The studies presented in this thesis focus on the synthesis of air-stable solid supported lipid bilayers by anhydrobiotic mechanisms. Supported lipid bilayers (SLBs) serve as platforms that mimic cellular membrane surfaces in appearance and behavior. One of the most attractive aspects of the SLB is that it exhibits two-dimensional fluidity that allows for individual components to rearrange as they would in actual cellular membranes. The one thing that would allow the SLB to become an ideal biosensor is the ability to remain stable in the absence of bulk water. As it stands now, unprotected SLBs are unstable in the presence of air causing the membrane to rearrange and delaminate from the surface. Several biological organisms utilize the process of anhydrobiosis to persevere in severe dehydrated states. Anhydrobiosis occurs when organisms employ large amounts of sugars, particularly disaccharides, to protect their cell membranes. The sugars, often released as a stress response, protect the membrane by replacing the water around the lipid headgroups while also interacting with other sugars to form a glass atop the bilayer. One of the most successful anhydrobiotic sugars has been trehalose, although other sugars have been evaluated and are capable of protecting lipid bilayers minimally. The experimental section of this thesis involves the creation of SLBs that are examined with and without the presence of sugar molecules. Essentially, the SLB was created, exposed to sugar solutions, dried, and subsequently rehydrated. Successful experiments occurred when rehydrated bilayers exhibited little damage and were mobile and functional. In addition to trehalose, several other mono- and disaccharides were used as were glycolipids, lipids with sugar headgroups. Upon the completion of all experiments it was clear that trehalose afforded the most protection of all species tested and that glycolipids do not sufficiently protect the membrane during rehydration. Therefore, the addition of a sugar such as trehalose to an SLB could allow for the creation of an air-stable biosensor that would be both practical and require little maintenance.

Filaments of the desiccation-tolerant cyanobacterium Nostoc commune are embedded within, and distributed throughout, a dense glycan sheath. Analysis of the glycan of field materials and of pure cultures of N. commune DRH1 through light and electron microscopy, immunogold-labelling and staining with dyes, revealed changes in the pattern of differentiation in glycan micro-structure, as well as localized shifts in pH, upon rehydration of desiccated field material. A Ca/Si rich external (pellicular) layer of the glycan acts as a physical barrier on the surface of N. commune colonies. A purified fraction (> 12 kDa) of an aqueous extract of the glycan from desiccated field material contained glucose, N -acetylglucosamine, glucosamine, mannose and galactosamine with ratios of 3.1 : 1.4 : 1 : 0.1 : 0.06, respectively. Ethanol extracts of N. commune contained trehalose and sucrose and the levels of both became undetectable following cell rehydration. Elemental analysis of glycan extracts showed a flux in the concentrations of salts in the glycan matrix following rehydration of desiccated colonies. Intracellular cyanobacterial trehalase was identified using immunoblotting and its synthesis was detected upon rehydration of desiccated field cultures. Water-stress proteins (Wsp; molecular masses of 33, 37, and 39 kDa are the most abundant proteins in glycan), a water soluble UV-AlB-absorbing pigment, the lipid-soluble UV-protective pigment scytonernim, as well as two unidentified cyanobacterial glycoproteins (75 kDa and 110 kDa), were found within the glycan matrix. No evidence was found for either glycosylation, phosphorylation or acylation of Wsp polypeptides. NH2-terminal sequence analysis of the three proteins of Wsp were identical: Ala-Leu-Tyr-Gly-Tyr-Thr-Ile-Gly-Glu-Gln-X-Ile-Gln- Asn-Pro-Ser-Asn-Pro-Ser-Asn-Gly-Lys-Gln. An unidentified 68-kDa protein, the second most abundant protein in aqueous extracts of the glycan, was isolated and its N-terminal sequenced was determined: Ala-Phe-lle-Phe-Gly-Thr-Ile-Ser-Pro-Asn-Asn-Leu-Ser-Gly- Thr-Ser-Gly-Asn-Ser-Gly-Ile-Val-Gly-Ser-Ala. Gene bank searches with these sequences, and an internal sequence ofWsp (Glu-Ala-Arg-Val-Thr-Gly-Pro-Thr-Thr-Pro-Ile-Asp), identified homologies with various carbohydrate-modifying enzymes. Purified Wsp polypeptides associate with 1,4-β-D-xylanxylanohydrolase activity that was inhibited specifically by Wsp antiserum. In the absence of salt, Wsp polypeptides, and the water-soluble UV -A/B-absorbing pigments, form multimeric complexes through strong ionic interactions. The role of the glycan, and the protein and pigments that reside within it, in the desiccation tolerance of N. commune is discussed with respect to structure/function relationships. / Ph. D.

The distribution of tardigrades within the layers of the cushion moss Grimmia alpicola Hedwig, 1801 was investigated. The aim of this study was to determine the tardigrade species present within the moss layers during both wet and dry periods and to determine if migration occured in response to changing moisture conditions. Samples of the moss were removed from concrete caps on brick fence posts before and after rainfall and separated into two sections (top and bottom). The tardigrades from each layer and moisture condition were identified to species. Data for each species were statistically analyzed with a two-way analysis of variance (ANOVA) to compare the numbers of individuals present in the top and bottom layers of the moss under both wet and dry conditions. Five tardigrade species were identified, including two species new to science: Macrobiotus sp. n.: Milnesium tardigradum Doyère, 1840: Echiniscus viridissimus Peterfi, 1956; Echiniscus perviridis perviridis Ramazzotti, 1959; and Echiniscus sp. n. The new species will be described in a forthcoming paper. No significant differences were found in the numbers of the individuals of four of the five species in each layer within the moss or for each moisture condition. Only one species, E. viridissimus, was significantly more frequent in the top layer of the moss, regardless of moisture condition. Migration within the moss cushion was not detected in any of these five species as a result of changes in moisture conditions. In xeric moss species, it may not be beneficial for tardigrades to migrate to avoid desiccation. Instead, they apparently undergo anhydrobiosis in both the top and bottom layers of the moss cushion.

This work provides a comparative and genetic analysis of the responses to water stress in desiccation-tolerant and desiccation-sensitive nematodes. Caenorhabditis elegans, a model organism for the study of development, aging, and cell biology was shown to be a desiccation-sensitive organism that survives relative humidities above 40\% for periods of up to seven days. Transcripts from the desiccation-tolerant species Panagrolaimus rigidus AF36 and sp. PS1579, which were expressed uniquely during separate desiccation and osmotic stresses, as well as during recovery from exposure to the dual stresses, were cloned. These sequences were used to search for similarities in the genome sequence data of C. elegans. Putative anhydrobiotic-related transcripts were identified that potentially encode heat shock protein 70, late embryogenic abundant protein, and trehalose-phosphate synthase. Other putative genes that were identified within eight separate libraries encode proteins involved in transcription (histones), protein biosynthesis (ribosomal proteins, elongation factors), protein degradation (ubiquitin, proteases), and transport and cell structure (actin, collagen). Gene ontology analysis of the cloned transcripts revealed that developmental processes are activated during exposure to the stresses as well as during recovery, which may suggest a "rejuvenation" process as a key to survival in Panagrolaimus nematodes. Genes that were up-regulated during desiccation stress in C. elegans were classified as belonging either to an early response (until 12 hours of stress), or to a late response (after 12 hours of stress). The early response was characterized by the up-regulation of a large number of genes encoding mono-oxygenases, which may suggest onset of oxidation stress during desiccation of C. elegans. The late response was characterized by the appearance of transcripts encoding proteins of the immune system, heat shock proteins (protein denaturation), and superoxide dismutases (oxidation damage). Genes in C. elegans that were down-regulated in response to desiccation stress include those encoding proteases and lysozymes (metabolic shutdown). Genes that encode channel proteins (water homeostasis) were found among the transcripts up-regulated during recovery of C. elegans. The up-regulation of gpdh-1 and hmit-1.1, two transcripts linked to hyperosmotic stress, suggest that osmotic stress is experienced by C. elegans. Comparison of these data with those obtained from exposure of C. elegans to a range of other stresses showing that the nematode C. elegans uses specific transcripts for the desiccation response; transcripts that are not induced in other stresses such as heat, anoxia or starvation. In addition, transcripts regulated during desiccation stress of C. elegans were also regulated during dauer formation, which may indicate common stress tolerant mechanisms. Recent studies in mammalian cells and C. elegans have shown that microRNAs are able to degrade and to sequester mRNA especially during stress in so called stress bodies. In this study, C. elegans microRNA knock-outs showed a significant decrease in desiccation stress survival compared to wild type C. elegans which may suggest the importance of microRNAs for stress survival in C. elegans and other organisms. / Ph. D.

The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis – life without water. This study examines the function of group 1 Late Embryogenesis Abundant (LEA) proteins, hydrophilic unstructured proteins which accumulate in the stress-tolerant cysts of A. franciscana. Group 1 LEA proteins were knocked down in cysts using RNA interference. Cysts without group 1 LEA proteins exhibited low survival following desiccation and/or freezing, suggesting a role for these proteins in tolerance of low water conditions. In contrast, cysts with or without group 1 LEA proteins responded similarly to hydrogen peroxide exposure , indicating little to no function in reducing damage due to oxidative stress. This is the first in vivo functional study of group 1 LEA proteins in an animal, and may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.

To persist in extreme environments, some meiofaunal taxa have adopted outstanding resistance strategies. Recent years have seen increased enthusiasm for understanding extreme-resistance mechanisms evolved by tardigrades, nematodes and rotifers, such as the capability to tolerate complete desiccation and freezing by entering a state of reversible suspension of metabolism called anhydrobiosis and cryobiosis, respectively. In contrast, the less common phenomenon of diapause, which includes encystment and cyclomorphosis, is defined by a suspension of growth and development with a reduction in metabolic activity induced by stressful environmental conditions. Because of their unique resistance, tardigrades and rotifers have been proposed as model organisms in the fields of exobiology and space research. They are also increasingly considered in medical research with the hope that their resistance mechanisms could be used to improve the tolerance of human cells to extreme stress. This review will analyse the dormancy strategies in tardigrades, rotifers and nematodes with emphasis on mechanisms of extreme stress tolerance to identify convergent and unique strategies occurring in these distinct groups. We also examine the ecological and evolutionary consequences of extreme tolerance by summarizing recent advances in this field.

O nematoide de vida livre Panagrolaimus superbus é uma espécie anidrobiótica, ou seja, possui a capacidade de sobreviver ao estresse hídrico extremo adentrando no estado de anidrobiose. Durante tal estado adquiri tolerância a extremos de temperatura (~0 K a +151 °C), pressões hidrostáticas (1,2 GPa) e radiação ionizante. Entretanto, não se sabe qual a tolerância a água deuterada, molécula que possui dois átomos de deutério (isótopo estável e natural do hidrogênio) ao invés do hidrogênio, que em altas concentrações afeta negativamente sistemas biológicos. Além disso, uma vez que o processo de anidrobiose depende do movimento de moléculas de água pela membrana plasmática da célula, não se sabe se os canais responsáveis por este transporte, as aquaporinas, de espécies anidrobióticas possuem alguma particularidade na sua estrutura. Em vista disso, o objetivo deste trabalho visa investigar se em concentrações de 10%, 40% e 99,9% D2O, tanto de modo crônico quanto agudo, P. superbus tem sua viabilidade, crescimento populacional e desenvolvimento alterados. Além disso, a comparação in silico da sequência de aminoácidos (estrutura primária) entre aquaporinas de espécies anidrobióticas e não anidrobióticas, com ênfase nas sequências genéticas de P. superbus foi feita. Os resultados encontrados demonstram a alta tolerância de P. superbus a concentrações relativamente elevadas de D2O, não tendo sua viabilidade e desenvolvimento alterados em nenhum cenário, mesmo com uma troca total, homogênea e instantânea de sua matriz aquosa. Efeitos negativos foram encontrados apenas no crescimento populacional após exposição 10%, 40% e 99,9% D2O, contudo não o inviabilizaram. Ademais, não foram encontradas grandes diferenças entre as sequências primárias de aquaporinas de anidrobiotos e não anidrobiotos, sugerindo que estes canais de água não divergem em estrutura terciária entre tais grupos. Dos dois ESTs encontrados em P. superbus (números de acesso no NCBI: GW411914.1 e GW408200.1) o primeiro deles é o provável representante do gene da aquaporina na espécie, enquanto que o segundo aparenta ser um transcrito não codificante de proteínas. / The free-living nematode Panagrolaimus superbus is an anhydrobiotic species, it means that this species has the capacity to survive extreme water stress entering into the state of anhydrobiosis. During such a state, it acquires tolerance to extremes of temperature (~ 0 K to +151 °C), hydrostatic pressures (1.2 GPa) and ionizing radiation. However, the tolerance to deuterium oxide is poorly investigated. This molecule has two atoms of deuterium (natural and stable isotope of hydrogen) rather than hydrogen and in high concentrations negatively affects biological systems. Furthermore, since the process of anhydrobiosis depends on the movement of water molecules across the cell membrane, it is unclear whether the channels responsible for this transport, aquaporins, of anhydrobiotic species have some particularity in their structures. In view of this, the work aims to investigate whether P. superbus has its viability, population growth and development altered at concentrations of 10%, 40% and 99.9% D2O in chronic and acute expositions. In addition, the in silico analyses of amino acid sequence (primary structure) of aquaporins between anhydrobiotic and non-anhydrobiotic species, with emphasis at the P. superbus genetic sequences, were performed. The results demonstrated the high tolerance of P. superbus at high concentrations of D2O, their viability and development did not change in any scenario, even with a total, homogeneous and instantaneous exchange of their aqueous milieu. Negative effects were found only on population growth after exposure to 10%, 40% and 99.9% D2O, although not hindering the procedure. Furthermore, no significant differences were found between the primary aquaporin sequences of anhydrobiotic and non-anhydrobiotic species, suggesting that these water channels do not differ in tertiary structure between such groups. Two ESTs found in P. superbus, (NCBI access numbers: GW411914.1 and GW408200.1): the first likely corresponds to the aquaporin gene in the species, while the second appears to be a noncoding transcript.

Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic, suspended animation state. Distributed to various taxa of life, these organisms have evolved strategies to efficiently protect their cell membranes and proteins against extreme water loss. At the molecular level, a variety of mutually non-exclusive mechanisms have been proposed to account particularly for preserving the integrity of the cell membranes in the desiccated state. Recently, it has been shown that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation for harsh desiccation (preconditioning), thereby allowing for a reversible desiccation / rehydration cycle. Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, the dauer larvae exhibit a systemic chemical response upon preconditioning by dramatically reducing their phosphatidylcholine (PC) content. The C. elegans strain daf-2 was chosen for these studies, because it forms a constitutive dauer state under appropriate growth conditions. Using complementary approaches such as chemical analysis, time-resolved FTIR-spectroscopy, Langmuir-Blodgett monolayers, and fluorescence spectroscopy, it is shown that this chemical adaptation of the phospholipid (PL) composition has key consequences for their interaction with trehalose. Infrared-spectroscopic experiments were designed and automated to particularly address structural changes during fast hydration transients. Importantly, the coupling of headgroup hydration to acyl chain order at low humidity was found to be altered on the environmentally relevant time scale of seconds. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates during lyotropic transitions and sub- headgroup H-bond interactions as compared to PLs from non-preconditioned larvae. The effects are related to the intrinsically different hydration properties of PC and phosphatidylethanolamine (PE) headgroups, and lead to a larger hydration-dependent rearrangement of trehalose-mediated H-bond network in PLs from preconditioned larvae. This results in a lipid compressibility modulus of ∼0.5 mN/m and 1.2 mN/m for PLs derived from preconditioned and non-preconditioned larvae, respectively. The ensemble of these changes evidences a genetically controlled chemical tuning of the native lipid composition of a true anhydrobiote to functionally interact with a ubiquitous protective disaccharide. The biological relevance of this adaptation is the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose- containing cells during fast rehydration. Finally, the thermo-tropic lipid phase behavior was studied by temperature-dependent ATR-FTIR and fluorescence spectroscopy of LAURDAN-labeled PLs. The results show that the adaptation to drought, which is accomplished to a significant part by the reduction of the PC content, relies on reducing thermo-tropic and enhancing lyotropic phase transitions. The data are interpreted on a molecular level emphasizing the influence of trehalose on the lipid phase transition under biologically relevant conditions by a detailed analysis of the lipid C=O H-bond environment. The salient feature of the deduced model is a dynamic interaction of trehalose at the PL headgroup region. It is proposed here that the location of trehalose is changed from a more peripheral to a more sub-headgroup-associated position. This appears to be particularly pronounced in PLs from preconditioned worms. The sugar slides deeper into the inter-headgroup space during hydration and thereby supports a quick lateral expansion such that membranes can more readily adapt to the volume changes in the swelling biological material at reduced humidity. The data show that the nature of the headgroup is crucial for its interaction with trehalose and there is no general mechanism by which the sugar affects lipidic phase transitions. The intercalation into a phosphatidylethanolamine-rich membrane appears to be unique. In this case, neither the phase transition temperature nor its width is affected by the protective sugar, whereas strong effects on these parameters were observed with other model lipids. With respect to membrane preservation, desiccation tolerance may be largely dependent on reducing phosphatidylcholine and increasing the phsophatidylethanolamine content in order to optimize trehalose headgroup interactions. As a consequence, fast mechanical adaptation of cell membranes to hydration-induced strain can be realized.

Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic, suspended animation state. Distributed to various taxa of life, these organisms have evolved strategies to efficiently protect their cell membranes and proteins against extreme water loss. At the molecular level, a variety of mutually non-exclusive mechanisms have been proposed to account particularly for preserving the integrity of the cell membranes in the desiccated state. Recently, it has been shown that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation for harsh desiccation (preconditioning), thereby allowing for a reversible desiccation / rehydration cycle. Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, the dauer larvae exhibit a systemic chemical response upon preconditioning by dramatically reducing their phosphatidylcholine (PC) content. The C. elegans strain daf-2 was chosen for these studies, because it forms a constitutive dauer state under appropriate growth conditions. Using complementary approaches such as chemical analysis, time-resolved FTIR-spectroscopy, Langmuir-Blodgett monolayers, and fluorescence spectroscopy, it is shown that this chemical adaptation of the phospholipid (PL) composition has key consequences for their interaction with trehalose. Infrared-spectroscopic experiments were designed and automated to particularly address structural changes during fast hydration transients. Importantly, the coupling of headgroup hydration to acyl chain order at low humidity was found to be altered on the environmentally relevant time scale of seconds. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates during lyotropic transitions and sub- headgroup H-bond interactions as compared to PLs from non-preconditioned larvae. The effects are related to the intrinsically different hydration properties of PC and phosphatidylethanolamine (PE) headgroups, and lead to a larger hydration-dependent rearrangement of trehalose-mediated H-bond network in PLs from preconditioned larvae. This results in a lipid compressibility modulus of ∼0.5 mN/m and 1.2 mN/m for PLs derived from preconditioned and non-preconditioned larvae, respectively. The ensemble of these changes evidences a genetically controlled chemical tuning of the native lipid composition of a true anhydrobiote to functionally interact with a ubiquitous protective disaccharide. The biological relevance of this adaptation is the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose- containing cells during fast rehydration. Finally, the thermo-tropic lipid phase behavior was studied by temperature-dependent ATR-FTIR and fluorescence spectroscopy of LAURDAN-labeled PLs. The results show that the adaptation to drought, which is accomplished to a significant part by the reduction of the PC content, relies on reducing thermo-tropic and enhancing lyotropic phase transitions. The data are interpreted on a molecular level emphasizing the influence of trehalose on the lipid phase transition under biologically relevant conditions by a detailed analysis of the lipid C=O H-bond environment. The salient feature of the deduced model is a dynamic interaction of trehalose at the PL headgroup region. It is proposed here that the location of trehalose is changed from a more peripheral to a more sub-headgroup-associated position. This appears to be particularly pronounced in PLs from preconditioned worms. The sugar slides deeper into the inter-headgroup space during hydration and thereby supports a quick lateral expansion such that membranes can more readily adapt to the volume changes in the swelling biological material at reduced humidity. The data show that the nature of the headgroup is crucial for its interaction with trehalose and there is no general mechanism by which the sugar affects lipidic phase transitions. The intercalation into a phosphatidylethanolamine-rich membrane appears to be unique. In this case, neither the phase transition temperature nor its width is affected by the protective sugar, whereas strong effects on these parameters were observed with other model lipids. With respect to membrane preservation, desiccation tolerance may be largely dependent on reducing phosphatidylcholine and increasing the phsophatidylethanolamine content in order to optimize trehalose headgroup interactions. As a consequence, fast mechanical adaptation of cell membranes to hydration-induced strain can be realized.

Background. The phylum Tardigrada consists of over 1,300 species that inhabit terrestrial, freshwater and marine environments throughout the world. In terrestrial habitats they live primarily in mosses, lichens, leaf litter and soil, whereas tardigrades in freshwater and marine environments are mainly found in sediments and on aquatic plants. More than 65 species have been previously reported in the state of Tennessee, USA. Methods. Tardigrades present in moss cushions (Grimmia sp.) collected from a xerothermic habitat on the East Tennessee State University campus, Johnson City, TN, USA, were extracted, mounted on slides, identified, and counted. Additional samples of fresh dried moss were used for integrative analyses, including morphological analysis with phase contrast (PCM) and scanning electron microscopy (SEM), as well as molecular analyses of COI, 18S rRNA, 28S rRNA, and ITS-2 of the Macrobiotus and Milnesium species. Results. Five species were found, including two species new to science: Viridiscus miraviridis sp. nov. and Macrobiotus basiatus sp. nov. Viridiscus miraviridis sp. nov. differs from other members of the genus mainly by having a different type of dorsal cuticle and some other, more subtle, morphometric characters. In addition to the two new species, Viridiscus perviridis and Viridiscus viridissimus were present, and males of Vir. viridissimus were found for the first time, the first record of males in the genus Viridiscus. Macrobiotus basiatus sp. nov. is most similar to Macrobiotus nelsonae, but it differs from Mac. nelsonae mainly by the stylet supports being situated in a more anterior position, shorter and narrower egg processes, and a smaller number of areoles around the egg processes. Moreover, the identification of Milnesium inceptum was confirmed as the first record for the USA by analysis of COI.