Doctor of Philosophy (Ph.D.), University of Dayton, 2024, Biology

Vertebrate freeze tolerance is an extraordinary phenomenon in which up to 70% of an animal's extracellular fluids are converted to ice while circulation, respiration, and neuronal function are all simultaneously suspended during freezing. Upon thawing, animals must tolerate the resumption of physiological function, restore intracellular fluid volumes, and repair injuries that occurred during freeze-thaw. The mechanisms that enable animal freeze tolerance vary by species and represent a myriad of biochemical, cellular, systems, and organismal strategies. Cope's gray treefrog Dryophytes (Hyla) chrysoscelis is a freeze tolerant anuran that repeatedly freezes and thaws each winter in part by utilizing a complex system of cryoprotectants including glycerol, glucose, and urea. Intracellular transport is facilitated by specialized aquaglyceroporin proteins that enable transmembrane movement of water and the cryoprotectants glycerol and urea during freeze-thaw. The physiological mechanisms that enable freeze tolerance in D. chrysoscelis are not entirely understood and cannot be explained by cryoprotectant accumulation alone. The aim of this dissertation is to explore the unknown physiological mechanisms of freeze tolerance in D. chrysoscelis by using an integrative perspective that incorporates all levels of biological organization. Novel experimental protocols were used to evaluate the ecophysiological effects of repeated freezing and thawing, characterize organismal responses to seasonal and cold acclimation, and determine the effects of cold acclimation and freeze-thaw cycles on membrane lipid composition using 1H-NMR analysis. The results from these studies emphasized the complexity of freeze tolerance in D. chrysoscelis and revealed several novel aspects of freeze tolerance in this species including dynamic blue and green dorsal coloration in frozen and thawing frogs, “freeze resistance” in a freeze tolerant vertebrate, evidence of seasonal (open full item for complete abstract)

Committee: Carissa Krane (Advisor); David Goldstein (Committee Member); Thomas Williams (Committee Member); Yvonne Sun (Committee Member); Amit Singh (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Ecology; Molecular Biology; Organismal Biology; Physiology

Master of Science, Miami University, 2014, Zoology

Rapid cold-hardening (RCH) is a highly conserved response in insects that induces physiological changes within minutes to hours of exposure to low temperature and provides protection from chilling injury. Recently, a similar response, termed drought-induced RCH, was described following acute desiccation. In this study, we investigated the limits and mechanisms of this response in larvae of the goldenrod gall fly Eurosta solidaginis. The cold-hardiness of larvae increased after as few as 2 h of desiccation and a loss of less than 1% fresh mass, as organismal survival increased from 8% to 41% following exposure to -18°C. We also demonstrated that drought-induced RCH occurs independently of neuroendocrine input, as midgut tissue desiccated ex vivo displayed improved freeze-tolerance relative to control tissue. Our results indicate that E. solidaginis larvae are extremely sensitive to desiccation stress, which is a triggering mechanism for one or more physiological pathways that confer enhanced freeze-tolerance.

Committee: Richard Lee PhD (Advisor); Jon Costanzo PhD (Committee Member); Paul Schaeffer PhD (Committee Member) Subjects: Entomology; Physiology

Master of Science, The Ohio State University, 2024, Horticulture and Crop Science

Sub-zero freezing temperatures cause 5-15% of annual crop losses to worldwide grapevine cultivation. Based on their cold hardiness, i.e., the ability to survive under low temperature conditions, grapevine genotypes can be classified as a) cold sensitive, such as Vitis vinifera (-18℃ to -22℃ critical range), and b) cold hardy, such as Vitis labrusca (-26℃ to -29℃). During spring, late-spring frost conditions can cause injury to young shoots emerging from dormant buds, affecting grapevine yield and wine quality. Enhancing cold hardiness and frost tolerance can improve grapevine's survivability under extreme low temperature conditions. Cold hardy wild grapevine species, such as native North American Vitis labrusca, are being utilized for the development of cold hardy hybrid cultivars, however, most of these species have low-chilling requirements, leading to early budburst in spring. Therefore, despite being cold hardy as dormant buds, it is unknown if the young shoots of Vitis labrusca have higher frost tolerance than those of Vitis vinifera cultivars. Our goal was to determine the difference in frost tolerance and transcriptomic response related to low temperatures between young shoots of cold hardy V. labrusca acc. ‘GREM4' and cold sensitive V. vinifera cv. ‘Cabernet Sauvignon'. Results showed that ‘GREM4' shoots had significantly higher frost tolerance than those of ‘Cabernet Sauvignon'. Transcriptomic analysis for chill (4℃) and freeze (-2℃) stress revealed that 'GREM4' shoots exhibited upregulation of genes encoding cell-wall-associated receptor kinases and extensin proteins under both chill and freeze stress. Moreover, genes encoding 3-ketoacyl-coenzymeA synthase (KCS), a key enzyme involved in wax biosynthesis, and genes related to sugar transport and metabolism were differentially expressed between ‘GREM4' and ‘Cabernet Sauvignon'. Interaction analysis between species and temperature treatments revealed that the gene encoding abscisic acid (ABA) degrading enzym (open full item for complete abstract)

Committee: Andrea Gschwend (Advisor); Imed Dami (Advisor); Eric Stockinger (Committee Member); Jonathan Fresnedo-Ramirez (Committee Member) Subjects: Genetics; Horticulture; Plant Sciences

Master of Sciences, Case Western Reserve University, 2023, Biology

Capturing the scope of biodiversity loss and future species distribution under global climate change requires considering how species will respond across spatial and temporal scales. Here, we address these gaps with a research program that considers physiological responses to warming under winter acclimation and exploring the potential for evolution of physiological traits at fine spatial scales. To do so, we use population comparative approaches of common arthropods between urban and non-urban habitats to assess differences in winter physiology, and within urban habitats to explore divergence at fine spatial scales. Our results suggest that understanding species responses to contemporary warming requires assessing physiology under ecologically relevant conditions and that multiple pathways of compensatory responses at fine spatial scales could be especially important in human impacted habitats. Future investigations could explore the variation of climatic conditions across seasons and determining the mechanisms of species responses to multiple selective pressures.

Committee: Ryan Martin (Advisor); Angela Dixon (Committee Chair); Jean Burns (Committee Member); Sarah Diamond (Committee Member) Subjects: Biology; Climate Change; Ecology; Evolution and Development

Doctor of Philosophy, Miami University, 2018, Zoology

When challenged by brief exposure to sub-lethal stresses, insects and other small ectotherms can make physiological adjustments that enhance their stress tolerance within minutes. Through these rapid acclimatory responses, insects adjust their physiology to track ambient conditions and counter the negative effects of perturbation by abiotic stresses. Although rapid acclimation is best studied as a response to brief chilling, diverse environmental cues, including high temperature, dehydration, and anoxia trigger similar responses. Further, recent evidence suggests that different cues for rapid acclimation trigger distinct mechanistic responses. This dissertation investigated the underpinning physiology and ecological importance of these diverse rapid acclimatory responses in three species of flies. The first project compared the physiological mechanisms triggered by brief chilling and dehydration in larvae of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis. Chilling produced solute accumulation in larval hemolymph and caused the activation of the second messenger p38 MAP kinase, while dehydration caused a redistribution of body water without significant accumulation of solutes and activated both p38 and Erk1/2 MAP kinase. Additionally, though neither treatment differed from control treatments, larvae that were chilled for 2 h maintained a higher metabolic rate at low temperatures than those that were dehydrated. The second project examined the effects that brief chilling and dehydration have on flight performance and fecundity in the flesh fly Sarcophaga bullata. Both triggers for rapid acclimation enhanced distance flown, peak velocity, increased flight time under stressful conditions, and preserved reproductive output. These results suggest that rapid acclimation helps insects to maintain basic behaviors when faced with stress in nature. The final project investigated the effects of brief exposure to diverse abiotic stresses in larvae of the Antarcti (open full item for complete abstract)

Committee: Richard Lee PhD (Advisor); Jon Costanzo PhD (Committee Member); Kathleen Killian PhD (Committee Member); Paul Schaeffer PhD (Committee Member); Jing Zhang PhD (Committee Member) Subjects: Physiology

Doctor of Philosophy, Miami University, 2013, Zoology

Low-temperature survival of the Antarctic midge, Belgica antarctica, is promoted by alternative strategies of freeze tolerance and freeze avoidance. Larvae are freeze tolerant year round. Yet, provided they avoid inoculative freezing, they could remain unfrozen by either supercooling (during acute exposure) or cryoprotective dehydration (during prolonged exposure). The first two projects in this dissertation compared each of these two strategies of freeze-avoidance to that of freeze-tolerance. The purpose of the first project was to compare the induction of the rapid cold-hardening (RCH) response in frozen and supercooled larvae. At the same induction temperature, RCH occurred more rapidly and conferred a greater cryoprotection in frozen versus supercooled larvae. Since the primary difference between these two groups is cellular dehydration, and dehydration without chilling significantly increased larval cold tolerance, it was speculated that cellular dehydration caused by freeze concentration promoted the rapid development of cryoprotection in frozen larvae. The second investigation examined the alternative overwintering strategies of freezing and cryoprotective dehydration. Freezing had little effect on larval body water content and hemolymph osmolality. In contrast, cryoprotective dehydration resulted in a progressive loss of body water, causing a four-fold increase in hemolymph osmolality. Also, freezing and cryoprotective dehydration produced distinctly different patterns of glycogen breakdown. However, post-recovery levels of glycogen were similar in these two groups as were total lipids. In summary, freezing and cryoprotective dehydration were both effective in promoting winter survival of larvae, with only minor differences in energetic costs. The use of cryoprotective dehydration as an overwintering strategy in B. antarctica is constrained by inoculative freezing from environmental ice. Thus, the last project investigated the effect of different microh (open full item for complete abstract)

Committee: Richard Lee Ph.D. (Advisor); A. John Bailer Ph.D. (Committee Member); Jon Costanzo Ph.D. (Committee Member); Kathleen Killian Ph.D. (Committee Member); Paul Schaeffer Ph.D. (Committee Member) Subjects: Biology; Entomology; Physiology; Zoology

Doctor of Philosophy, The Ohio State University, 2012, Horticulture and Crop Science

Grapes are temperate crops, frequently damaged by freezing temperatures. The economic losses that result from freezing injury are major problems for grape and wine industries in cold regions. Our central hypothesis is that soluble sugars in grape tissues provides increased freezing tolerance (FT) and the larger sugar, raffinose, accumulates earlier and to higher amounts in cold-tolerant than in cold-sensitive cultivars in response to cold temperature and short-day (SD) photoperiod. The central objective of this dissertation was to characterize the morphological, physiological and biochemical changes in response to cold acclimation (CA) and SD photoperiod. The specific objectives were to: 1) characterize the morphological, physiological and biochemical changes induced by low temperature in cold-tolerant and cold-sensitive Vitis species under controlled conditions; 2) characterize the morphological, physiological and biochemical changes induced by SD photoperiod in cold-tolerant and cold-sensitive Vitis species under controlled conditions and 3) characterize the morphological, physiological and biochemical changes induced by low temperature and SD photoperiod in cold-tolerant and cold-sensitive Vitis species under field conditions. In response to temperature under controlled environs, grape (Vitis spp) cultivars ‘Frontenac', ‘Couderc 3309', ‘Concord', ‘Cabernet Franc', ‘Traminette' and ‘Seyval' were evaluated. Shoot growth slowed under cold temperature regimes in all cultivars except ‘Concord'. Under the non-acclimating temperature regime, raffinose concentrations were low and similar among cultivars, whereas under CA temperature regimes raffinose accumulation was generally higher and cold-tolerant cultivars accumulated higher concentrations than did cold-sensitive cultivars. Basal leaves and buds accumulated the most raffinose. In evaluating responses to SD, experiments were conducted in greenhouse using ‘Cabernet Franc', ‘Couderc 3309' and ‘Concord' cultivars. Shoo (open full item for complete abstract)

Committee: Imed Dami PhD (Advisor); James Metzger PhD (Committee Member); Eric Stockinger PhD (Committee Member); Albert Miller PhD (Committee Member); Rebecca Lamb PhD (Committee Member) Subjects: Agriculture; Anatomy and Physiology; Horticulture; Plant Biology

Doctor of Philosophy, Miami University, 2010, Zoology

The demands of winter can be especially onerous to organisms inhabiting temperate and polar regions. In addition to contending with low relative humidity, organisms often experience subzero temperatures and risk freezing of their body fluids. A small group of frogs, turtles, and invertebrates tolerate freezing, during which they rapidly redistribute water and cryoprotectants across their cell membranes to prevent osmotic damage. Despite the importance of water and cryoprotectant movement, the mechanisms underlying redistribution of these molecules are not fully known. Recent evidence suggests that transmembrane, water-permeable proteins called aquaporins play a role in protecting cells during osmotic stress. This dissertation describes two studies that characterized the physiological role of aquaporins during desiccation and freezing in the goldenrod gall fly, Eurosta solidaginis. During autumn, E. solidaginis larvae prepare for winter by accumulating high levels of cryoprotectants (e.g. glycerol) and developing freeze tolerance. In the first study, I examined the abundance of aquaporin-like proteins from July through January to determine whether it is correlated to seasonal cold-hardening. Immunoblots of proteins from larvae collected during this period demonstrated a significant seasonal increase in the abundance of two aquaporin-homologues. Additionally, one of these homologues was regulated in response to short-term acclimation. These results support the hypothesis that aquaporins are closely tied to the seasonal acquisition of freeze and desiccation tolerance. In the second study, I sought to clone and functionally characterize an AQP from E. solidaginis and understand how it promotes tolerance of environmental stresses. We identified EsAQP1, described its molecular structure and predicted its phylogenetic relationship to other known aquaporins. Permeability measurements with a Xenopus laevis oocyte swelling assay determined that EsAQP1 was permeable to water (open full item for complete abstract)

Committee: Richard Lee PhD (Committee Chair); Jon Costanzo PhD (Committee Member); Paul James PhD (Committee Member); Michael Robinson PhD (Committee Member); Gary Lorigan PhD (Committee Member) Subjects: Biology; Zoology

Doctor of Philosophy, Miami University, 2005, Zoology

In this dissertation, I have examined possible links between physiological parameters associated with survival at low temperature and water balance of an overwintering insect. The first study provided a seasonal characterization of cold-tolerance and desiccation resistance in overwintering larvae of the goldenrod gall fly, Eurosta solidaginis. From September 20 to October 30 larvae exhibited a gradual increase in cold-tolerance that was associated with increases in cryoprotectants. In contrast, larvae exhibited a two-phase increase in desiccation resistance. The first was a dramatic six-fold reduction in rate of water loss that occurred between October 3 and October 16 as the gall tissue senesced. The second, more subtle reduction occurred between October 16 and December 11 and was associated with cryoprotectant production. The second study examined cues for the rapid, seasonal increase in desiccation resistance of E. solidaginis larvae associated with senescing of the gall tissue. Desiccation resistance increased dramatically within three days of removal from the gall, and was primarily due to reductions in respiratory water loss as larvae entered dormancy. This study illustrated that dormancy in overwintering insects that was primarily thought to be an adaptation to conserve metabolic fuels, also may be essential for water conservation. I also examined cold-tolerance and desiccation resistance in three widely separated populations of overwintering E. solidaginis larvae from Michigan, Ohio and Alabama. Larvae from the most northern population had higher concentrations of the cryoprotectant glycerol, were more cold-tolerant, and had lower rates of overall water loss after acclimation to 5 °C. In contrast, southern larvae had lower rates of metabolism and transpiratory rates of water loss after acclimation to 20 °C. Lastly, I examined possible links between extracellular solute regulation and cell volume maintenance in larvae subjected to dehydration and freezing. Af (open full item for complete abstract)

Committee: Richard Lee (Advisor) Subjects: Biology, Animal Physiology

Doctor of Philosophy (Ph.D.), University of Dayton, 2013, Biology

Cope's gray treefrog, H. chrysoscelis is a freeze tolerant anuran that accumulates glycerol as a cryoprotectant during cold acclimation. Aquaporins, members of the major intrinsic protein (MIP) family of transmembrane water pores, may play an important role in the mechanism of freeze tolerance by mediating glycerol and water transport across cell membranes. Thus, we hypothesize that HC-3, an ortholog of mammalian aquaglyceroporin AQP3, enhances membrane permeability to glycerol, facilitating the cellular response to osmotic gradients formed when extracellular water freezes. To address the hypothesis, erythrocytes from H. chrysoscelis were used as an in vitro cell culture model to study the regulation of HC-3 protein expression. Compared with warm-acclimated frogs, erythrocytes from cold-acclimated frogs showed abundant HC-3 protein and enhanced plasma membrane localization of HC-3. Erythrocytes, regardless of the original acclimation state, exhibited time and temperature-dependent regulation of HC-3 expression and an increase in the abundance of high molecular weight immunoreactive species within 24 hr of culture at 20°C. Likewise, erythrocytes cultured in glycerol-containing media consistently expressed relatively more glycosylated HC-3 than erythrocytes cultured in normal cell culture media. Thus, part of the regulation of HC-3 expression that occurs naturally during cold-acclimation is cell-based. Erythrocyte protein, when subjected to deglycosylation resulted in downward shift of high molecular weight HC-3 protein, demonstrating that HC-3 is post-translationally modified by N-linked glycosylation. In the absence of genomic knockout tools, a novel method of antisense HC-3 morpholino delivered in to cultured suspension erythrocytes via a peptide mediated Endo-Porter was developed where HC-3 protein expression was reduced by 94% in morpholino targeted cells (as assessed by Western blotting) as compared to controls. In addition, immunocytochemistry revealed a substa (open full item for complete abstract)

Committee: Carissa Krane (Advisor); Amit Singh (Committee Member); Tsonis Panagiotis (Committee Member); Shirley Wright (Committee Member); David Goldstein (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Molecular Biology; Physiology