Poly- and perfluoroalkyl substances (PFAS) are ubiquitous contaminants across the globe, can bioaccumulate in aquatic taxa, and potentially biomagnify in food webs. Consequently, research examining the influence of PFAS on wildlife is warranted. Amphibians are sensitive to contaminants such as PFAS because of their porous skin and associations with aquatic habitats where contaminants accumulate. Because PFAS tend to bioaccumulate and can adversely affect the endocrine system, there is a need to examine uptake rates to inform ecotoxicology studies, as well as a need to examine sublethal effects. To address these knowledge gaps I conducted two experiments. First, I exposed larval northern leopard frogs (<i>Rana pipiens</i>), American toads (<i>Anaxyrus americanus</i>), and eastern tiger salamanders (<i>Ambystoma tigrinum</i>) to PFAS chemicals perfluorooctanoic acid (PFOA) or perfluorooctane sulfonate (PFOS) at concentrations of 10 or 1000 ppb for 10 days and sampled them every 48 hours during the exposure period. In the next experiment, I examined the effects of PFAS exposure via contaminated substrate on the survival and growth of post metamorphic amphibians of the same species. I found that, for all species, body burdens often reached steady state within 48 to 96 h of exposure. Steady-state body burdens of PFOA ranged from 3,819–16,481 ng/g dry weight among treatments and species (corresponding BCFs of 0.5 to 2.5), while PFOS body burdens ranged from 6,955–489,958 ng/g dry weight (corresponding BCFs of 47–259) among treatments and species. These data suggest that steady state is rapidly reached in larval amphibians exposed to PFAS, particularly regarding PFOS. This reflects a high potential for trophic transfer of PFAS within food webs because amphibians are often low in trophic position and are important prey for many aquatic and terrestrial species. In post-metamorphic amphibians, there was no influence of PFAS on survival or mass. However, significant effects on snout-vent length were observed in all species, and body condition differences were observed for two of my species. I found that all leopard frogs increased in scaled mass index (SMI) when exposed to a PFAS treatment, indicating an increased body condition. Toads exhibited a more variable SMI pattern across treatments, with no outstanding trends, and tiger salamanders did not differ significantly across treatments. These data suggest that sublethal effects vary greatly depending on the species, possibly due to life history traits. Future research examining biomagnification potential is warranted to determine the influence of PFAS on food webs. Additionally, there is a need to determine the physiological mechanisms underlying the observed effects of PFAS exposure.<br>

<p>Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. </p><p>The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development.</p><p>In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease.</p><p>In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. </p><p>In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.</p>

Unlike in the greenhouse, plants need to cope with many environmental stresses under natural conditions. Among these conditions are drought, waterlogging, excessive or too little light, high or low temperatures, UV irradiation, high soil salinity, and nutrient deficiency. These stress factors can affect many biological processes, and severely retard the growth and development of higher plants, resulting in massive losses of crop yield and wood production. Plants have developed many protective mechanisms to survive and acclimate to stresses, such as the rapid induction of specific molecular chaperones and proteases at the molecular level. Molecular chaperones mediate the correct folding and assembly of polypeptides, as well as repair damaged protein structures caused by stress, while proteases remove otherwise non-functional and potentially cytotoxic proteins. The Clp/Hsp100 family is a new group of chaperones that consists of both constitutive and stress-inducible members. Besides being important chaperones, many Clp/Hsp100 also participate in protein degradation by associating with the proteolytic subunit ClpP to form the Clp protease complex. Higher plants have the greatest number and complexity of Clp proteins than any other group of organisms, and more than 20 different Clp isomers in plants have been identified (Paper I). Because of this diversity, we have adopted a functional genomics approach to characterise all Clp proteins in the model plant Arabidopsis thaliana. Our ongoing research strategy combines genetic, biochemical and molecular approaches. Central to these has been the preparation of transgenic lines for each of the chloroplast Clp isomers. These transgenic lines will be analysed to understand the function and regulation of each chloroplast Clp protein for plant growth and development. In Paper II, an Arabidopsis thaliana cDNA was isolated that encodes a homologue of bacterial ClpX. Specific polyclonal antibodies were made and used to localise the ClpX homologue to plant mitochondria, consistent with that predicted by computer analysis of the putative transit peptide. In addition to ClpX, a nuclear-encoded ClpP protein, termed ClpP2, was identified from the numerous ClpP isomers in Arabidopsis and was also located in mitochondria. Relatively unchanged levels of transcripts for both clpX and clpP2 genes were detected in various tissues and under different growth conditions. Using β-casein as a substrate, plant mitochondria possessed an ATP-stimulated, serine-type proteolytic activity that could be strongly inhibited by antibodies specific for ClpX or ClpP2, suggesting an active ClpXP protease. In Paper III, four nuclear-encoded Clp isomers were identified in Arabidopsis thaliana: ClpC1 and ClpP3-5. All four proteins are localized within the stroma of chloroplasts, along with the previously identified ClpD, ClpP1 and ClpP6 proteins. Potential differential regulation among these Clp proteins was analysed at both the mRNA and protein level. A comparison between different tissues showed increasing amounts of all plastid Clp proteins from roots to stems to leaves. The increases in protein were mirrored at the mRNA level for most ClpP isomers but not for ClpC1, ClpC2 and ClpD and ClpP5, which exhibited little change in transcript levels. Potential stress induction was also tested for all chloroplast Clp proteins by a series of brief and prolonged stress conditions. The results reveal that these proteins, rather than being rapidly induced stress proteins, are primarily constitutive proteins that may also be involved in plant acclimation to different physiological conditions. In Paper IV, antisense repression transgenic lines of clpP4 were prepared and then later characterised. Within the various lines screened, up to 90% of ClpP4 protein content was specifically repressed, which also led to the down-regulation of ClpP3 and ClpP5 protein contents. The repression of clpP4 mRNA retarded the development of chloroplasts and the differentiation of leaf mesophyll cells, resulting in chlorotic phenotypes. The chlorosis was more severe in young than in mature leaves due likely to the developmental expression pattern of the ClpP4 protein. Chlorotic plants eventually turned green upon aging, accompanied by a recovery in the amount of the ClpP4 protein. The greening process could be affected by the light quantity, either by altering the photoperiod or light intensity.

Establishing plant communities is essential for the restoration of contaminated land. As potential cover materials, fly ash and sewage sludge can prevent formation of acid mine drainage from sulfidic mine waste. The aim of the thesis was to i) screen for plants that can be established in, and prevent leakage of metals and nutrients from sludge on top of ash and tailings, and ii) investigate root growth into sealing layers of ash and sludge. Analyses were performed under laboratory, greenhouse and field conditions using selected plant species to examine the release of Cd, Cu, Zn, N, and P from the materials. Plant physiological responses and interactions with fly ash were also investigated. The data show that plants can decrease metal and nutrient leakage from the materials, and lower the elemental levels in the leachate, but with varying efficiencies among plant species. Plants capable of taking up both nitrate and ammonium were more efficient in preventing N leakage compared with those taking up primarily ammonium. Fast growing plants could raise the pH in acidic sludge leachate, but the initial pH decrease and N leakage was not counteracted by plants. Germination in fresh sludge was problematic, but enhanced by aeration of the sludge. In general, the accumulation of metals in plant shoots was low, especially if ash was located below the sludge. Fresh ash was phytotoxic (e.g., high alkalinity, salinity and metal levels) and induced the activity of stress-related enzymes in shoots. In sealing layers of aged and cured ash, roots could grow if the penetration resistance was low, or into the surface of stronger layers if the surface had become pulverized. The roots caused dissolution of calcium-rich minerals, possibly by exudation of saccharides. Addition of sludge to an ash layer increased root growth, likely due to decreased bulk density and pH, and nutrient addition. In conclusion, with selected plant species and a properly constructed cover, metal and nutrient leaching from the materials and root growth into the sealing layer can be restricted.

The Hsp70 family of molecular chaperones is essential for protein folding, re-folding misfolded client proteins, clearance of aberrant client proteins, and can also inhibit programmed cell death. There are two major cytosolic members of this family: the constitutive Hsc70, and the inducible Hsp72. Under stress conditions the Hsp70 family protects the cell from protein related damage by the induction of Hsp72. Hsc70 and Hsp72 are highly homologous with minor differences in substrate binding. In cancers, Hsp72 is commonly induced and this induction is thought to aid in cancer cell survival. In these studies we demonstrate the differential regulation of the prosurvival kinase Akt by Hsc70 and Hsp72. We demonstrate that of the two cytosolic forms, Hsp72 is the primary Akt regulator. Using a phenothiazine class inhibitor of Hsp70-family activity, methylene blue, we demonstrate dose dependent decreases in the levels of Akt; produced breast cancer specific cell death. This cell death could be rescued by the use of an Hsp70 family ATPase stimulating compound, SW02. We also demonstrate a similar phenotype with a rhodacyanine class Hsp70 family inhibitor, YM-1, also capable of reducing Akt and causing cancer specific cytotoxicity. The resulting Akt decreases were sufficient to block a tamoxifen-resistance pathway, allowing previously resistant cells to regain sensitivity to tamoxifen. These results demonstrate the capabilities of Hsp70 family inhibitors as potent compounds for the treatment of breast cancer.

Norway spruce (Picea abies) responds to stress by biosynthesis of chemical substances, which can deter invading insects or pathogens. Some of these substances are volatile and can be emitted to the surroundings while others are accumulated within the tree. Information about the susceptibility of individual plants to infestation, their volatile emissions and chemical defence is of interest, for example, in selecting plants for tree breeding programs. The first part of this research focused on volatiles emitted by Norway spruce plants. Collection of headspace volatiles by SPME and subsequent separation and identification with GC-MS was used to investigate Norway spruce plants of different ages and stress conditions as well as trapping semiochemicals like nepetalactone emitted by the spruce shoot aphids. It was even possible to analyse the emission of single needles in vivo and obtain spatial localisation of the stress reaction to methyl jasmonate or spruce spinning mites. Seedlings of different ages showed differences in chemical composition of emitted volatiles, with the pine weevil repellent, (4S)-(-)-limonene, one of the main compounds. Wounded phloem of conventional plants emitted high amounts of monoterpenes while the phloem of mini plants emitted (3Z)-hexenal and (3Z)-hexen-1-ol. In addition, a method to separate and identify the four diastereomers of nepetalactone by GC-MS and characteristic m/z-fragments was accomplished. The second part of the research deals with the chemical response of Norway spruce roots to inoculation with Heterobasidion annosum. Terpene concentrations increased after inoculation or wounding but the composition was mainly associated with clone identity and not to susceptibility or treatment. In contrast, inoculation with H. annosum induced a treatment-specific alteration of phenol composition. The constitutive phenol composition differed between more and less susceptible clones. The phenols astringin and astringin dimers (piceasides) as well as the terpene α-longipinene may be suitable markers of low susceptibility for P. abies to Heterobasidion. / QC 20110314

Terpenes and phenols are of importance in conifer defense against insects and pathogens. Knowledge about tree chemical defense is vital for developing practical methods to maintain healthy forests. With the aims of characterizing the defensive chemical induction in Norway spruce Picea abies and demonstrating its ecological function to spruce bark beetle Ips typographus, we measured the terpenoid and phenolic content in the bark of mature Norway spruce trees suffering windstorm, inoculated with Ceratocystis polonica, or treated with methyl jasmonate (MeJA), and investigated the colonization and pheromone emission of I. typographus. All three stressors altered the chemical profile in the bark of Norway spruce. Trees damaged by windstorm had lower proportions of (+)-3-carene and two unidentified stilbenes, and a higher taxifolin glycoside content than trees without apparent windstorm damage; C. polonica inoculation induced extremely strong quantitative terpene accumulation in the wound reaction zone, but only increased the levels of (+)-3-carene, sabinene and terpinolene in the bark near the reaction zone; MeJA treatment generally elicited quantitative terpene accumulation, but the induction differed extensively between individual trees. In addition, logs from MeJA-treated tree showed much stronger wounding response compared to control logs. The chemical profile of Norway spruce affected the colonization and pheromone emission of I. typographus. In response to fungal inoculation, terpene present in the reaction zone inhibited the colonization of I. typographus in a dose-dependent manner. Host defense elicited by MeJA treatment reduced emissions of 2-methyl-3-buten-2-ol and (S)-cis-verbenol, the two key aggregation pheromone components of I. typographus, and altered the ratio between the two components. / QC 20110503

Genetic material of Lithuanian origin varieties, breeding lines and wild ecotypes representing P. pratense, P. bertolonii and P. phleoides species were studied for the most important agro-morphological and feeding value indicators as well as at the genetic level employing different biochemical–molecular markers. The water stress experiment clearly demonstrated the existence of different levels of water stress response in Phleum species, suggesting that P. phleoides might have evolved under conditions of limited water availability. “Blind” mapping by HRM was used successfully to map water stress response genes of timothy in the perennial ryegrass mapping population. In total, 12 putative water stress response genes were mapped in seven perennial ryegrass linkage groups. / Pasitelkus agromorfologinius, kokybės ir biocheminius – molekulinius metodus ištirtos lietuviškos kilmės pašarinių, žemaūgių bei stepinių motiejukų veislės, selekcinės linijos bei laukiniai ekotipai. Fiziologiniai sausros atsparumo tyrimų rezultatai įrodė, kad stepiniai motiejukai turi geriau išvystytą atsparumo sausrai mechanizmą. Pirmą kartą motiejukuose aptikti ekspresuojami atsparumo sausrai kandidatiniai genai HRM metodu sužymėti daugiametės svidrės genolapyje.

The doctoral dissertation analyses and compares general endotracheal and combined endotracheal epidural anesthesia’s impact on organ systems and describes the systems parameters in laparoscopic colorectal surgery. Comparing two perioperative analgesia techniques used in laparoscopic colorectal surgery the hemodynamic and respiratory parameters trends; the impact of anesthesia and postoperative analgesia methods on patients’ tracheal extubation time, intestinal motility recovery rate, duration of hospitalization and inflammatory response have been determined. Laparoscopic colorectal resection, even being a minimally invasive technique for laparoscopic surgery, stimulates the body's response to stress and pro-inflammatory mediator’s secretion. Perioperative pain management may also influence the immune response. The doctoral dissertation analyses the impact of epidural analgesia method on the body stress response, investigating variations of cortisol and interleukin-6 levels. The results showed that analgesia and patient satisfaction using epidural analgesia method for perioperative pain management were better. Tracheal extubation time was significantly shorter. Recovery of intestinal motility using epidural analgesia was significant and much prior than using intravenous analgesia. The use of epidural analgesia in laparoscopic colorectal surgery caused less stress response – less cortisol levels increase. It has not showed the increase in number of complications. / Disertacijoje analizuojama ir lyginama bendrosios endotrachėjinės ir kombinuotos endotrachėjinės epiduralinės anestezijos įtaka atskiroms organų sistemoms ir tas sistemas apibūdinantiems rodikliams laparoskopinių kolorektalinių operacijų metu. Darbe nagrinėjama dviejų perioperacinių skausmo malšinimo būdų įtaka hemodinamikos ir kvėpavimo sistemos parametrų kitimo tendencijoms, pacientų trachėjos ekstubacijos laikui, žarnyno motorikos atsinaujinimo greičiui, hospitalizacijos trukmei bei organizmo uždegiminiam atsakui. Laparoskopinės storosios žarnos rezekcinės operacijos, net ir būdamos minimaliai invazinės dėl laparoskopinės operacijos technikos, sužadina stresinį organizmo atsaką bei uždegimo mediatorių išskyrimą. Perioperacinis skausmo valdymas taip pat gali daryti įtaką imuniniam atsakui. Disertacijoje nagrinėjama epiduralinės analgezijos metodo įtaka organizmo stresiniam atsakui tiriant kortizolio kiekio kitimus ir interleukino-6, kaip vieno pagrindinių uždegimą skatinančių citokinų, koncentracijos kitimą taikant epiduralinę analgezijos metodiką. Gauti rezultatai parodė, kad analgezijai pasitelkiant epiduralinį skausmo malšinimo metodą, perioperacinis pacientų skausmo valdymas ir pasitenkinimas yra geresnis, trachėjos ekstubacijos laikas patikimai trumpesnis, žarnyno peristaltikos atsitaisymas ankstyvesnis, sukeliamas stresinis organizmo atsakas mažesnis (mažesnis kortizolio koncentracijos padidėjimas) ir nenustatyta komplikacijų padaugėjimo.

Disertacijoje analizuojama ir lyginama bendrosios endotrachėjinės ir kombinuotos endotrachėjinės epiduralinės anestezijos įtaka atskiroms organų sistemoms ir tas sistemas apibūdinantiems rodikliams laparoskopinių kolorektalinių operacijų metu. Darbe nagrinėjama dviejų perioperacinių skausmo malšinimo būdų įtaka hemodinamikos ir kvėpavimo sistemos parametrų kitimo tendencijoms, pacientų trachėjos ekstubacijos laikui, žarnyno motorikos atsinaujinimo greičiui, hospitalizacijos trukmei bei organizmo uždegiminiam atsakui. Laparoskopinės storosios žarnos rezekcinės operacijos, net ir būdamos minimaliai invazinės dėl laparoskopinės operacijos technikos, sužadina stresinį organizmo atsaką bei uždegimo mediatorių išskyrimą. Perioperacinis skausmo valdymas taip pat gali daryti įtaką imuniniam atsakui. Disertacijoje nagrinėjama epiduralinės analgezijos metodo įtaka organizmo stresiniam atsakui tiriant kortizolio kiekio kitimus ir interleukino-6, kaip vieno pagrindinių uždegimą skatinančių citokinų, koncentracijos kitimą taikant epiduralinę analgezijos metodiką. Gauti rezultatai parodė, kad analgezijai pasitelkiant epiduralinį skausmo malšinimo metodą, perioperacinis pacientų skausmo valdymas ir pasitenkinimas yra geresnis, trachėjos ekstubacijos laikas patikimai trumpesnis, žarnyno peristaltikos atsitaisymas ankstyvesnis, sukeliamas stresinis organizmo atsakas mažesnis (mažesnis kortizolio koncentracijos padidėjimas) ir nenustatyta komplikacijų padaugėjimo. / The doctoral dissertation analyses and compares general endotracheal and combined endotracheal epidural anesthesia’s impact on organ systems and describes the systems parameters in laparoscopic colorectal surgery. Comparing two perioperative analgesia techniques used in laparoscopic colorectal surgery the hemodynamic and respiratory parameters trends; the impact of anesthesia and postoperative analgesia methods on patients’ tracheal extubation time, intestinal motility recovery rate, duration of hospitalization and inflammatory response have been determined. Laparoscopic colorectal resection, even being a minimally invasive technique for laparoscopic surgery, stimulates the body's response to stress and pro-inflammatory mediator’s secretion. Perioperative pain management may also influence the immune response. The doctoral dissertation analyses the impact of epidural analgesia method on the body stress response, investigating variations of cortisol and interleukin-6 levels. The results showed that analgesia and patient satisfaction using epidural analgesia method for perioperative pain management were better. Tracheal extubation time was significantly shorter. Recovery of intestinal motility using epidural analgesia was significant and much prior than using intravenous analgesia. The use of epidural analgesia in laparoscopic colorectal surgery caused less stress response – less cortisol levels increase. It has not showed the increase in number of complications.