Moluscos têm sido empregados como bioindicadores em estudos de contaminação ambiental. Nesse contexto, o caramujo de água doce Biomphalaria glabrata tem sido avaliado como um bom modelo laboratorial, e estudos prévios apontaram sua aplicação na pesquisa ambiental. A proteína HSP70 é uma molécula de 70 kDa, pertencente a uma família de proteínas com papel na manutenção da homeostase dos seres vivos: as proteínas de choque térmico (HSPs); e vem sendo estudada como potencial biomarcador de dano ambiental, indicando estresse e protegendo os organismos dos danos às proteínas. Neste trabalho, foi caracterizada a proteína HSP70 de B. glabrata pelo Western blot, com o objetivo de seu emprego em aplicações ambientais futuras. Para isso, caramujos de 5-6 meses de idade, com diâmetro de concha de 14,4 (±1,7) mm, foram expostos ao calor e ao cloreto de cádmio (CdCl2) a fim de se verificar a resposta desta proteína frente a esses estresses. Os animais foram dissecados para investigação da indução da HSP70. As proteínas foram extraídas dos tecidos com tampão RIPA, separadas em eletroforese desnaturante em gel de poliacrilamida, transferidas para uma membrana de nitrocelulose e detectadas com anticorpo específico para HSP70. A CL50/96h foi determinada como sendo 0,34 (0,30-0,37) ppm para o CdCl2 e serviu de referência para os experimentos de indução da proteína. Foi observado que a exposição a temperaturas subletais aumentou a resistência dos caramujos à temperatura letal de 42 °C. Exposições prévias ao calor de 33 °C e ao CdCl2 a 0,22 ppm aumentou a sobrevivência dos caramujos B. glabrata à concentração letal de CdCl2 (0,7 ppm) e à temperatura letal (42 °C), respectivamente. Os achados do Western blot apontaram para um possível papel da HSP70 nesse processo. Os resultados mostraram relação entre a proteína HSP70 e o aumento na sobrevivência aos estímulos letais após prévia exposição a estresses moderados. O Western blot mostrou uma indução da HSP70 nos grupos pré-expostos, se comparados aos grupos controles. A glândula digestiva foi o tecido mais responsivo, no que concerne à indução da proteína HSP70, comparando com tecidos de cabeça/pé e ovoteste. Foi encontrado o pico de indução da HSP70 nos caramujos B. glabrata após 48 horas de exposição ao calor de 33 °C, e após 96 horas de exposição ao CdCl2 a 0,22 ppm. Apesar do bem conhecido papel da HSP70 na termotolerância e tolerância a outros agentes estressores nos organismos vivos, esta foi a primeira vez que isto foi demonstrado no B. glabrata, oferecendo subsídios para a sua aplicação em estudos de monitoramento ambiental. Os resultados apresentados aqui abrem o caminho para estudos futuros dessa proteína no molusco, e fornecem mais bases para o conhecimento do B. glabrata. / Molluscs have been employed as bioindicators in studies of environmental stress. In this way the freshwater snail Biomphalaria glabrata has been evaluated as a good laboratory model, and previous studies have pointed for its application in environmental research. The HSP70 protein is a molecule of 70 kDa from a family of proteins with role in maintaining homeostasis: the Heat Shock Proteins (HSPs), and it has been studied as a potential biomarker for environmental injury indicating stress and providing protection against the protein damage. In this work, the protein HSP70 was characterized in B. glabrata by Western blotting aiming its employment in future environmental applications. To this purpose, 5-6 months old snails, with shell diameter of 14,4 (±1,7) mm, were exposed to heat and to cadmium chloride (CdCl2) in order to verify the response of this protein to the stresses. Animals were dissected to investigate induction of HSP70. Proteins were extracted from tissues with RIPA buffer, fractionated in denaturing polyacrilamide gel electrophoresis, transferred to nitrocellulose membrane, and detected with a HSP70-specific antibody. The CL50/96h was determined as 0,34 (0,30-0,37) ppm for CdCl2 and served as reference in the experiments for protein induction. It was observed that exposure to sublethal temperatures improved the resistance of snails B. glabrata to the lethal temperature of 42 ºC. Previous sublethal exposure to heat at 33 °C and to CdCl2 at 0,22 ppm improved the survival of snails B. glabrata to a lethal concentration of CdCl2 (0,7 ppm) and to a lethal temperature (42 ºC), respectively. The findings of Western blot pointed to a possible role of HSP70 protein in this process. Results showed a correlation between HSP70 and the improvement of survival to lethal stimuli after a previous exposure to mild stresses. The Western blot showed an induction of HSP70 protein in the preexposed groups as compared to the control ones. The digestive gland was the most responsive tissue to stress regarding the HSP70 protein induction compared with heat/foot and ovotestis. An induction peak of HSP70 was found after 48 hours of exposure to heat at 33 °C, and after 96 hours of exposure to CdCl2 at 0,22 ppm. Despite of the well known role of HSP70 in thermotolerance and tolerance to other stress agents in living organisms, it was the first time it was shown in B. glabrata, supporting its application in environmental monitoring studies. The results presented here open a way to future studies of this protein in the mollusc, and provide more basements to knowledge of B. glabrata.

<p>Natural stress and disturbances are important factors affecting the structure and function of ecosystems. However the magnitude of stress has escalated due to anthropogenic activities. Environmental monitoring and toxicity assessments try to protect ecosystems from unwanted human alterations. The aim of this Doctoral thesis was to increase the understanding of the complex effects that environmental stress has on individuals and invertebrate populations. The low saline environment in the Baltic Sea is perceived as stressful for most organisms living there. In Paper I, it was found that Baltic blue mussels living in the less saline northern Baltic Proper (~5 psu) had lower basal metabolism and were more susceptible to toxic exposure than the mussels in the south (~7 psu). There was no genetic differentiation between the mussels from the northern and southern areas while there were genetic differences between mussels from sites within the respective areas (Paper III), indicating that there is not a simple relationship between the health of the mussels and genetic diversity in the microsatellite loci studied. In Paper IV it was found that the heat tolerance of the intertidal dogwhelk <i>Nucella lapillus</i> is oxygen dependent. Increased oxygen levels resulted in higher survival rate. Protein expression profiles also became more similar to those of the controls, compared to the whelks exposed to high temperature and normal oxygen levels. In Paper V and VI it was found that exposure to a single toxicant for more than one generation decreased the genetic diversity in exposed copepod populations even though abundances remained unaltered. In Paper VI, exposure to naturally contaminated sediments, which contained of a mixture of toxicants, did not decrease genetic diversity. However the genetic divergence (<i>F</i><i>ST</i>) within the treatments was very high, probably due to small effective population sizes in the replicates. Likewise in Paper III, the very low blue mussel abundance in the north together with the stressful environment suggests a small effective population in the northern Baltic Proper. In conclusion, my studies show that, measuring effects on several levels, including both functional and structural endpoints will both increase the sensitivity of the tests and increase their ecological relevance. </p>

<p>This thesis is an attempt to describe how different environmental factors influence life history traits in different populations, sexes and developmental stages in the moor frog, <i>Rana arvalis. </i>The studied populations are located along 1100 km latitudinal gradient, with pH varying between 4.0 and 8.5. I have used data from both natural populations and common garden experiments. </p><p>Reproducing moor frogs were larger and older at high latitudes, indicating a selective advantage of large size at high latitudes and/or earlier reproduction at low latitudes. When controlling for age I found that frogs were older and smaller at low pH, which may be a result of a reduced growth rate due to acid stress. The both sexes respond differently to different environments, with the lowest sexual dimorphism in body size found in the acid environments. This is possibly caused by a trade-off between growth and reproduction. Being large is considered to be advantageous, in females due to increased fecundity, and in males due to higher ability to compete for mates, while the cost of high growth is a reduced possibility to survive until the next mating season. Moor frog embryos originating from an acid population survived better under acid stress than embryos from a neutral population. Using quantitative genetic techniques I found strong maternal effects and small additive genetic variation for the traits in acid and non acid populations. The variation in acid stress tolerance owed largely to non-genetic effects. Females from acid localities lay larger eggs, which probably improves the performance of tadpoles under acid conditions. The trade-off between egg size and fecundity was stronger in acid populations indicating that females in acid populations reduced fecundity to increase offspring size. Finally, frogs from acidified environments were more asymmetric in skeletal traits further indicating the developmental stress created by acidification.</p>

This thesis is an attempt to describe how different environmental factors influence life history traits in different populations, sexes and developmental stages in the moor frog, Rana arvalis. The studied populations are located along 1100 km latitudinal gradient, with pH varying between 4.0 and 8.5. I have used data from both natural populations and common garden experiments. Reproducing moor frogs were larger and older at high latitudes, indicating a selective advantage of large size at high latitudes and/or earlier reproduction at low latitudes. When controlling for age I found that frogs were older and smaller at low pH, which may be a result of a reduced growth rate due to acid stress. The both sexes respond differently to different environments, with the lowest sexual dimorphism in body size found in the acid environments. This is possibly caused by a trade-off between growth and reproduction. Being large is considered to be advantageous, in females due to increased fecundity, and in males due to higher ability to compete for mates, while the cost of high growth is a reduced possibility to survive until the next mating season. Moor frog embryos originating from an acid population survived better under acid stress than embryos from a neutral population. Using quantitative genetic techniques I found strong maternal effects and small additive genetic variation for the traits in acid and non acid populations. The variation in acid stress tolerance owed largely to non-genetic effects. Females from acid localities lay larger eggs, which probably improves the performance of tadpoles under acid conditions. The trade-off between egg size and fecundity was stronger in acid populations indicating that females in acid populations reduced fecundity to increase offspring size. Finally, frogs from acidified environments were more asymmetric in skeletal traits further indicating the developmental stress created by acidification.

Natural stress and disturbances are important factors affecting the structure and function of ecosystems. However the magnitude of stress has escalated due to anthropogenic activities. Environmental monitoring and toxicity assessments try to protect ecosystems from unwanted human alterations. The aim of this Doctoral thesis was to increase the understanding of the complex effects that environmental stress has on individuals and invertebrate populations. The low saline environment in the Baltic Sea is perceived as stressful for most organisms living there. In Paper I, it was found that Baltic blue mussels living in the less saline northern Baltic Proper (~5 psu) had lower basal metabolism and were more susceptible to toxic exposure than the mussels in the south (~7 psu). There was no genetic differentiation between the mussels from the northern and southern areas while there were genetic differences between mussels from sites within the respective areas (Paper III), indicating that there is not a simple relationship between the health of the mussels and genetic diversity in the microsatellite loci studied. In Paper IV it was found that the heat tolerance of the intertidal dogwhelk Nucella lapillus is oxygen dependent. Increased oxygen levels resulted in higher survival rate. Protein expression profiles also became more similar to those of the controls, compared to the whelks exposed to high temperature and normal oxygen levels. In Paper V and VI it was found that exposure to a single toxicant for more than one generation decreased the genetic diversity in exposed copepod populations even though abundances remained unaltered. In Paper VI, exposure to naturally contaminated sediments, which contained of a mixture of toxicants, did not decrease genetic diversity. However the genetic divergence (FST) within the treatments was very high, probably due to small effective population sizes in the replicates. Likewise in Paper III, the very low blue mussel abundance in the north together with the stressful environment suggests a small effective population in the northern Baltic Proper. In conclusion, my studies show that, measuring effects on several levels, including both functional and structural endpoints will both increase the sensitivity of the tests and increase their ecological relevance.

Vienas esminių gyvybės bruožų – gebėjimas prisitaikyti prie kintančių aplinkos sąlygų. Dėl aplinkos poveikio kinta tiek prokariotiniai, tiek vienaląsčiai ir daugialąsčiai eukariotiniai organizmai, todėl nenuostabu, kad tam tikri gyvų ląstelių pasitelkiami atsako į aplinką ar poveikį būdai yra universalūs. Vienas tokių universalių su atsaku į aplinką susijusių reiškinių yra signalinių molekulių ir signalo perdavimo kelių egzistavimas įvairiausiose gyvybės formose, nuo bakterijų iki žinduolių ląstelių. Signalo perdavimo sistemos elementai kinta signalo perdavimo metu, todėl, analizuojant signalo perdavimo kelių elementus – signalines molekules, galima įvertinti ar ląstelė „pajuto“ aplinkos poveikį, ar aktyvintos atsako į poveikį sistemos. Taigi signalo perdavimo elementų analizė gali būti naudojama kaip ląstelės atsako į poveikį vertinimo įrankis. Šioje apžvalgoje nagrinėjama: • Eukariotų signalinių kelių komponentai – Ras šeimos baltymų funkcijos mejozėje; • Prokariotų signalinių kelių komponentai – bakterijų atsako į rūgštinį aplinkos stresą sistema; • Sutrikusių funkcijų (vėžinių) ląstelių atsakas į taikomą terapiją – signalinių elementų atsako į terapiją vertinimui paieška; • Signalinių kelių komponentų tyrimai pogenominėje eroje – kokybiškai naujas tyrimų etapas. Išvados 1. MAPK – universalus Ras aktyvinamo ląstelių dalijimosi signalinio kelio komponentas pagal kurio fosforilinimo laipsnį galima spręsti apie ląstelių aktyvinimą dalijimuisi; 2. asr RNR – universalus... [toliau žr. visą tekstą] / One of the fundamental features of the live species is the ability to adapt to the changing environment. Both prokaryotes and eukaryotes change due to the environmental stress, therefore it is obvious, that common mechanisms to cope the environmental stresses are universal among all the live organisms. One of such universal mechanisms related to the environmental stresses – signal transduction pathways and signal molecules existing among all kingdoms of live species. Elements of signal transduction systems change during the signal transduction, therefore the analysis of signal molecules allows the evaluation of the state of cellular response and the analysis of signaling elements could be employed as a tool to evaluate the cellular response. Following topics are discussed in this review: • Components of eukaryotic signal transduction system – function of Ras proteins in meiosis; • Components of prokaryotic signal transduction system – components of bacterial response to acid stress; • Response to the used therapy of transformed cells – search for the signal elements to evaluate the response to therapy; • Investigations of signal transduction components in post genomic era – qualitatively new stage of investigations. Conclusions 1. MAPK is a universal component of Ras signal transduction pathway to evaluate, according to the stage of phosphorylation of MAPK, the activation of the cell for division 2. asr RNA is a universal component of adaptation of enterobacteria to... [to full text]

<p>Environmental stress affects plant development and productivity. Sulfur deficiency is a key nutrient deficiency that adversely affects crop yield. The model plant Arabidopsis thaliana has played an informative role in deciphering the mechanisms involved in sulfur assimilation, as well as, the response to limited conditions. Using Arabidopsis thaliana as a model to investigate gene expression in the root, microarray data sets have been generated. These data sets consist of whole root sections for 6 time points across 72 hours, and enriched populations of 5 radial cell-types and 4 sections of 3 developmental zones of the root at 3 hrs on sulfur limited conditions. With these data it was determined which cellular tissues and developmental zones were affected most by sulfur limited conditions. Furthermore, a novel phenotype was characterized that occurs in roots after growth on low sulfur conditions. Cellular inclusions build up within the cytoplasm of mature cortical root cells. These inclusions have been termed "sulfur pox" and their composition remains to be determined.</p> / Dissertation

Environmental stress cracking (ESC) is one of the main, and probably the most common, failure mechanisms involved in polymer fractures. This type of failure is critically important as it occurs suddenly, without any visible pre-fracture deformation. Such failure can be catastrophic and costly in cases where structural integrity is required. In polyethylene (PE), ESC occurs through a slow crack growth mechanism. Cracks initiate from stress-concentrated imperfections, propagate through the bulk of PE, and ultimately result in a brittle fracture. In order to predict the environmental stress cracking resistance (ESCR) of PE, it is necessary to fully understand the molecular structure of the resin. In this thesis, attempts were made to find relationships between molecular structure characteristics and material responses, mainly inter-lamellar entanglements and strain hardening behaviour of PE resins, through mechanical and rheological experiments. Inter-lamellar entanglements are believed to be the main factor controlling slow crack growth of PE. Extent of entanglements and entanglement efficiency were investigated by monitoring the strain hardening behaviour of PE resins in the solid state through a uniaxial tensile test, and in the melt state, through extensional rheometry. ESCR is usually assessed by unreliable and time consuming testing methods such as the notch constant load test (NCLT) on notched PE specimens in the presence of an aggressive fluid and elevated temperatures. In this thesis, a practical, yet reliable, tensile test was developed for the evaluation and prediction of ESCR. The developed test offers a more reliable and consistent ESCR picture without the drawbacks of the subjective notching process and presence of aggressive fluids. Through this test, a factor called ???corrected hardening stiffness (cHS)??? was developed, which can easily be used for a relative ranking of ESCR of different PE resins. Studies were next extended to the melt state via shear and extensional rheometry. Through studies in the shear mode, a molecular weight-normalized average characteristic relaxation time (??N) was found to be efficient in predicting the extent of chain entanglements in resins. This provided a potential melt indicator for a relative measure of ESCR, for linear low density polyethylene (LLDPE), with different short chain branching levels. Extensional studies were conducted to evaluate the strain hardening behaviour in the melt state. An inverse correlation was obtained between ESCR and the melt strain hardening coefficient (MSHC), found from Sentmanat Extensional Rheometry (SER). This indicated an inverse relationship between ESCR and chain extensibility in the melt. In addition, a new factor called ???melt hardening stiffness (mHS)??? was developed from the slope of a stress-strain line, obtained from SER. This factor, analogous to cHS, can be used for a practical and reliable ranking of ESCR of PEs. ESCR is usually associated with classical crystalline phase property indicators, such as crystallinity and lamella thickness. In this thesis, the effect of processing and post processing temperature on the extent of inter-lamellar entanglements were investigated, evaluated, and correlated to ESCR. Also, analysis of the lamella surface area (LSA) was pursued since LSA reflects changes in phase interconnectivity more precisely. The focus of this part of the study was on the effect of temperature on LSA to identify the optimum processing and post-processing conditions which yield a higher LSA. It was reasonable to presume that PE with larger lamella lateral surface areas will have more inter-lamellar entanglements, hence higher ESCR. Finally, a well-controlled ultraviolet (UV) photoinitiated reactive extrusion (REX) process was developed for selective formation of long chain branches in the PE structure. This was conducted to impose restrictions against stretching of the polymer chain, which consequently enhanced ESCR.

L’huître creuse Crassostrea gigas est le produit aquacole le plus commercialisé dans le monde. Cependant, en France et dans d’autres régions du monde, des mortalités estivales massives affectent de façon alarmante les populations naturelles et cultivées de cette espèce, surtout aux stades juvéniles. Ces évènements sembleraient être liés à un déséquilibre entre les acteurs de la triade hôte – agent pathogène – environnement, favorisant l’affaiblissement des mécanismes de défense de l’hôte, et par conséquent, l’apparition et/ou l’augmentation de maladies. Parmi les facteurs environnementaux pouvant contribuer à ce déséquilibre, les contaminants chimiques sont connus pour moduler les capacités de défense de divers organismes aquatiques. Les phénoloxydases (PO) sont les enzymes-clés d’une cascade biochimique responsable de la production de mélanine et sont impliquées dans les défenses immunitaires et dans la reconnaissance du non-soi chez les invertébrés. Récemment, une activité de type PO a été détectée chez C. gigas, et a été proposée comme biomarqueur potentiel de la contamination chimique. Cependant, de nombreuses inconnues existent quant aux différents types d’activité PO présents chez C. gigas, et à leur implication dans les mécanismes de défense chez cette espèce. Dans ce contexte général,les objectifs de cette thèse ont été d’ 1) identifier les différents types de PO présents dans différents tissus de l’huître 2)évaluer le potentiel des PO en tant que biomarqueurs dans des expériences d’exposition in vivo à des contaminants organiques, 3) évaluer le potentiel des PO en tant que biomarqueurs dans des études de biosurveillance in situ. Nous avons pu montrer, pour la première fois chez cette espèce, l’existence de deux types d’activité PO : une activité catécholase et une activité laccase. De plus, nous avons pu mettre en évidence in vitro un effet bactéricide lié à l’activité des PO contre deux souches pathogènes de l’huître, Vibrio splendidus et V. aesturianus, dont leur présence a été fréquemment associée aux phénomènes de mortalités estivales. Enfin, grâce à une analyse multi-biomarqueurs sur différents tissus de C. gigas,l’ensemble des résultats ont permis de proposer que les PO pourraient être utilisées comme biomarqueurs de stress environnemental, plutôt que de contamination, dans la surveillance de la qualité des eaux côtières et estuariennes. / The Pacific oyster Crassostrea gigas is the leading aquaculture product at the worldwide level. However, massive summer mortalities affect dramatically cultivated and natural oyster populations, especially at young life stages. These events could be linked to an unbalance between the different actors of the triad host – pathogen agent – environment, which could favour a weakening of defence mechanisms in the host, and consequently, the emergence and/or increase of diseases. Among environmental factors that could contribute to this unbalance, chemical contaminants are known to modulate defence capacities in different aquatic organisms. Phenoloxidases (POs) are the key enzymes of a biochemical cascade responsible for the production of melanin and are implicated in immune defences and in self/non-self recognition in invertebrates.Recently, a PO-like activity was detected in C. gigas, and was suggested as a potential biomarker of chemical contamination. However, little is known on the different PO activities present in C. gigas, and on their implication indefence mechanisms in this species. In this general context, the aims of this work were to 1) identify the different types of PO present in different tissues of C. gigas 2) evaluate their potential as biomarkers in experiments of in vivo exposure to organic contaminants, 3) evaluate their potential as biomarkers in in situ biomonitoring studies. We showed, for the first time in this species, the existence of two PO-type activities: a catecholase activity and a laccase activity. Moreover, we observed an in vitro bactericidal effect associated to PO activities against two oyster pathogens, Vibrio splendidus and V.aesturianus, which have often been associated to massive summer mortalities in oysters. Finally, based on a multiple biomarkeranalysis in different tissues of C. gigas, results suggest that POs could be used as biomarkers of environmental stress, rather than of contamination, for biomonitoring studies in coasts and estuaries.

Organisms with complex life cycles may experience diverse stressors during their development. Stressors experienced in early life stages may influence the quantity and quality of individuals in later life stages. However, it is unclear if these effects persist later in life and how they may influence population dynamics. This dissertation uses two amphibian species, the Western spadefoot toad (Pelobates cultripes) and the spotted salamander (Ambystoma maculatum) to explore how biotic and abiotic factors experienced in aquatic and terrestrial environments influence phenotype and survival. We use a combination of field mesoscosm studies, laboratory studies and modeling to explore how early life history stressors persist in diverse environmental contexts. In Chapter 1, pond drying and larval density negatively influence multiple aspects of phenotype in the Western spadefoot toad. In Chapter 2, reduced body size due to larval stressors persisted in the first year of life in spotted salamanders in both high and low terrestrial resource environments. Additionally, there was no relationship between size at metamorphosis and post-metamorphic terrestrial survival. In Chapter 3, low terrestrial moisture levels reduced post-metamorphic growth rates by reducing food intake in juvenile spotted salamanders from both high and low larval density treatments. In Chapter 4, we scale up the effects of reduced body size in the Western spadefoot toad to explore how reduced body size due to pond drying may influence population densities using a stage specific matrix model. Reduced body size, by delaying maturity, may reduce adult densities in the Western spadefoot toad. This dissertation suggests that life stages are highly interconnected, as stressors experienced early in life stage may persist through their effects on phenotype in the absence of compensatory mechanisms. Variation in later life stages may also influence phenotype, but may not completely erase effects of early environments. Stressors experienced early in life may also translate to population level consequences, especially when organisms experience multiple stressors across life stages.