Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 143-153). / xvi, 175 leaves, bound 29 cm

University, Diss., 2007--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.

Thesis (Ph.D. (Zoology & Entomology)) - Rhodes University, 2009.

The aim of this PhD research was to study multi-element composition in wetland plants from contrasting habitats, including hot springs, temporary wetlands, and metal-rich areas. Triglochin maritima L. (seaside arrowgrass) was chosen for the study because this species is common in alkaline/saline soils and is adapted to diverse habitats. Eleocharis rostellata, Juncus balticus, Salix exigua, S. boothii, and S. wolfii were also included in the study. Field studies and greenhouse experiments were conducted to study the multi-element composition in plants. In the greenhouse experiment the effects of temperature and soil biota on multielement uptake in T. maritima were studied. Root-zone soils and plant samples were analyzed for 32 - 50 elements using inductively coupled plasma OES/MS spectrometry. The expected outcomes from this research were: 1) the development of multi-element fingerprints for T. maritima and other plant species from contrasting habitats, and 2) a better understanding of the effects of temperature and soil biota on multi-element uptake in T. maritima. Habitat specific element concentration patterns in T. maritima were observed; concentrations of Mn, Li, and B were high in plants from hot spring influenced wetlands, whereas Ca, P, Mg, Fe, Sr, Ba, Ti, and Cu were higher in the plants of temporary wetlands. J. balticus and Salix species from mine impacted and uncontaminated sites revealed distinct differences in multi-element fingerprints. J. balticus showed high concentrations of S, K, Mn, Fe, Cu, Al, As, and Cd at contaminated sites compared to un-contaminated sites. Multi-element fingerprints of Salix species showed that S. boothii had higher concentrations of Mn, Fe, Al, and Ti compared to S. exigua and S. wolfii. To our knowledge for the first time the association of mycorrhizal fungus in T. maritima was confirmed, and significant effects of temperature on element concentrations, contents, and their translocation in plants were observed. Generally, the distribution of the total contents of P, Na, Mn, B, Cu, Mo, Li, Sr, Ti, and Cs in both roots and leaves were lower at 40 °C compared to 20 and 30 °C, but their distribution and translocation from root to leaves were higher at 40°C. Even though the biological and physiological functions of Li, Sr, Ba, Rb, and Ti in plants are not fully understood, these elements were substantially taken up by T. maritima, and significant positive correlations of these elements were found with elements that have known biological functions. Overall, concentrations of Ca, P, Mg, Mn, B, Sr, and Ba in T. maritima showed variation due to differences in habitats, temperature, and experimental growing conditions (greenhouse and field condition). Concentration patterns of Na, K, and Zn were species specific and affected by temperature. Li concentrations varied due to habitat differences, growth conditions, and species differences. Future research directions could include: 1) identification of the fungal species associated with T. maritima and studies to elucidate their possible role in survival of T. maritima in the elevated temperature of hot springs, 2) the effects of soil factors, such as salinity and 3) seasonal variation in uptake and translocation, particularly for the less studied elements with yet unrecognized but potential biological functions in plants. / NIH Grant Number P20 RR016471 from the INBRE Program of the National Center for Research Resources / North Dakota State University. Department of Biological Sciences / Robert H. Levis II Cross Ranch Fellowship / North Dakota State University. Environmental and Conservation Sciences program

Ruppia maritima is a euryhaline hydrophyte found as a cosmopolitan inhabitant of shallow water habitats. In Chesapeake Bay, Ruppia maritima L. (Potamogetonaceae) and Zostera marina L. (Zosteraceae) form an important submerged aquatic vegetation community. Research in Chesapeake Bay has focused primarily on Zostera marina. Ruppia maritima occurs abundantly in large monospecific stands as well as in mixed stands with Zostera marina. Recent surveys have shown that natural revegetation in some areas has occurred and Ruppia maritima was the primary colonizer in the natural revegetation of some areas. The objective of this study was to investigate the reproductive biology of Ruppia maritima including the possible function of seed banks and vegetative and sexual propagules on the colonization of new habitats, and the plant's ecological impact around Goodwin Islands, York County, Virginia. Ruppia maritima rapidly colonized experimental plots that have historically been mixed beds or have been monospecific beds of Zostera marina because it utilized a combination of sexual and asexual reproduction. Ruppia maritima colonized plots by rapid rhizome growth. Seed reserves were probably more important in re-establishing populations than in "maintenance" of populations. Ruppia produces energy costly Post-reproductive shoots. These shoots which produce inflorescences (and then seeds) remain viable after seeds mature and can detach, disperse, and colonize sites. First year plants were not found to produce an inflorescence. This is significant in the establishment of new habitats. If a fledgling population is distressed by poor water quality or sediment disturbance, the possibility of producing seeds seems to be eliminated unless the plants have been established for one full growing season. This may explain the ephemeral nature of some Ruppia populations. / Ph. D.

Increasing human pressure combined with sea level rise and increased storminess is threatening coastal ecosystems around the world. Among these ecosystems, saltmarshes are particularly endangered due to their position in temperate areas with low wave action where human density is often high (e.g. estuaries). Around the UK, centuries of land reclamation have led to a substantial decrease of the area of saltmarsh. Over the past decades, restoration schemes have been implemented in numerous coastal locations in an attempt to counteract this loss. Such schemes involve allowing sea water to inundate a previously embanked area and letting the vegetation develop naturally, thereby reverting to saltmarsh through natural colonisation. However, surveys of restored areas that have looked at the recovery of plant species diversity or functional characteristics often show that restored saltmarshes do not reach the state of a natural saltmarsh ecosystem. While there is much data at the species level, recovery of plant intra-specific diversity (genetic diversity) has not been assessed in restored saltmarsh although this component of biodiversity is receiving increasing attention for its effect on ecosystem function. This thesis represents the first attempt to (1) characterize the nation-wide genetic structure of two important north-west European saltmarsh plant species, the common saltmarsh grass (Puccinellia maritima) and the sea arrowgrass (Triglochin maritima) and (2) compare levels of genetic diversity and structure between restored and natural ecosystems. Microsatellite molecular markers were developed for both species. Using innovative methods to analyse the genetic data obtained for these two polyploid species, this thesis highlights that genetic diversity at the national scale is organised regionally for both species, although gene-flow is still restricted between populations within the same region. Gene-flow between populations is determined by different processes depending on the species. While coastal processes mainly influence gene dispersal in P. maritima, overland routes of dispersal are involved for T. maritima. These differences are believed to be due to differences in dispersal ecology between the two species. Although gene-flow exists between distant saltmarshes, the genetic analysis of P. maritima and T. maritima colonists arriving on restored sites highlighted their local origin and reaffirmed that it is preferable to restore saltmarsh where a nearby natural saltmarsh can act as a source of colonists. A multiple paired-site comparison identified similar genetic diversity between restored and natural saltmarshes indicating that restoration of local genetic diversity is rapid for both species. A single site comparison at Skinflats in the Forth estuary compared fine-scale spatial genetic structure between the restored and natural saltmarsh. Interestingly, no structure was detected for T. maritima either in restored or natural saltmarsh. In contrast, a strong genetic structure organised along the elevation gradient was observed in the natural saltmarsh for P. maritima but was absent in the restored saltmarsh. The origin of this structure is not clear but could be due to restricted gene-flow between individuals from different elevations due to strong post-zygotic selection, as suggested in previous work. In any case, this lack of structure in the restored saltmarsh indicates that genetic recovery is incomplete in this respect for P. maritima. This thesis introduces the growing field of restoration genetics to saltmarsh ecology and identifies the principal population genetic trends in two of the species dominating the vegetation of north-west European saltmarshes community. The information given here will be useful for restoration practitioners and provides a strong foundation for future work characterizing the importance of genetic diversity for saltmarsh function.

Remirea maritima Aubl. (Cyperacea) is a species known as "pinheirinho-da-praia" and popularly used to cure diarrhea, fever, kidney diseases, pain and inflammation. Many studies have shown the presence of many secondary metabolites of R. maritima, however, scientific reports about its pharmacological properties are still scarce. Therefore, this work aimed to perform the chemical composition of extracts of R. maritima, to evaluate the antitumor and immunomodulator effects, to investigate the antioxidant activity, cytotoxicity as well as to perform preclinical studies of toxicity of hydroalcoholic extract 40% of R. maritima. The chemical composition of the aquous extract (AE) and hydroalcoholic extracts 40% (40HA) and 70% (70HA) were assessed by HPLC and LC-MS/MS and isovitexina, vitexin, luteolin and caffeyol compounds were identified. Larger quantities of isovitexin and vitexin were found in AE, while luteolin and caffeyol higher percentages were found in the 40HA. In vitro antitumor activity was determined using three human tumor cell lines by the MTT assay and only the 40HA presented potential antitumor property with IC50 of 27.08, 46.62 and 50 μg/mL for OVCAR-8 (ovarian adenocarcinoma), NCI-H385M (bronchus-human alveolar carcinoma) and PC-3 (human prostate carcinoma) human cell lines, respectively. AE and 70HA did not show any significant in vitro antitumor activity. In this context, only 40HA was submitted to others assays. The antitumor activity in vivo was assessed in Sarcoma 180-bearing mice. Body weight loss, leukogram, biochemistry (AST, ALT, Urea and Creatinine), tumor weight and organ weights (liver, spleen, and kidney) were evaluated. Inhibition rate of tumor was 57.16-62.57%, at the doses 25 and 50 mg/kg of 40HA (v.o.), respectively, and the tumor histological sections presented large number of apoptotic cells by Tunel assay. Moreover, 40HA also demonstrated immunomodulary activity with increase production of antibodies ovoalbumin specific by ELISA test and the spleen histology. The determination of TPC was measured by the Folin-Ciocalteu reagent, and the 40HA showed high amount of phenols. The redox activity of 40HA was evaluated with different reactive species generated in vitro and its cytotoxic effect against fibroblast cells (L929) and melanoma (B16F10) was tested by Neutral Red assay. The total reactive antioxidant potential (TRAP) of 40HA showed significant antioxidant capacity in the concentrations tested; 40HA was also effective at reducing hydroxyl radicals, iron and nitric oxide. The lipoperoxidation in vitro was also decreased by 40HA and data showed protection from oxidative damage. Additionally, the 40HA presented decreased of 50% and 30% of cell viability at concentrations of 80 μg/mL for L929 and B10F16, respectively, and although it presented antiproliferative effect, the 40HA was not selective for melanoma. Pre-clinical toxicity studies performed according to ANVISA and OECD. In acute toxicity, the animals received a single dose of 2000 mg/kg, while in subacute toxicity test, the animals received daily doses of 100, 200 and 400 mg/kg of 40HA. Urinalysis was also evaluated. The 40HA showed no indications of toxicity change. In conclusion, the 40HA presented in their chemical constitution the presence of flavonoids and high total phenols content. In pharmacological study, the 40HE presented in vitro and in vivo antitumor effect, potential of inducing apoptosis, immunomodulator and antioxidant properties without presenting substantial toxicity. / A Remirea maritima Aubl. (Cyperacea) é uma espécie conhecida "pinheirinho-da-praia" e usada popularmente para curar diarreia, febre, doenças renais, dor e inflamação. Estudos têm mostrado a presença de muitos metabólitos secundários ativos, entretanto, relatos científicos sobre suas propriedades farmacológicas ainda são escassos na literatura. Desta forma, este trabalho teve como objetivo caracterizar quimicamente extratos da R. maritima, avaliar os efeitos antitumoral e imunomodulador, averiguar a atividade antioxidante, a citotoxicidade assim como realizar estudos pré-clínicos de toxicidade do extrato hidroalcoólico 40% da R. maritima. A composição química do extrato aquoso (EA) e os extratos hidroalcoólicos 40% (EH40) e 70% (EH70) foi avaliada por HPLC e LC-MS/MS e os compostos isovitexina, vitexina, luteolina e cafeoil foram identificados. Maiores quantidades de isovitexina e vitexina foram encontrados no EA, enquanto que a luteolina e o cafeoil foram encontradas em maiores porcentagens no EH40. A atividade antitumoral in vitro foi avaliada em três culturas de células tumorais humanas pelo ensaio do MTT e apenas o EH40 apresentou efeito antitumoral in vitro com IC50 de 27,08; 46,62 e >50 μg/mL para as linhagens de células de OVCAR-8 (adenocarcinoma de ovário humano), NCI-H385M (carcinoma brônquio-alveolar humano) e PC-3M (carcinoma de próstata humano), respectivamente. O EA e o EH70 não apresentaram nenhuma atividade antitumoral in vitro. Diante desse resultado, apenas o EH40 foi conduzido aos ensaios subsequentes. A atividade antitumoral in vivo foi avaliada utilizando camundongos transplantados com Sarcoma 180. A evolução do peso, o leucograma, a análise bioquímica (AST, ALT, uréia e creatinina), o peso do tumor e o peso relativo dos órgãos (fígado, baço e rim) foram avaliados. A taxa de inibição do tumor foi de 57,16 62,57%, nas doses de 25 e 50 mg/kg de EH40 (v.o.), respectivamente, e secções histológicas do tumor apresentou elevado número de células apoptóticas através do teste de Tunel. Adicionalmente, o EH40 demonstrou atividade imunomoduladora com aumento da produção de anticorpos específicos para a ovoalbumina mensurada pelo teste de ELISA e pela histologia do baço. A determinação de Fenóis totais foi mensurada pelo reagente de Folin Ciocalteu e o EH40 mostrou alto teor de fenóis. A atividade redox do EH40 foi avaliada com diferentes espécies reativas geradas in vitro e a citotoxicidade contra células de fibroblastos (L929) e de melanoma (B16F10) testada por meio de ensaio do Vermelho Neutro. O potencial total antioxidante reativo (TRAP) mostrou significativa capacidade antioxidante do EH40 nas concentrações testadas; também foi eficaz em reduzir radical hidroxila, ferro e a formação de óxido nítrico. A lipoperoxidação in vitro também foi diminuída pelo EH40 e os dados mostraram proteção aos danos oxidativos. Adicionalmente, o EH40 apresentou diminuição da viabilidade celular em 50% e 30% na concentração de 80 μg/mL para L929 e B10F16, respectivamente, e embora tenha apresentado efeito antiproliferativo, o EH40 não foi seletivo para melanoma. Estudos pré-clínicos de toxicidade foram realizados segundo a ANVISA e a OECD. Na toxicidade aguda, os animais receberam uma dose única de 2000 mg/kg, enquanto que na toxicidade subaguda, os animais testes receberam doses diárias de 100, 200 e 400 mg/kg de EH40. A urinálise também foi avaliada. O EH40 não apresentou alterações que indicassem toxicidade. Em suma, o EH40 apresentou em sua constituição química a presença de flavonoides e altor teor de fenóis totais e, em estudo farmacológico, o EH40 apresentou atividade antitumoral in vitro e in vivo, com potencial indutor de apoptose, propriedade imunomoduladora e antioxidante sem apresentar toxicidade substancial.

Quatre espèces halophyles, Silene maritima, Carpobrotus edulis, Senecio cineraria et Limonium latifolium, ont fait l’objet d’études phytochimiques en procédant par fractionnement bioguidé. Nous avons ainsi identifié une trentaine de métabolites bioactifs, capables de limiter la production d’espèces réactives de l’oxygène qui interviennent dans le stress radicalaire et/ou inflammatoire et/ou de favoriser la lipolyse adipocytaire. La visualisation de l’impact du biotope sur le métabolome de Silene maritima nous a permis d’identifier des marqueurs jouant un rôle important dans la capacité adaptative de cette halophyte sous l’effet de stress environnementaux. L’analyse de profils métaboliques montre clairement qu’un même individu, récolté à des moments différents et/ou dans des biotopes différents, possède une composition phytochimique variable. Afin de tester l’effet d’éliciteurs sur la capacité biosynthétique de suspensions cellulaires de Silene maritima, nous avons eu recours à la biotechnologie végétale. Nous avons également développé des conditions de culture in vitro permettant l’initiation de cals à partir d’une halophyte protégée, Crambe maritima. Ces cals pourront in fine servir à initier des suspensions cellulaires, valorisables industriellement. / Four halophile species, Silene maritima, Carpobrotus edulis, Senecio cineraria and Limonium latifolium have been studied phytochemically by bioguided fractionation. This work led to the discovery of thirty biologically active compounds able to reduce the production of reactive oxygen species that occur during inflammatory and/or radical stress, and to promote the adipocytary lipolysis. The visualization of the biotope’s impact on the halophyte Silene maritima metabolome helped us to identify markers playing an important role in the adaptative capacity of plants under environmental stress. Analysis of metabolite profiles clearly shows that the same individual harvested at a different moment and/or from a different biotope possesses an extremely variable chemical composition. In order to test elicitor effect, we used plant biotechnology. Finally, we were able to develop in vitro culture conditions permitting initiation of callus from the protected halophile Crambe maritima. The obtained callus could serve to initiate cell suspension, which is suitable for industrial purposes.

La bactérie hyperthermophile Thermotoga maritima a été cultivée dans un fermenteur dans lequel la concentration en O2 a été rigoureusement contrôlée. A 80°C et pH 7, il a été démontré que T. maritima pouvait survivre à des expositons de durées variables à l’O2 et qu’elle était capable de le consommer. La vitesse spécifique de consommation de l’O2 a été estimée à 73.6 µmoles O2.min-1.g protéines-1 lors d’une courte exposition à l’O2 (30 min). De longues expositions à l’O2 (20 h) nous ont permis de démontrer que la présence d’O2 ralentissait la croissance de T. maritima et conduisait à un shift du métabolisme vers la production de lactate aux dépens de l’acétate et à un arrêt de production d’H2. Dans ces conditions, il a été constaté que 73% du glucose était consommé selon un métabolisme partiellement oxydatif faisant intervenir simultanément les deux voies Embden-Meyerhof et Entner-Doudoroff de la glycolyse. En l’occurrence, l’oxydation incomplète du glucose est corrélée à la réduction de l’O2 en eau. Les études transcriptomiques ont montré que cette réduction de l’O2 résultait d’une cascade de réactions intermédiaires faisant intervenir des enzymes de type peroxydases [activation de l’expression des enzymes Ahp (alkyl hydroperoxyde réductase), Bcp1 et Bcp2 (thiol peroxydase thioredoxin-dépendante)] qui acheminent les électrons libérés via les radicaux libres. D’autres enzymes comme la rubréryhtrine et la neelarédoxine interviendraient pour détoxiquer les espèces réactives d’O2. Les électrons libérés seraient au final utilisés pour réduire l’O2 en H2O par l’enzyme FprA, dont l’expression varie en fonction du potentiel redox du milieu de culture. Ce schéma est proposé comme un des éléments essentiels du dispositif enzymatique permettant la consommation de l’O2 et la protection des cellules contre les effets des espèces réactives de l’oxygène chez T. maritima. / Batch cultures of the hyperthermophilic bacterium Thermotoga maritima were performed in a bioreactor where O2 concentrations in the gas phase were strictly controlled. At 80°C and pH 7, we demonstrated that T. maritima survived despite being exposed to oxygen at different times and that it consumed it. O2 uptake rate was estimated at 73.6 µmoles O2 min-1g proteins-1 during a short exposure to O2 (30 minutes). A long time exposure of T. maritima cultures to oxygen (20h) led to a drastic reduction in growth, together with a shift in glucose metabolism towards lactate instead of acetate production and a stop in H2 production. Under these conditions, it has been observed that 73% of glucose was partially oxidised by using both Embden-Meyerhof and Entner-Doudoroff glycolytic payhways. Uncomplete oxidation of glucose is correlated to a reduction of O2 to H2O. Transcription analyses revealed that this reductive process of O2 involved enzymes like peroxidases [activation of alkyl hydroperoxide reductase (ahp), bcp1 and thioredoxin-dependent thiol peroxidase (bcp 2)]. Moreover, genes encoding reactive oxygen species (ROS)-scavenging systems (neelaredoxin and rubrerythrin), were found to be upregulated during oxygen exposure. The oxygen reductase FprA, which expression was shown to depend on the redox level of the culture medium, is proposed as a primary consumer of O2. All these enzymes are essential for T. maritima to consume O2 consumption and to fight against the toxic effects of ROS in cells.

Eutrophication, particularly due to nitrogen (N) and phosphorus (P) input, has been massively altered by anthropogenic activities. Thus it is important to understand the impact on salt marsh plants; however studies on salt marsh plants within Galveston Bay, Texas are limited. In this study, the effects of repeated nutrient additions in monospecific plots of Spartina alterniflora, Batis maritima¸ and Salicornia virginica as well as mixed plots of B. maritima and S. virginica were studied over 15 months. Results showed that nutrient loading led to an increase in height, biomass, growth rate and percent nitrogen (N) within all three species studied, but were species specific more than dose dependent. Nitrogen content in leaves had a positive correlation with P content in leaves but a negative correlation with carbon (C) content. Nutrient loading lead to a significant increase in total chlorophyll in the fertilized plots of S. alterniflora and S. virginica one month into the study. Nutrient addition to two succulent species, B. maritima and S. virginica in mixed plots did not reveal a distinct superior competitor within the 15 month study in terms of growth and nutrient use efficiencies; however using the maximum growth rates of the monospecific plots, the Monod model was used to determine which species would dominate at high nutrient loads. Based on height data S. alterniflora would dominate, while B. maritima would dominate according to the Monod model based on biomass.