Overlooked for a long time, wood falls on the deep oceanic floor are now recognized as extreme, reducing ecosystems based on chemosynthesis, in the same way that hydrothermal vent, cold seeps and whale falls with which they share important physicochemical and faunistic similarities. In these ecosystems, bacterial chemosynthesis using reduced compounds (H2S, CH4) and/or digestion of refractory organic compounds (cellulose and lignin in wood, organic matrix of bone) by heterotrophic bacteria play an essential trophic role, and appear to promote establishment of mutualistic symbioses between bacteria and metazoans for the exploitation of environmental resources. Beyond the description of the taxonomic composition of fauna associated with these sites, current studies aim to understand the functioning and evolutionary links of these ecosystems. Digestive and/or chemoautotrophic associations with microorganisms that could be the key to survive in these habitats have been described in a number of molluscs and annelids but only few crustaceans. The latter distinguish from the other groups by the fact they do not realize intracellular symbioses (endosymbioses) but mainly carry ectosymbioses on their integument (epidermis and cuticle) even in the digestive tract that is partly lined by epidermis (in stomodeum and proctodeum). Moreover, up to now, nothing was known about crustaceans from deep-sea wood falls, their feeding habits and associations with microorganisms. The present work focused on the crustaceans and especially 15 species of decapods from wood falls in the South Pacific Ocean. The specimens were recovered during several French cruises (Salomon2, BOA1, SantoBOA, SalomonBOA3) organized near the Vanuatu, Solomon Island and New Caledonia by the Muséum National dHistoire Naturelle of Paris. The investigation of the feeding biology and microbial associations of the decapod species combined three complementary approaches : 1) a morphological approach using light and electron microscopy to describe the external and gut structures as well as the gut content and microflora, 2) a trophic approach based on stable isotopes analyses (C and N) together with the nature of the gut content and 3) a molecular approach based on 16S rRNA gene analyses and FISH labeling to identify and locate the bacteria in the gut content (ingested bacteria) and on the gut lining (resident bacteria or symbionts). Taxonomic identification and morphological observations of the species provided a first non-exhaustive inventory of the best represented decapod species in wood accumulations in the deep South Pacific and point out three interesting informations. 1) Most decapods from the sunken woods belong to Reptantia. 2) Some species (e.g. Munidopsis spp.) belong to deep sea taxa and exhibit typical characteristics while others (e.g. Xylopagurus) much more resemble shallow-water species, indicating that the colonization of wood falls by decapods may have occurred from both deep and coastal habitats. 3) Association degree with sunken wood could be important for some species that appear endemic (e.g. Pylochelidae, Xylopagurus) or which probably have their complete life cycle on wood falls (most of the gravid females having big eggs with a direct larval development). From a trophic point of view, morphological observations of the digestive system, examinations of gut contents and stable isotope analyses carried out on 15 species, allow us to classify the decapod crustacean from deep-sea sunken woods into two major groups, detritivores and predators/scavengers, and four trophic guilds (trophic levels) depending on two primary food sources, the wood and the particulate organic matrix (marine snow). The four guilds were identified as bacteriovorous detritivores (M. nitida, M. pilosa, M. bispinoculata, Munidopsis sp.1), xylophagous detritivores (M. andamanica, R. amboinensis), omnivores (X. caledonicus) and predators/scavengers (pylochelid species, Munida spp, Axiidae sp.1, M. cylindrophthalma). A fifth guild could be represented by limivorous detritivores (C. acutirostella, Alpheidae sp.1). This breaks with the widespread idea that decapod crustaceans from wood falls are all scavengers or predators. In addition, these results have evidenced of some special dietary strategies and highlight the importance of xylophagous decapods in the ecosystem, by their mechanical degradation of the substrate and their production of feces which should enrich the sediment and have a significant impact on the composition of wood and sediment microbiota (bacteria and fungi). Microscopic observations also allowed us to identify potentially symbiotic associations with resident (and transient) microorganisms (bacteria and/or trichomycetes) in the hindgut of five detritivorous species. Owing that, three of these species have a wood-based diet (M. andamanica, M. nitida, R. amboinensis) and considering the location of these microflora, it is strongly suggested that the resident microorganisms are directly related to the xylophagous diet of the host and most particularly involved in the digestion of wood. More detailed in two xylophagous species, M. andamanica and R. amboinensis, not only these microflora can be distinguished on the basis of their morphology, location and probable genetic differences but also by their roles and involvement in the digestion of the woody substrate. The digestive bacteria of M. andamanica could act as trophic intermediates while those of R. amboinensis probably only help in its digestion. From the stable isotopes ratio, it is likely that R. amboinensis directly assimilates the wood digestion products while M. andamanica feeds on wood with a trophic intermediate. Cloning and sequencing 16S rRNA gene from the gut bacteria of M. andamanica revealed its resident hindgut microflora is largely dominated by two phylotypes (OTUs) of possibly symbiotic bacteria that belong to Firmicutes and Alphaproteobacteria. Surprisingly, these OTUs are very close to gut bacteria isolated from a coastal thalassinid shrimp N. californiensis and from the Chinese crab E. sinensis. The results raise the question of the development and evolution of digestive symbioses in decapods and crustaceans in relation to the diet and/or adaptation to a special habitat, owing that among galatheid crabs the symbiotic relationships appear closer in certain species (M. andamanica) than in others (M. nitida, M. bispinoculata) and that similar differences appear between taxonomic groups. On the other hand, the genetic proximity of bacteria from distant crustacean species raise the question of an eventual co-evolution of linked hosts and symbionts contrasting with the permanent re-acquisition of symbionts from the medium.

Phylogeny and morphology were examined for the cyanobionts from the water fern Azolla and the cyanobacterial genus Nostoc originating from symbioses with different host plants (genera Gunnera, Cycas, Dioon, Encephalarthos, Macrozamia, and Anthoceros), the lichen genus Pannaria, and free-living Nostoc isolates from different habitats. Nostoc isolates of Pannaria formed a closely related group, but, in general, no monophyletic nature was attributed to the genus Nostoc, in contrast to the cyanobionts from Azolla which were contained in a unique monophyletic group. No correlation was detected between the diversity of the studied cyanobacteria and their geographical origin, while high host specificity was proved for the Azolla cyanobionts and the Nostoc isolates from the bipartite Pannaria lichen. Two patterns of evolution leading to symbiotically competent heterocystous cyanobacteria were distinguished, one comprising symbiotic Nostoc species and the other comprising cyanobacteria in association with the water fern Azolla. The production of the non-protein amino acid BMAA, a potential neurotoxin, was also examined. A rapid and sensitive method involving the lysis and extraction of amino acids from cyanobacteria combined with an HPLC assay for fluorescence detection of BMAA was developed. To determine whether the plant or the cyanobacterium was the origin of the BMAA in the cyanobacterium-Azolla symbiosis, the cyanobacterium-free Azolla pinnata var imbricata strain 511 was examined. HPLC analysis demonstrated a significant BMAA production in the absence of the cyanobacterium. However, PCR and cloning revealed the presence of bacteria of the genus Ochrobactrum in the plant.

The aim of this s tudy is to investigate the three great existential questions—anxiety, love and death—in the works of Stig Dagerman. Emphasis is placed on the novels Ormen [The Snake] (1945), De dömdas ö [The Island of the Doomed ] (1946), Bränt barn [A Burnt Child] (1948), and Bröllopsbesvär [Wedding Worries] (1949), as well as the play Den dödsdömde [The Condemned] (1948). To this end, interpretation is used in the sense given to it by Paul Ricoeur—interpretation that leads to an understanding of a double meaning. In the present case, this means that the texts' symbolic level is revealed and accorded as great an importance as the story at the surface, manifest level . The theme of anxiety is most apparent in the first novel, while the question of eroticism is central to De dömdas ö and Bränt barn. Each novel expresses a pessimistic view of erotic love as a realizable possibility. Death, which has concrete, motivational and abstract aspects , occurs in every text. Murder and suicide interact with inner, spiritual deadness. This state means acquiring a lowered or threatened inner vitality in which the psyche becomes "icebound". Inner deadness is a form of anxiety. Analysis reveals that anxiety, the complications of love and the problem of death are related to a mother figure. The mother figures in the texts are alternately the objects of destructive hatred and excessive love. Behind these mother figures is concealed the experience of a mother who has betrayed, and who is there fore the object of both aggression and longing. This longing, according to Freudian-Lacanic theory, is expressed in a desire for the mother's body, i.e. a longing fo r the lost unity with the mother in original symbiosis. Thus, the mother figure in the texts also has mythical dimensions—Mother and death are in a sense equivalent. To die is to return to the mother, but death, or the proximity of death thus also provides the opportunity for a symbolic rebirth. Both the proximity of concrete death—as in thwarted suicide attempts—and a symbolic death — as in the form of extreme self-degradation—give rise to an increased feeling of life, if only for a moment. The psychologist Robert Jay Lifton has shown that murder can be a conscious step in a process of self- destruction. Murder is in this case a phase in a search for revitaliza-tion, which is thus the ultimate aim of suicide. This phenomenon appears on the symbolic level in Dagermans works, especially in the play Den dödsdömde. The interpretation reveals that the existential question that dominates Dagerman's texts is the struggle against the threat of inner deadness. The fixation with death in the texts is, in the final analysis , an expression of a desire to live. / digitalisering@umu

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

Recent work has shown that microorganisms are surprisingly like animals in having sophisticated behaviours such as cooperation, communication, and recognition, as well as many kinds of symbioses. Here we show first that the social amoeba Dictyostelium discoideum has a primitive farming symbiosis that includes dispersal and prudent harvesting of the crop. About one-third of wild-collected clones engage in husbandry of bacteria. Instead of consuming all bacteria in their patch, they stop feeding early and incorporate bacteria into their fruiting bodies. They then carry bacteria during spore dispersal and can seed a new food crop, which is a major advantage if edible bacteria are lacking at the new site. However, if they arrive at sites already containing appropriate bacteria, the costs of early feeding cessation are not compensated, which may account for the dichotomous nature of this farming symbiosis. We also observed farmer Dictyostelium discoideum clones carry bacteria that they do not use as food. We hypothesized that these bacteria may play a defensive role against other D. discoideum clones. In our second study, we investigated the impact of these bacteria-carrying farmers on non-farming D. discoideum clones. We found that the presence of farming clones reduces spore production in non-farmers. Furthermore, this effect increases with frequency of farming clones, demonstrating the vulnerability of non-farming clones to farmers though in this experiment we had not separated the effects of the farmer clone and the bacteria they carry. In our third study we exposed non-farmers to a filtered supernatant from the most common non-food carried bacterium, Burkholderia xenovorans . This supernatant is likely to carry whatever the bacteria are producing. We treated Dictyostelium clones at the beginning of the social stage and found that the supernatant enhanced spore production of farming clones and hurt spore production of non-farming clones. This study shows that the effects of the bacteria can be restricted to a filtered supernatant alone. This discovery of symbiosis of D. discoideum with bacteria, and its impact on social interactions among D. discoideum clones will provide a fertile ground for further experiments on the evolution of sociality.

A collaborative approach to industry-environment issues is acknowledged as a key aspect of sustainable development. Sincerely, resource sharing among firms offers the potential to increase stability of operations, especially in supply-constrained areas, by ensuring that access to important inputs such as water, energy and raw materials are guaranteed. Industrial Symbiosis (IS), a sub-field of Industrial Ecology, is primarily concerned with the cyclical flow of resources through networks of industrial units as a means of cooperatively approaching environmentally sustainable industrial activity. In line with this principle, a critical assessment of the change in environmental performance brought about by industrial symbiosis (IS) was conducted in nineteen selected eco-industrial park case studies identified in all regions of the world with the exception of the African continent. Case study selection criteria were based on models of eco-industrial parks proposed by Chertow (2000). A description of the type of material exchanges that go on in each case study was carried out which revealed evidence of implemented synergies in respective case studies. A comparative assessment of cross-case patterns which is a semi-quantitative matrix used to quantify the degree of environmental performance showed that there was a clear evidence of improved environmental performance among respective case studies investigated where water, energy and material flows served as indicators. Results obtained from the study showed a common pattern of industrial presence in respective case studies reflecting the occurrence of heavy process industries such as oil refineries, cement industries, petrochemical industries, and steel industries. The principle of “anchor tenant” proposed by some experts in the field of industrial ecology was strongly supported by the obtained results. Symbiotic cooperation among participating firms in respective case studies were mainly on areas like cogeneration, re-use of materials, recycling and wastewater treatment and re-use.

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

<p>Lichen-forming fungi employ a successful mode of nutrition as symbiotic partners with green algae and/or cyanobacteria (the photobiont). Nearly one fifth of all known fungi are obligate lichen formers, yet we know little of how they find compatible partners and establish long-lived symbiotic relationships. The combined growth of these symbionts forms a body (thallus) with emergent properties unlike either of the symbionts individually grown. Based on other well-studied eukaryotic systems, the development of a lichen thallus must rely upon the successful identification and collaboration of these two very different organisms. Identifying the molecular basis of microbe recognition and interactions remains one of the greatest challenges in studying symbiotic systems. </p><p>In this thesis, I determine the stage in which to begin looking for lichen symbiosis specific genes, and then examine mycobiont and photobiont genes that, when compared to the aposymbiotic state, are upregulated in the symbiotic state. Using the symbiosis between the mycobiont <italic>Cladonia grayi</italic> and the photobiont <italic>Asterochloris</italic> sp., as well as scanning electron microscopy observations of the earliest stages of contact between <italic>C. grayi</italic> and <italic>Asterochloris</italic> sp., I determined that the mycobiont undergoes a specific change in phenotypic growth in response to <italic>Asterochloris</italic> sp. This change is particular to the lichen symbiosis, and is not observed with algal shaped inanimate objects or algae other than <italic>Asterochloris</italic>. I then used this phenotypically defined stage that is exclusive to lichen symbiosis to begin studying the the genetic and molecular mechanisms underlying the development of a stratified lichen thallus. Using suppression subtractive hybridization to determine differential gene expression, fungal and algal libraries were made of upregulated genes in the first 2 stages of lichen symbiosis. The symbiotic expression levels of select genes were then verified using quantitative PCR. Lastly, a candidate gene for involvement in lichen symbiosis was transformed into <italic>Saccharomyces cerevisiae</italic> to test for protein function.</p><p>Further results of this study show that the fungal protein products of genes upregulated in lichen symbiosis show significant matches to proteins putatively involved in fungal self and non-self recognition, lipid metabolism, negative regulation of glucose repressible genes, an oxidoreductase, a dioxygenase, and a conserved hypothetical protein. Algal genes that are upregulated in lichen symbiosis include a chitinase-like protein, an amino acid metabolism protein, a dynein related protein, and a protein arginine methyltransferase. Furthermore, genes that are expressed in the early stages of lichen symbiosis are common varying metabolic pathways. Furthermore stages 1 and 2 of development are marked not by a drastic change in transcriptional products, but instead by an overall change in genes that are already expressed. Finally, the <italic>Cladonia~grayi Lip3</italic>was cloned in its entirety from genomic DNA and cDNA, was predicted to be secreted using signal peptide prediction software, and shown to be a functioning secreted extracellular lipase in yeast.</p><p>I conclude that many genes are involved in the interactions of symbionts and the development of a stratified lichen thallus, and that many more genes remain to be discovered. Furthermore, the possibility that genes exist in either symbiont that are specific to lichen symbiosis remains, and that their discovery awaits the creation of better genomic tools for \textit{Cladonia~grayi} and <italic>Asterochloris</italic>.</p> / Dissertation

Symbiosis is commonly present in marine invertebrates. Many corals and sponges have symbiotic algae or bacteria. In the previous studies of the sponge Cinachyra australiensis, 85% of the bacteria associated with the sponge have high similarity (88.65%) with the symbiotic chemoautotrophic sulfur-oxidizing bacteria of the deep-sea hydrothermal vent mussel, Solemya reidi. This study aims to investigate the localization of the chemoautotrophic sulfur-oxidizing bacteria associated with Cinachyra australiensis. The Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) large-subunit genes for autotrophic organisms were amplified by polymerase chain reaction from the sponge samples. The phylogenetic relationship of the RubisCO large subunit genes was analyzed. A total of 26 clones were selected and sequenced. They could be divided into two groups. One (9 clones) belongs to form I type IB (cynobacteria and green algae). The other (17 clones) belongs to form II type IA (chemoautotrophic symbiotic bacteria). The location of the sulfur-oxidizing chemoautotrophic bacteria was shown to be intracellular symbiosis within the mesoglial cells by fluorescence in situ hybridization.

Nitrogen fixation is an ecologically important microbial process that can contribute bioavailable combined N to habitats low in N. Shipworms, or wood-boring bivalves, host N2-fixing and cellulolytic symbiotic bacteria in gill bacteriocytes, which have been implicated as a necessary adaptation to an N-poor C-rich (wooden) diet. Shipworm symbionts are known to fix N within the gill habitat and newly fixed N is subsequently incorporated into non-symbiont containing host tissue. The presence of N2-fixation in gill bacteriocytes presents a conundrum because N2-fixation is tightly regulated by oxygen in most other diazotrophic microbes. Also, the direct evidence of new N being incorporated into the host tissue indicates that there are potentially complex nutrient cycles in this symbiosis, which have not been investigated. We used the cultivated symbiont Teredinibacter turnerae, which has been isolated from many shipworm species, as a model organism to elucidate controls on N2-fixation and N release in the shipworm symbiosis. Our results indicate that headspace oxygen concentration does not control biomass specific N2-fixation and respiration activity in T. turnerae, but it does influence the magnitude of the growth rate and timing of culture growth. Also, we examined the controls of oxygen on inorganic nutrient uptake rates, and documented a small amount of dissolved inorganic nitrogen release. While the N budget is only partially balanced, we provide indirect evidence for the allocation of fixed N to the excretion of exopolymeric substances and dissolved organic nitrogen; future studies that measure these additional N sinks are necessary to close the N budget. Although there are limitations of using pure cultures to investigate a complex symbiotic system, this study provides direct experimental evidence that T. turnerae has adaptations that are conducive to N2-fixation in gill bacteriocytes.