistance, we used site-directed mutagenesis to insert three newt mutations identified in neural subtype Nav1.6 into the TTX-sensitive mouse ortholog and examined their effects on TTX binding by heterologous expression and electrophysiological recording in Xenopus laevis oocytes. We found that each individual mutation increased TTX resistance to varying degrees, but the triple mutant was extremely resistant to TTX concentrations exceeding 100 μM. Taken together, our results indicate that TTX is derived from the skin microbiome in the extremely toxic rough-skinned newt and that multiple adaptations in newt Navs were required for the nervous system to adapt to TTX toxicity. Overall, this research contributes to a growing understanding that symbiotic microbes can affect the physiology of animal hosts and their nervous systems, and that evolution by natural selection may target genetic variation across both host and symbiont genomes, collectively termed the ‘hologenome’.
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Title: Mechanisms of southern grasshopper mouse (Onychomys torridus) muscle resistance to the paralytic and lethal toxins in Arizona bark scorpion (Centruroides sculpturatus) venom
Creator: Parigi, Abhijna A.
Date: 2018
Collection: Electronic Theses & Dissertations
Description: Voltage-gated sodium ion channels (Nav¬¬¬¬¬¬) are transmembrane proteins responsible for initiating electrical signals in excitable cells. Because Nav channels play a crucial role in neuromuscular coordination, they are targeted by a diverse array of neurotoxins produced across the animal kingdom. Arizona bark scorpions (Centruroides sculpturatus) produce toxins that disrupt Nav channel function, causing pain, muscle paralysis and respiratory failure. Southern grasshopper mice (Onychomys...
Show moreVoltage-gated sodium ion channels (Nav¬¬¬¬¬¬) are transmembrane proteins responsible for initiating electrical signals in excitable cells. Because Nav channels play a crucial role in neuromuscular coordination, they are targeted by a diverse array of neurotoxins produced across the animal kingdom. Arizona bark scorpions (Centruroides sculpturatus) produce toxins that disrupt Nav channel function, causing pain, muscle paralysis and respiratory failure. Southern grasshopper mice (Onychomys torridus) hunt bark scorpions. In response to selection by scorpion venom, grasshopper mice have evolved physiological resistance to toxins that cause pain and death. Although previous work identified modifications in one grasshopper mouse Nav channel (Nav1.8) that provide resistance to venom pain, mechanisms underlying resistance to muscle paralysis remain unknown. In skeletal muscle, Nav1.4 channels regulate muscle contraction. Previous studies showed that toxins in C. elegans and C. vittatus venoms disrupt Nav1.4 gating mechanisms. Thus, I tested the hypothesis that C. sculpturatus venom contains toxins that target Nav1.4, and that grasshopper mice are resistant to the effects of these toxins via molecular changes to their Nav1.4. Using molecular and electrophysiological analyses, I compared the structural and functional properties of grasshopper mice Nav1.4 channels to those of house mice and rats (rodents that are both sensitive to scorpion venom) and found that grasshopper mice Nav1.4 has evolved reduced sensitivity to C. sculpturatus venom. Further, I identified amino acid changes in the grasshopper mice Nav1.4 protein that contribute to reduced toxin sensitivity. Finally, I show that the beta subunits (accessory proteins that modulate Nav1.4 channel gating kinetics) of grasshopper mice do not have species-specific effects on channel function in the presence or absence of venom. My results demonstrate that highly conserved proteins can be evolutionarily modified with minimal effects to their baseline functional properties.
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Title: Studying the effects of sampling on the efficiency and accuracy of k-mer indexes
Creator: Almutairy, Meznah
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Searching for local alignments is a critical step in many bioinformatics applications and pipelines. This search process is often sped up by finding shared exact matches of a minimum length. Depending on the application, the shared exact matches are extended to maximal exact matches, and these are often extended further to local alignments by allowing mismatches and/or gaps. In this dissertation, we focus on searching for all maximal exact matches (MEMs) and all highly similar local...
Show more"Searching for local alignments is a critical step in many bioinformatics applications and pipelines. This search process is often sped up by finding shared exact matches of a minimum length. Depending on the application, the shared exact matches are extended to maximal exact matches, and these are often extended further to local alignments by allowing mismatches and/or gaps. In this dissertation, we focus on searching for all maximal exact matches (MEMs) and all highly similar local alignments (HSLAs) between a query sequence and a database of sequences. We focus on finding MEMs and HSLAs over nucleotide sequences. One of the most common ways to search for all MEMs and HSLAs is to use a k-mer index such as BLAST. A major problem with k-mer indexes is the space required to store the lists of all occurrences of all k-mers in the database. One method for reducing the space needed, and also query time, is sampling where only some k-mer occurrences are stored. We classify sampling strategies used to create k-mer indexes in two ways: how they choose k-mers and how many k-mers they choose. The k-mers can be chosen in two ways: fixed sampling and minimizer sampling. A sampling method might select enough k-mers such that the k-mer index reaches full accuracy. We refer to this sampling as hard sampling. Alternatively, a sampling method might select fewer k-mers to reduce the index size even further but the index does not guarantee full accuracy. We refer to this sampling as soft sampling. In the current literature, no systematic study has been done to compare the different sampling methods and their relative benefits/weakness. It is well known that fixed sampling will produce a smaller index, typically by roughly a factor of two, whereas it is generally assumed that minimizer sampling will produce faster query times since query k-mers can also be sampled. However, no direct comparison of fixed and minimizer sampling has been performed to verify these assumptions. Also, most previous work uses hard sampling, in which all similar sequences are guaranteed to be found. In contrast, we study soft sampling, which further reduces the k-mer index at a cost of decreasing query accuracy. We systematically compare fixed and minimizer sampling to find all MEMs between large genomes such as the human genome and the mouse genome. We also study soft sampling to find all HSLAs using the NCBI BLAST tool with the human genome and human ESTs. We use BLAST, since it is the most widely used tool to search for HSLAs. We compared the sampling methods with respect to index size, query time, and query accuracy. We reach the following conclusions. First, using larger k-mers reduces query time for both fixed sampling and minimizer sampling at a cost of requiring more space. If we use the same k-mer size for both methods, fixed sampling requires typically half as much space whereas minimizer sampling processes queries slightly faster. If we are allowed to use any k-mer size for each method, then we can choose a k-mer size such that fixed sampling both uses less space and processes queries faster than minimizer sampling. When identifying HSLAs, we find that soft sampling significantly reduces both index size and query time with relatively small losses in query accuracy. The results demonstrate that soft sampling is a simple but effective strategy for performing efficient searches for HSLAs. We also provide a new model for sampling with BLAST that predicts empirical retention rates with reasonable accuracy."--Pages ii-iii.
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Title: Transmissibility and localization of tetrodotoxin in the rough-skinned newt, Taricha granulosa
Creator: Wegener, Sarah (Graduate of Michigan State University)
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "Tetrodotoxin (TTX) is a powerful neurotoxin that prevents the propagation of action potentials, leading to paralysis and sometimes death in nearly all animals. However, a diverse group of marine and freshwater animals possess TTX, which they use for offense, defense, and communication. One of most studied TTX-mediated interactions is the predator-prey arms race between the rough-skinned newt (Taricha granulosa) and common garter snake (Thamnophis sirtalis). Variation in toxicity among...
Show more"Tetrodotoxin (TTX) is a powerful neurotoxin that prevents the propagation of action potentials, leading to paralysis and sometimes death in nearly all animals. However, a diverse group of marine and freshwater animals possess TTX, which they use for offense, defense, and communication. One of most studied TTX-mediated interactions is the predator-prey arms race between the rough-skinned newt (Taricha granulosa) and common garter snake (Thamnophis sirtalis). Variation in toxicity among populations of newts matched by TTX-resistance in predatory snakes has captured the focus of much research centered on the hypothesis that the arms race is the sole driver of variation. Nevertheless, recent studies suggest a more complex dynamic. Explanations of the dramatic variation in TTX among different populations of newts can only be constructed once fundamental questions about the origin, function, and transmission of TTX in newts have been more thoroughly explored. In this study, I took two approaches to address the origin, function, and transmission of TTX: 1) a cohabitation experiment in which I paired toxic and non-toxic newts to test whether toxicity can be acquired through contact, and 2) an experiment to determine the distribution and concentration of TTX in different tissues. The cohabitation experiment revealed no detectable change in the toxicity of non-toxic male newts, suggesting that a physical or physiological impediment prevents non-toxic newts from becoming toxic. The tissue toxicity experiment demonstrated that TTX is present throughout the body in structurally and functionally diverse tissues, which has many implications for the involvement of TTX in communication and reproduction in addition to defense."--Page ii.
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