Search

Abdominal pain and discomfort are common symptoms of Inflammatory bowel disease (IBD) and diagnostic criteria for irritable bowel syndrome (IBS) that significantly impair quality of life. Pain in IBD is thought to be mediated by the activation of pain-sensing nerves (nociceptors) that innervate the bowel by mechanical modalities, such as the distention of visceral organs or by mediators released in response to gut inflammation. Pain management in these conditions is challenging due to the side effects associated with commonly used analgesics, and so a significant unmet clinical need exists for the development of new visceral analgesics. GPR35 is a G- protein-coupled receptor (GPCR) which preferentially signals through the activation of Gαi/o subunits. GPR35 is designated as an "orphan" GPCR due to the ambiguity of its cognate ligand. However, a few synthetic (e.g., zaprinast and cromolyn) and endogenous agonists (e.g., kynurenic acid) have been identified, which have facilitated research into its function (O'Dowdl et al., 1998; Divorty et al., 2015). In recent years, many of these agonists have been shown to be anti-nociceptive in experimental studies of pain signalling. These effects are abolished in GPR35 -/- mice, thereby providing target validation for the analgesic potential of GPR35 agonists (Ohshiro et al., 2008; Cosi et al., 2011; Alexander et al., 2015). In this thesis, I investigated the role of the GPR35 receptor in the regulation of visceral nociception. Our *in-silico* analysis of previously published transcriptomic data reveals significant co-expression of GPR35 with noxious transducer TRPA1 in the nociceptive neuronal population of colonic DRG neurons. We showed that stimulation of TRPA1 vigorously excites colonic afferents, induces afferent mechanosensitivity and releases neuropeptide Substance-P (SP) from the colonic tissues, which exerts excitatory effects on colonic afferents. Application of the GPR35 receptor agonists cromolyn or zaprinast attenuates TRPA1-induced afferent excitation, relieves mechanosensitivity, and inhibits the release of SP from colonic tissues, thereby restricting the afferent excitation and colonic contractility induced by SP. GPR35 agonists also inhibited the excitatory action of the disease-relevant mediator PGE2. Finally, the involvement of GPR35 as a molecular determinant of cromolyn or zaprinast action was confirmed by repeated experiments in GPR35-/- animal tissues. These findings suggest that GPR35 represents a high-value target for the development of visceral analgesics.

Rhinovirus (RV) is the leading cause of exacerbations of airway disease, during which the airways become hypersensitive and manifests clinically with troublesome bouts of cough. The mechanism responsible for respiratory virus induced cough in humans is unknown. This research aimed to determine whether RV directly infects and alters neural function or exerts its effect indirectly via the release of inflammatory mediators from infected airway epithelium. A human sensory neuronal model was derived from differentiation of human dental pulp stem cells towards a neuronal phenotype, termed peripheral neuronal equivalents (PNEs). PNEs were infected for 2, 6, 24, 48 and 72 hrs with major group RV-A16, or minor group RV-A1b, at a range of multiplicities of infection (MOI) (0.01 - 100). Primary bronchial epithelial cells (PBEC) from chronic cough, COPD and healthy patients, were infected for 24 hrs with RV-A16 and at MOI 1. PNEs expressed intracellular adhesion molecule 1 (ICAM-1), the entry receptor of major group RV. Viral titrations, Western Blotting (WB), immunocytochemistry (ICC) and flow cytometry confirm RV-A16 entry and replication in PNE when infected at MOI 1 for 24, 48 and 72 hours. RV-A1b infection at the same MOI and infection times, failed to infect PNE cells to detectable levels. A human antibody array indicated upregulation of several cytokines following RV-A16 infection, most notably IL-1β at 24 hours post infection (h.p.i) and IL-4 at 48 h.p.i. Viral titrations, WB and ICC confirmed RV-A16 replication in PBEC at MOI 1 at 24 h.p.i. Significant increases IL-1β were observed in chronic cough and COPD PBEC infected at MOI 1. Treatment of PNE with IL-1β for 24 hrs resulted in TRPA1 channel sensitivity in response to TRPA1 agonist, cinnamaldehyde. We report for the first time the susceptibility of an airway neuronal model to RV infection. Findings confirm RV-A16 entry and replication in PNE, as well as a pro-inflammatory cytokine release.

In idiopathic pulmonary fibrosis (IPF), human lung myofibroblasts (HLMFs) are observed in "fibroblastic foci" which are thought to represent abnormal persistence of myofibroblasts, in part because of failure of normal apoptosis. Oxidative stress is observed in many diseases including IPF. To date however, therapeutics directed specifically at "oxidative stress" have had no important clinical benefit for patients. Part of this failure is our lack of a nuanced understanding of the specific redox signalling and homeostasis abnormalities in disease. Some members of the transient receptor potential cation channels (TRP) are sensitive to redox status and may contribute to important cellular activities in response to oxidative stress. I describe the production and validation of new tools for the study of expression of redox-sensitive TRP cation channels family members, ankyrin 1 (TRPA1) and melastatin 2 (TRPM2). I describe the mRNA, protein, and functional ion current expression of these channels in cells derived from human lungs, including HLMFs. Importantly I highlight problems with the sensitivity and specificity of anti-TRPA1 antibodies widely used in the literature. Transforming factor β1(TGFβ1) is strongly implicated in driving IPF pathogenesis, and I show that TGFβ1 reduces TRPA1 expression in HLMFs. TRPA1 activation was found to play a role in HLMF cell death induced by H2O2 and other agonists, and TGFβ1 exposure induced resistance to this agonist-dependent HLMF death via the downregulation of TRPA1. This may go towards explaining the persistence of myofibroblasts in IPF and other fibrotic diseases. Finally, I study for the first time the H₂O₂ elimination dynamics in HLMFs using genetically encoded redox sensors. These novel tools have been adapted to enable the future study of specific interactions between TRP channels and other genes with cellular redox homeostasis.

Painful stimuli are detected at the peripheral terminals of specialised primary sensory neurons known as nociceptors. Nociceptors detect noxious (painful) stimuli to evoke pain sensation via the activation of Transient Receptor Potential Ankyrin 1 (TRPA1). TRPA1 activity can be modulated by G-Protein Coupled Receptors (GPCR), leading to sensitisation and hyperalgesia (enhanced pain sensation), typically seen in inflammatory pain conditions. Human Mas-related G-protein-coupled receptor X1 (MRGX1) is a promising target for pain relief, mainly because of its restricted expression in nociceptors. To date however, the role MRGX1 plays in inflammatory pain regulation in humans remains unclear as functional nociceptors from humans are not available and rodents do not harbour MRGX1-encoding genes. Here, we generated in vitro models of human nociceptors from 1) neural crest stem cells using a previously validated method and 2) human neural progenitors using magnetic-activating cell sorting (MACs) technology, both derived from human dental pulp as well as an ex-vivo model of trigeminal nerves. Immunofluorescent staining revealed MRGX1 expression ex-vivo in dental afferents, which was more intense in inflamed versus healthy dental pulps. Endogenous MRGX1 protein expression was confirmed in both in-vitro models of human nociceptors. The functionality of MRGX1 and its interaction with TRPA1, as well as the mechanisms involved was confirmed by calcium imaging. Results showed that MRGX1 indirectly activates TRPA1 via IP3- mediated store release of calcium ions and sensitises TRPA1 to agonist stimulation via PKC. MRGX1 modulates TRPA1 activity via two distinct xxi mechanisms suggesting an important nociceptive role. MRGX1 therefore is a potential pain relief target with minimal side effects due to restricted neuronal expression.

This thesis examined how and why TRPV1 function is being modulated in sensory neuropathy and explored the potential of its rescue in the urinary bladder of STZ-­‐induced diabetic rats. Diabetes induced a rapid decline in TRPV1 function and changes in neurogenically mediated electrically-­‐evoked responses together with a gradual decline in muscarinic function. Diabetic bladder was also deficient in muscarinic and TRPV1 organ bath temperature-­‐induced changes but not in those affecting spontaneous contractile activity. Exposure to a potential neuropathy causative agent, methylglyoxal was studied and its mechanism of action explored through the use of TRPA1 ligands. Methylglyoxal exposure mimicked some of the effects of diabetes on TRPV1, neurogenic electrically evoked responses and muscarinic function. Methylglyoxal effects were seen to be partly through TRPA1 receptor activation but other as yet undefined pathways were also involved. Use of TRPA1 ligands revealed an unexpected complexity of the interaction of the TRPA1 receptor with TRPV1. Finally the potential of reversing the diminished TRPV1 response was examined through the use of three known sensitising agents, bradykinin, NGF and insulin. Bradykinin was the only agent seen to reverse the TRPV1 diminished response back up to to control equivalent levels and through the use of bradykinin selective ligands, it was seen that the dual activation of BK-­‐1 and BK-­‐2 receptor was necessary to rescue the TRPV1 response. The likely mechanism of action of bradykinin was through prostaglandin production as indomethacin blocked TRPV1 rescue. In the acute stage of diabetes, TRPV1 function is downregulated and may be caused by exposure to a neuropathy-­‐causing metabolite such as methylglyoxal. The TRPV1 function still retains plasticity at this acute stage because function could be enhanced back to control levels by bradykinin receptor activation : a potential for early therapeutic intervention.

Mechanotransduction is fundamental to many sensory processes, including balance, hearing and motor co-ordination. However, for such an essential feature, the mechanism(s) that underlie it are poorly understood. The mechanotransducing stretch receptors that relay information on the tonicity and length of skeletal muscles have been well-defined, particularly at the gross anatomical level, in a wide variety of species, encompassing both vertebrates and invertebrates. To date, there exists a wealth of data describing them, anatomically, as well as good electrophysiological data from stretch receptors of some larger organisms. However, comparatively few studies have succeeded in identifying putative mechanotransducing molecules in such systems. Nonetheless, this class of sensory mechanotransducers perhaps offer the best means of identifying molecules that permit the stretch-sensitivity of such endings, revealing new information about the underlying mechanisms of stretch receptors, and mechanoreceptors more generally. However, a different approach is clearly needed; a theoretical approach, utilising mathematical modelling, offers a powerful means of pooling the current wealth of knowledge on the reported electrophysiological behaviour of muscle stretch receptors. This study, therefore, develops an extended theoretical model of a stretch receptor system in order to reproduce, in silico, the reported behaviour of both vertebrate and invertebrate stretch receptors, within the same modelling environment, thus enabling the first quantitative framework for comparing these data, and moreover, making predictions of the likely roles of specific molecular entities within a stretch receptor system. Subsequently, this study utilises a model in vivo system to test these theoretical predictions. The genetic toolbox of D. melanogaster offers a wide range of tools that are extremely suitable for identifying mechanotransducing molecules in stretch receptors. However, very little is currently known about such endings in this organism. This study, therefore, firstly characterises a putative stretch receptor organ in larval Drosophila, the dbd neuron, via a novel experimental approach. It is shown that this neuron exhibits known properties of stretch receptors, as previously observed in other, similar organs. Furthermore, these observations bear out the predictions of the mathematical model. Having defined the dbd neuron as a muscle stretch receptor, pharmacological and genetic assays in this system, combined with predictions from the mathematical model, identify a key role for the recently-discovered DmPiezo protein as an amiloride-sensitive, mechanically-gated sodium channel (MNaC) in dbd neurons, with TRPA1 also acting in this system in a supporting role. These data confirm the essential role of an MNaC in mechanosensory systems, but also supply important evidence that, whilst the electrophysiological mechanisms in stretch receptors are remarkably similar across taxa, different species likely employ various molecular mechanisms to achieve this.

Blood-seeking mosquitoes primarily rely on thermal and chemical cues as they navigatetowards hosts. Mosquitoes display specific preferences for target host temperature whileavoiding harmful ambient temperature. This behavior known as thermotaxis is in partregulated by the nociceptor transient receptor potential ankyrin 1 (TRPA1), which isexpressed in thermosensitive sensilla of mosquitoes. TRPA1 of female mosquitoes isknown to detect both noxious temperatures and chemicals; when activated by thesestimuli, TRPA1 triggers avoidance behaviors. Therefore, TRPA1 is considered apotential biochemical target for mosquito repellents and antifeedants. One aspect ofTRPA1 channels from mosquitoes and other insects that has not been fully studied is thepotential interactions between temperature and chemical agonists. In this study, Iexamined whether high ambient temperatures that activate Aedes aegypti TRPA1(AaTRPA1) influence the sensitivity of the channel and behavior of mosquitoes torepellent TRPA1 chemical agonists. First, I expressed AaTRPA1 heterologously inXenopus laevis oocytes and used whole-cell two-electrode voltage clamping to measureTRPA1 activity. I found that the electrophysiological response of AaTRPA1 to twochemical agonists (catnip oil, citronellal) was significantly reduced while the channel wasactivated by a thermal stimulus ( ~38°C). Moreover, in behavioral bioassays, I found that adult female Ae. aegypti were less repelled by catnip oil when exposed to a noxioustemperature (~50°C). Collectively, my results suggest that TRPA1-agonizing repellents,such as catnip oil, may be less efficacious during extreme heat events, which arebecoming more common as global climate change progresses.

The remarkable diversity of animal behaviors have long attracted biologists from different fields. However, the genetic architecture and molecular changes behind the diversification of these behavioral traits have remained elusive due to technical limitations, preventing deeper understanding of evolutionary processes of behaviors. My dissertation work aims to dissect the evolution, genetic architecture, and molecular basis of behavioral traits using a herbivorous drosophilid fly, Scaptomyza flava, with a particular emphasis on the evolution of its chemosensory behaviors that are specifically tuned to the specialized chemicals produced by mustard plants in the order Brassicales.In Chapter 1, I briefly summarized the historical context and recent trends in the study of behavioral evolution in animals. I argue that the evolution of chemosensory behavior in insects provides an excellent model for uncovering the genetic architecture and molecular basis of behavioral change across lineages. I further introduced S. flava as a unique system to address such questions that allows us to bridge genomic and functional approaches by leveraging the power of model organisms in genetics, the vinegar fly Drosophila melanogaster and the model mustard Arabidopsis thaliana.In Chapter 2, I investigated transcriptional evolution in the chemosensory organs, in the genus Scaptomyza with a particular emphasis on six major insect chemosensory gene families. I conducted a comparative bulk RNA-sequencing between S. flava and its microbe-feeding relative, S. pallida, and searched for genes experiencing larger expression divergence between species as candidates behind chemosensory evolution. This approach successfully illuminated evolutionary patterns across gene families and narrowed down the candidates that provide insight into specific sensory changes aided the colonization of Brassicales host plants.In contrast to the exploratory analyses in the previous chapter, Chapter 3 conducted a detailed investigation on a single gene and its products, TrpA1, which encodes a chemosensory ion channel detecting electrophilic toxins, including mustard-derived isothiocyanates (ITCs). Again, I took a comparative approach between the mustard-feeding S. flava and the microbe-feeding relatives to address how the functional evolution of TRPA1 has been intertwined with the dietary and chemosensory evolution among the drosophilids. I integrated multiple experimental approaches spanning from genomics and genetics to electrophysiology, and found that the reduced chemical sensitivity of the TRPA1 channel and the modified composition of alternatively spliced isoforms were likely two major molecular changes behind the weak avoidance toward ITCs in the S. flava lineage. However, I also found that the TRPA1 activity from the distantly-related generalist D. melanogaster was even weaker than S. flava, despite the strong behavioral aversion to ITCs, suggesting that the evolutionary relationship of the TRPA1 channel function and the gustatory preferences is more complicated than it seems, and requires careful investigations on multiple characteristics of the gene.

Little is known about the pharmacological activity of Monarda fistulosa L. essential oils. To address this issue, we isolated essential oils from the flowers and leaves of M. fistulosa and analyzed their chemical composition. We also analyzed the pharmacological effects of M. fistulosa essential oils on transient receptor potential (TRP) channel activity, as these channels are known targets of various essential oil constituents. Flower (MEOFl) and leaf (MEOLv) essential oils were comprised mainly of monoterpenes (43.1% and 21.1%) and oxygenated monoterpenes (54.8% and 77.7%), respectively, with a high abundance of monoterpene hydrocarbons, including p-cymene, γ-terpinene, α-terpinene, and α-thujene. Major oxygenated monoterpenes of MEOFl and MEOLv included carvacrol and thymol. Both MEOFl and MEOLv stimulated a transient increase in intracellular free Ca2+ concentration ([Ca2+]i) in TRPA1 but not in TRPV1 or TRPV4-transfected cells, with MEOLv being much more effective than MEOFl. Furthermore, the pure monoterpenes carvacrol, thymol, and β-myrcene activated TRPA1 but not the TRPV1 or TRPV4 channels, suggesting that these compounds represented the TRPA1-activating components of M. fistulosa essential oils. The transient increase in [Ca2+]i induced by MEOFl/MEOLv, carvacrol, β-myrcene, and thymol in TRPA1-transfected cells was blocked by a selective TRPA1 antagonist, HC-030031. Although carvacrol and thymol have been reported previously to activate the TRPA1 channels, this is the first report to show that β-myrcene is also a TRPA1 channel agonist. Finally, molecular modeling studies showed a substantial similarity between the docking poses of carvacrol, thymol, and β-myrcene in the binding site of human TRPA1. Thus, our results provide a cellular and molecular basis to explain at least part of the therapeutic properties of these essential oils, laying the foundation for prospective pharmacological studies involving TRP ion channels.

Thermosensory nociception alerts organisms to potential environmental dangers, thereby serving as a protective mechanism for driving adaptive behavioral responses to safeguard against incipient damage. Transient receptor potential (TRP) channels play a key role in thermosensation and some can be activated directly or indirectly by changes in temperature. Protostome, and many deuterostome, TRPA1s have been associated with high-temperature sensing. For example, Drosophila TRPA1 is known to function in Class IV (CIV) polymodal nociceptor neurons for high heat (as well as mechanical and chemical) nociception. Conversely, some deuterostome TRPA1s (including those of mice and humans) have been identified as noxious cold sensitive. However, little is known regarding how TRPs mechanistically function in regulating noxious cold detection and whether there is molecular conservation across phyla. Our previous studies identified Class III (CIII) multidendritic (md) sensory neurons as cold nociceptors that are required for noxious cold-evoked contraction (CT) behavior in larvae. We further demonstrated via CIII-specific neurogenomic studies that TRPA1 is enriched in these neurons, suggesting it may contribute to noxious cold sensing. In this work, analyses of multiple TrpA1 whole-animal mutants revealed severe impairment of cold-evoked CT behavior, which we likewise observed with CIII-specific TrpA1 knockdown. Behavioral studies of TrpA1 mutations revealed isoform-specific requirements for noxious cold sensing that are distinct from isoforms linked to noxious heat sensing via CIV neurons. Electrophysiological recordings demonstrate that TRPA1 is required in CIII neurons for cold-evoked neural activity. TrpA1 appears to function in cold-evoked sensory transduction, and CIII-specific expression of TrpA1 rescued nocifensive behavior in whole animal TrpA1 mutants. Further, behavioral deficits were also rescued by ectopic expression of human TrpA1. Moreover, ectopic expression studies reveal TrpA1 is sufficient to confer cold sensitivity on otherwise non-cold-sensing neurons. Collectively, these findings shed new light on evolutionarily ancient properties of TRPA1 in thermal sensing and raise interesting questions regarding the molecular and mechanistic underpinnings of TRPA1 polymodality

Since the dawn of human existence, humankind has relied on nature’s bounty for food, raiment, and medicine. Countless populations from prehistoric beginnings to the present have proven the therapeutic properties of plants to treat all manner of afflictions heaped upon them by the “unseen” organisms of this world and the pains that accompany life. The expansion of human knowledge regarding the causes of disease, organic biochemistry, medicine, and more, have led to our modern understanding and application of chemical matter to relieve pain, treat infections, kill cancer cells, and control mosquito populations.Natural products, those organic molecules obtained from living organisms (e.g., plants, bacteria, marine sponges), are a keystone to drug discovery and modern medicine. As part of an ongoing effort, at The Ohio State University College of Pharmacy, in a drug discovery project from natural sources, three spirotetronate-containing polyketides, namely, abyssomicin 4 (2.142), neoabyssomicin B (2.143), and abyssomicin 2 (2.144), were isolated from a Streptomyces sp. obtained from the liverwort, Bazzania trilobata (L.) S. F. Gray. This represents the first report of isolated and characterized compounds from a liverwort-associated, endophytic bacterium.Cinnamosma fragrans Baill. has been used in traditional medicine practice for many indications and has been shown to produce an array of unsaturated dialdehyde sesquiterpenoids with diverse biological activities. Notably, unsaturated dialdehyde sesquiterpenoids show promising insecticidal and repellent activity and may serve as lead compounds for the development of much-needed mosquitocidal agents capable of controlling mosquito populations and mitigating the spread of mosquito-borne diseases. The compound, cinnamodial (3.20), was obtained in large quantities from the root bark of C. fragrans and used as a scaffold for semi-synthetic analog generation. The prepared analogs were used to probe the structure-activity relationship of cinnamodial and the results obtained confirm the importance of the dialdehyde moiety for mosquitocidal activity and the α,ß-unsaturated carbonyl for antifeedant activity. The improved larvicidal activity observed in a prepared methyl ketone (3.32) derivative and a hydroquinone (3.35) derivative of cinnamodial (3.20) suggest that the development of more stable and effective insecticidal derivatives from this lead compound is possible.

Over the past decade, the transient receptor potential ankyrin 1 (TRPA1) receptor has been extensively studied because of its wide distribution across the human body and its roles in many aspects of sensation, some of which are physiological and others of which are pathophysiological. A member of the TRP channel family, TRPA1 is most commonly found in somatosensory neurons present in nociceptors, especially the dorsal root ganglia and trigeminal ganglia, but its expression has been reported in tissues as disparate as the cells of the inner ear, non-neuronal cells in the small and large intestines and lungs, and in the beta islet cells of the pancreas. Physiologically, TRPA1’s primary role is as a nociceptive chemosensor detecting exogenous agonists that are irritants, such as AITC from wasabi, and endogenous agonists that are indicators of oxidative stress, such as H2O2. However, when TRPA1 becomes dysregulated, its physiological roles can become distorted. Research has demonstrated that TRPA1 is involved in many pathologies including chronic pain and inflammation, neuropathic pain, itch, and respiratory diseases, but to date, few TRPA1-targeting therapeutics have been successful, and it is evident that greater understanding of the receptor is required to address this need. Recently, the discovery of optovin revealed that a synthetic photoreactive compound could confer photosensitivity onto vertebrate TRPA1, but much is still unknown about photo-dependent TRPA1 activation. One way to approach this question is through identification of additional chemotypes that act similarly to optovin. In this dissertation, I describe the process of identifying such compounds and the characterization of their properties. Analysis of data from a large-scale, behavior-based chemical screen conducted in larval zebrafish established a small subset of compounds that induced a strong behavioral response to light. Structural analysis showed that the hit compounds were in fact structurally diverse, and photochemical, behavioral, and toxicity studies uncovered characteristics unique to each compound. Experiments using TRPA1 mutant zebrafish confirmed that a subset of these hits are TRPA1-dependent. Together, these structurally, photochemically, and pharmacologically diverse compounds form a tool set that can be used to modulate the TRPA1 receptor, allowing future researchers to gain a better understanding of the receptor’s characteristics and mechanisms, research its roles in sensation and photo-behaviors, and identify therapeutics to address pathologies caused by its dysregulation.

TRPA1 - also known as the ‘wasabi receptor’ - is activated by pungent natural products from mustard and allium plants and is responsible for the sinus clearing or eye stinging pain that one experiences when eating horse radish or chopping an onion. More importantly, TRPA1 is a receptor for an astoundingly broad class of volatile environmental irritants and endogenous pro-algesic agents that elicit or exacerbate tissue damage, pain, and neurogenic inflammation. Many of these irritants are chemically reactive electrophiles that activate TRPA1 through a fascinating mechanism involving covalent modification of the channel’s cytoplasmic N-terminus, a phenomenon that is not well understood at the biochemical or structural level. TRPA1 also functions as a ‘receptor-operated’ channel that is activated downstream of phospholipase C signaling pathways, such as those involved in the transduction of pruritic signals. Together, these actions make TRPA1 a major contributor to a broad range of chemo-nociceptive actions include pain and itch.In this work we have exploited natural product pharmacology, functional recording, and structural methods to understand how TRPA1 is activated by a range of stimuli (reactive and non-reactive, direct and indirect). Collectively, these data draw a detailed mechanistic picture of how this important chemo-nociceptive ion channel detects physiological stimuli, transduces this information into neuronal excitation, and is regulated by cellular events. In light of TRPA1’s well recognized role in acute and persistent pain and itch, our findings are of both investigational and therapeutic import. Beyond TRPA1, they are of general relevance to illuminating the gating mechanisms other sensory TRP channels, several of which are also regulated downstream of metabotropic receptors and/or by cellular calcium.

Female mosquitoes employ a diverse array of sensory cues when finding hosts for blood feeding. Among these cues, the mosquito’s ability to detect body heat while host seeking has been appreciated for decades. This thermosensory system is largely understood to detect convective air currents around warm surfaces that activate thermosensory neurons in the mosquito antenna. Because these convective currents occur quite physically close to the warm surface, we chose to investigate whether mosquitoes can also sense infrared radiation emitted from hosts as well, which could function at a much greater distance. To do this we developed a novel behavioral paradigm that exposes mosquitoes to infrared cues and records their behavioral responses. Using this new paradigm, we found that mosquitoes detect infrared while exposed to other host cues and use this sense to navigate towards host objects. Furthermore, this sense is influenced by both the IR intensity of the source and environmental conditions. Lastly, we found that mosquitoes lacking the thermally activated ion channel TRPA1 completely lose their ability to detect thermal infrared. This work provides the first demonstration that IR is a cue used by mosquitoes for host seeking.

Bacterial keratitis is a devastating, sight-threatening infection of the cornea. However, in order to truly grasp how bacterial keratitis ensues, it is first imperative to understand how the healthy eye is maintained. Only then can we truly decipher the intricacies of how a cornea becomes susceptible to potentially dangerous microorganisms.Bacteria surround us and inhabit almost every niche in our environment and on our bodies. Thus, the question arose: are bacteria present on the ocular surface, and if so, what role do they play in maintaining health? This is the first question I sought to address in my dissertation. I used bacterial labeling techniques to be able to identify live microbes and gain a spatial understanding of the bacteria on the mouse ocular surface. Interestingly, I found that live bacteria are rarely present on the cornea. This was in stark contrast to the conjunctiva, which does host bacterial inhabitants, including long filamentous bacteria. The cornea’s ability to prevent a microbiome is dependent on IL-1R and MyD88 signaling, an important finding to demonstrate the role of this pathway during homeostasis and not just as a response to pathogens.It is fortunate that the cornea is an expert at preventing bacterial colonization considering that it is constantly exposed to the environment and all of the surrounding microbes. Thus, I wanted to gain a further understanding of how the cornea prevents a microbiome on its surface. Tear fluid has previously been shown to be an important factor towards bacterial defense of the ocular surface. Several constituents that make up basal tears are antimicrobial and impede bacterial virulence. One such component is DMBT1, a glycoprotein that inhibits bacterial twitching. Twitching motility is a surface movement important for epithelial traversal and virulence. How DMBT1 inhibits Pseudomonas aeruginosa twitching, a common cause of contact lens related corneal infections, was investigated. It was found that N-glycosylation of DMBT1 contributes to this defense mechanism.The cornea is the most innervated tissue in the body. Not only are nerves important for sensory transduction, but they also can have immunomodulatory roles. The contributions of sensory nerves in regard to corneal defense has previously not been explored. Thus, I sought to unravel the role of polymodal nociceptors, TRPA1 and TRPV1, in corneal defense against bacteria. I found that these neuronally expressed ion channels contribute to preventing bacterial colonization on the cornea and communicate with immune cells when the healthy cornea is challenged with P. aeruginosa.After exploring several factors involved in keeping the cornea free of bacteria, I next wanted to gain a better understanding of how common ocular modulations affect its ability to withstand bacteria. First, I explored the impact of dry eye disease, a common debilitating disease with an unknown pathogenesis, on susceptibility to bacterial adhesion. Dry eye disease is associated with a myriad of factors that hinder corneal defense such as altered tear film composition, reduction of antimicrobial peptides, and poor epithelial integrity. I used an experimentally induced mouse model of dry eye disease and determined if environmental bacteria could colonize the corneas. Interestingly, I found that dry eye disease did not increase susceptibility to bacterial colonization on the cornea and no changes occurred on the conjunctiva. Thus, local bacteria on the ocular surface are unlikely to play a role in the pathogenesis of dry eye disease and induction of the disease does not hamper the ability of the cornea to ward off unwanted inhabitants.Finally, the effects of contact lens wear on the cornea was studied. Progress to decipher how a contact lens alters the corneal environment and any implications this may have, has previously been hindered by limitations of human studies and a lack of animal models. Our lab developed a silicon-hydrogel contact lens to fit on mice that mimics a human lens wearer. We found that a contact lens on a healthy cornea initiates a parainflammatory response observed as an infiltration of dendritic cells at 24 h after lens wear and an influx of neutrophils after 5 days of lens wear. Interestingly, this increase in immune cells in the cornea did not disrupt the macroscopic morphology and the cornea remained clear. Colonization of environmental bacteria on the contact lens was also detected. This understanding of how the lens affects the healthy cornea is imperative to grasp what is being altered that increases susceptibility to bacterial keratitis.Overall, this dissertation focuses on bacterial interactions with the ocular surface. First, I determined that the cornea does not host a microbiome. Several factors contribute to keeping the cornea free of bacteria including tear fluid, and glycoprotein DMBT1, IL-1R and MyD88 dependent signaling, and polymodal nociceptors. Dry eye disease does not increase the susceptibility to bacterial colonization but contact lens wear is associated with bacterial adhesion. Whether these bacteria on the lens contribute to the infiltration of immune cells observed with lens wear remains to be understood. The results from this dissertation increases our general understanding of how the ocular surface maintains health and prevents bacterial colonization. Studies also give insights into mechanisms impacted by lens wear that may contribute to the increased risk of bacterial keratitis.

Neuropathic pain affects up to 50% of the 29 million diabetic patients in the United States. Neuropathic pain in diabetes manifests as a disease of the peripheral and central nervous systems. The prevalence of type 2 diabetes is far greater than type 1 (90%), yet the overwhelming focus on type 1 models this has left the mechanisms of pain in type 2 diabetes largely unknown. Therefore I aimed to improve the current mechanistic understanding of pain associated with type 2 diabetes using two preclinical rodent models: Zucker Diabetic Fatty rats and db/db mice. In addition, I highlight the translational importance of simultaneous measurement of evoked/sensory and non-evoked/affective pain-related behaviors in preclinical models. This work is the first to show a measure of motivational-affective pain in a model of type 2 diabetes. I used methodological approaches including: (1) immunohistochemical and calcium imaging to assess stimulus-evoked sensitization; (2) measurement nociceptive behaviors and evoked sensory thresholds as well as pain affect using novel mechanical conflict avoidance and conditioned place preference/aversion assays; (3) pharmacological and genetic manipulation of methylglyoxal, TRPA1, AC1, and PPARγ. I hypothesized that the thiazolidinedione class of peroxisome proliferator-activated receptor gamma (PPARγ) agonists would reduce neuropathic pain-like behavior and spinal neuron sensitization in traumatic nerve injury and type 2 diabetes. As PPARγ is a nuclear receptor, and already targeted clinically to promote cellular insulin sensitization to reduce hyperglycemia, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. In two separate research aims, I challenged this view and tested whether the PPARγ agonist pioglitazone would (1) rapidly alleviate neuropathic pain through a non-genomic mechanism and (2) reduce painful sensitization in nociceptive and neuropathic pain models independent from lowering blood glucose. I aimed to investigate the contribution of the glucose metabolite methylglyoxal to painful type 2 diabetes. I tested the hypothesis that methylglyoxal produces nociceptive, evoked, and affective pain that is dependent on activation of the sensory neuron cation channel TRPA1 and the secondary messenger enzyme AC1. I also tested whether pioglitazone or the novel methylglyoxal scavenging peptide GERP10 could alleviate painful type 2 diabetes.

Itch is defined as an unpleasant sensation that evokes a desire to scratch. In contrast to acute itch that is transient, chronic itch is a persistent, debilitating condition for which there are few treatment options. Chronic itch accompanies a number of skin diseases and systemic conditions, as well as a variety of neurological disorders. However, little is known about the molecules and cell types that mediate acute or chronic itch in primary sensory neurons and skin.We have established an essential role for the ion channel, TRPA1, in multiple forms of both acute and chronic itch. We have shown that TRPA1 is required for neuronal activation and itch behaviors in mice downstream of acute exogenous and endogenous itch compounds. Similarly, TRPA1 is required for itch behavior and itch-evoked expressional changes in both sensory neurons and skin in a mouse model of dry skin. We have also identified a novel itch-causing compound: Thymic Stromal Lymphopoietin (TSLP). Numerous studies suggest that the epithelially-derived cytokine TSLP acts as a master switch that triggers both the initiation and maintenance of the chronic itch disease atopic dermatitis. Our work demonstrates that TSLP activates sensory neurons directly and leads to acute itch behaviors. TRPA1 is required for both TSLP-evoked neuronal activation and itch behaviors. Taken together, our work shows that TRPA1 is a master regulator of itch signaling.

Pain is a major personal and community burden throughout the world with currently limited treatment options for persistent pain due to unacceptable side effects, dependence or frank inefficacy. It is necessary to understand the anatomical and molecular pathways leading to pain to better cope with the current challenge of treating it.

Aims: Propofol, clinically named as Diprivan is an intravenous anesthetic known to cause hypotension in patients presenting for surgery. We have investigated the vasodilatory signaling cascade by which propofol causes hypotension using both in vivo and in vitro experimental approaches.Methods and Results: Using high-fidelity microtip transducer catheter, mean arterial blood pressure (MAP) was measured in control, transient receptor potential ankyrin subtype 1 knock-out (TRPA1-/-), transient receptor potential vanilloid 1 knock-out (TRPV1-/-) and TRPA1-TRPV1 double-knockout mice (TRPAV-/-) in the presence and absence of L-NAME (an endothelial nitric oxide synthase inhibitor) and penitrem A [a big-conductance calcium gated (BKCa) channel inhibitor]. To further support our in-vivo data, murine coronary microvessels were isolated and cannulated for vasoreactivity studies. Furthermore, NO production from endothelial cells isolated from mouse aorta was also measured and immunocytochemical (ICC) studies were performed to show the intracellular localization of TRPA1 and TRPV1. Our in-vivo data shows that the characteristic propofol-induced depressor response is dependent on TRPA1-NO-BKCa pathway. Interestingly, vasoreactivity studies in isolated murine left anterior ascending (LAD) arteries demonstrate that TRPA1 and TRPV1 communicate with each other and propofol-induced vasodilation is dependent on both TRPA1 and TRPV1. Moreover our data also suggest that NO production and BK channel activation are the downstream mediators in this pathway. Finally, we demonstrate that NO production is attenuated in primary endothelial cells isolated from TRPAV-/- mice. ICC data also shows the co-localization of these channels in mouse aortic endothelial cells.Conclusions:This is the first study which has shown that propofol-induced vasodilation involves TRPA1 in-vivo and also there is an implication of cross-talk between TRPA1 and TRPV1 in the coronary bed. Furthermore by understanding the mechanisms by which this anesthetic causes hypotension and coronary dilation will help to mitigate the potential harmful side-effects of anesthesia in patients with little cardiovascular reserve. This will in turn ensure a better and faster post-operative recovery in patients, especially benefiting those suffering from diabetes and other cardiovascular disorders.

Pain is a major personal and community burden throughout the world with currently limited treatment options for persistent pain due to unacceptable side effects, dependence or frank inefficacy. It is necessary to understand the anatomical and molecular pathways leading to pain to better cope with the current challenge of treating it.