Measurement of soil phytase activity (PA) and delineation of the impact of this important phosphomonoesterase on the P-cycling process in soil and sediments suffer from the lack of a reliable assay. A method for measuring PA in soil that promises to be accurate and reliable has been recently published. The method involves the use of a novel chromophoric analog of phytic acid, referred to as T(tethered)InsP5 (5-O-[6-(benzoylamino)hexyl]-D-myo-inositol-1,2,3,4,6-pentakisphosphate). This study was conducted to measure PA in a Clymer forest soil, which contained over twice the amount of soil organic C as previously tested soils, using the TInsP5 PA assay. This investigation specifically addresses: (1) the development of a soil dilution technique for determining maximal PA, (2) identification of previously unsubstantiated soil-produced dephosphorylated intermediate probe species, (3) the impact of increasing assay buffer pH on soil PA and (4) testing stability of the probe's amide bond in a highly (bio)active forest soil. PA assays were conducted by measuring dephosphorylation of TInsP5 in citrate-acetate buffered (pH 4.2) active and autoclaved (Control) soil suspensions. Phosphorylated probe intermediates (i.e., TInsP4, TInsP3, TInsP2 and TInsP1) and T-myo-inositol were extracted from samples of soil suspension following incubation. Probe species were quantified using reversed phase high-performance liquid chromatography (RPHPLC) with UV detection. PA was calculated based on a mass balance approach. A soil dilution technique was developed to address the challenge of determining maximal PA in soils containing higher organic matter content. In the initial report on use of the TInsP5 method for measuring PA in soil, two "soil-generated" UV-adsorbing compounds (designated Y and Z) were observed, but never confirmed as probe species. The experimental evidence presented in this report supports inclusion of compound Y as a phosphorylated probe intermediate species (i.e. TInsPy), based primarily on its UV adsorption spectra (diode-array detection analysis). Compound Z could not be substantiated as a probe species based on the evidence presented in this study. PA of Claymer forest soil decreased with an increase in assay buffer pH. Further, the probe's amide bond linkage was stable in a forest soil exhibiting high PA. / Master of Science

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No. of bitstreams: 1 biancasartinidoespiritosanto.pdf: 3527781 bytes, checksum: c9a6ba355ca58ea44e1eb59dcc0ca070 (MD5) Previous issue date: 2012-02-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / No presente estudo foi avaliada a influência da poluição orgânica sobre a composição e estrutura da comunidade de ciliados peritríqueos em ambientes lóticos no município de Juiz de Fora, Minas Gerais sujeitos a diferentes níveis de poluição orgânica, bem como os aspectos ecológicos da relação epibiótica entre esses ciliados e moluscos gastrópodes. A presente dissertação está dividida em duas seções. Na primeira seção foram analisadas a composição e estrutura da taxocenose de protistas ciliados peritríqueos encontrados em nove córregos afluentes do rio Paraibuna, Juiz de Fora, MG, que recebem diferentes cargas de esgoto doméstico. Além disso, foi avaliado o potencial destes microorganismos como indicadores da qualidade da água desses ambientes. Os ciliados peritríqueos foram obtidos com o emprego de substratos artificiais em cinco coletas, com intervalos mensais, realizadas entre junho e outubro de 2010. Amostras de água foram coletadas para avaliar os parâmetros físico-químicos: condutividade elétrica, oxigênio dissolvido, pH e nutrientes. No laboratório, os peritríqueos foram quantificados em microscópio com contraste interferencial diferencial e fixados para realização de técnicas identificação por impregnação pela prata. Foram registradas 19 espécies de ciliados peritríqueos nas estações amostrais analisadas. A espécie Carchesium polypinum (Linné, 1785) ocorreu com maior freqüência e abundância nos ambientes com grande quantidade de poluição sendo considerada como indicadora desses ambientes. Foi observada correlação positiva entre a concentração de nutrientes e a abundância e densidade de peritríqueos. Os valores mais elevados de dominância, densidade e abundância de espécies ocorreram em ambientes com altos níveis de poluição orgânica enquanto que, os maiores valores de riqueza e diversidade foram observados nas estações com níveis intermediários de poluição orgânica. Na segunda seção, foi realizada a avaliação da composição e estrutura da comunidade e a distribuição espacial de peritríqueos epibiontes sobre a concha de moluscos da espécie Physa acuta (Draparnaud, 1805) coletados em um dos nove córregos analisados na seção 1. Dos 140 moluscos analisados, 60.7% estavam colonizados por pelo menos uma das sete espécies de peritríqueos epibiontes. A espécie Epistylis sp. foi considerada dominante, representando 70.4% da abundância total de epibiontes. Além disso, os epibiontes se distribuíram por toda a superfície da concha, apresentando maior abundância na superfície ventral do que na dorsal. O presente estudo contribui para a ampliação do conhecimento sobre a ecologia de protistas ciliados peritríqueos neotropicais. / In the present study, was evaluated the influence of the organic pollution on both the composition and structure of the taxocenose of peritrich ciliates found in lotic systems at Juiz de Fora, Minas Gerais State, with different loads of domestic sewage, as well the ecological aspects of the epibiotic relation between gastropods mollusks. The present dissertation is divided into two parts. In the first one, the influence of the organic pollution on both the composition and structure of the taxocenose of peritrichs ciliates found at nine tributary streams of the Paraibuna river, Juiz de Fora, MG, wich receive different levels of domestic sewage. Furthermore, the potential of these microorganisms as water quality indicators in these environments was evaluated. Peritrichs ciliates were obtained with the use of artificial substrates in five monthly sampling carried out between June and October 2010. Water samples were collected for assessment of the physico-chemical parameters: electrical conductivity, dissolved oxygen, pH and nutrients. At the laboratory, the peritrichs ciliates were quantified in microscope equipped with Differential Interferential Contrast, and then fixed to conduct identification techniques for silver impregnation. Nineteen peritrichs ciliates species were recorded at sampling stations studied. The peritrich Carchesium polypinum (Linné, 1785) was species more frequent in environments with high organic pollution load. The abundance and density of peritrichs ciliates was positively correlated with nutrients. It was also observed the highest dominance, density and abundance of species in environments with high levels of organic load, and on the other hand, the highest richness and diversity in environments with intermediary levels of organic pollution. In the second, the composition, spatial distribution and taxocenose structure of the peritrich as epibiont on the shell of the Physa acuta (Draparnaud, 1805) snail were evaluated. Of the 140 molluscs analyzed 60.7% were colonized by at least one of the seven species of peritrich epibiont. Epistylis sp. was considered as dominant species, representing 70.4% of the epibiont total abundance. In addition, the epibiont were distributed over the entire surface of the shell, with higher abundance in the ventral surface. This study contributes to the expansion of knowledge about the ecology of protists peritrichs ciliates in neotropical lotic systems.

Coral reefs are being negatively impacted by various causes worldwide, and direct intervention is often warranted following disturbance to restore or replace lost ecosystem structure and function. An experimental coral reef restoration study involving standardized artificial reef modules (ReefballsTM) was conducted in Mexico’s Yucatan Peninsula in the towns of Puerto Morelos and Akumal. The purpose was to explore the use of artificial structure for restoration and mitigation applications in a highly diverse and dynamic Caribbean coral reef environment by applying and evaluating the performance of select experimental treatments hypothesized to accelerate development of the associated biota. The first treatment consisted of invertebrate enhancing artificial substrate padding material, which provided structurally complex refuge space for mobile epifaunal/infaunal invertebrates and other benthic organisms. The second treatment consisted of coral transplants, intended to provide additional structural complexity and kick-start development of stony coral populations. The third treatment consisted of settlement plates which were intended to provide data on coral recruitment and survival rates. Multiple hypotheses relating to the interactions between experimental treatments and the resulting macroalgal, non-coral invertebrate, stony coral, and coral reef fish assemblages were examined, and comparisons were made between natural and artificial substrates. In Puerto Morelos there were 40 modules; 10 controls and 10 of each of 3 treatments: substrate pads, coral transplants, and settlement plates. In Akumal there were 12 modules; 6 controls and 3 of each of 2 treatments: substrate pads and settlement plates. Following module deployment, 6 biannual monitoring trips were made over the course of three years to assess the development of the biota, with a final 7th trip made six years post-deployment. Divers conducted non-destructive visual surveys to evaluate total abundance, species richness, size class distribution, and assemblage structure of coral reef fishes. Other monitoring work included coral recruit surveys, mobile epifaunal invertebrate collections from substrate pads, and digital imaging of coral transplants, natural reef reference corals, and benthic quadrat areas. Hurricane Dean compromised the Akumal study site during the first year of the study, but Puerto Morelos was unaffected. There the modules developed biotic assemblages that differed from what was found on the natural reef, and the data suggests that the substrate pads may have had an effect on the development of faunal assemblages. Lobophora variegata macroaglae and Desmapsamma anchorata sponge were the major contributors to benthic community composition, and both had significantly greater coverage on the substrate pads treatment modules. Lobophora grew rapidly and peaked within the first year, while sponges increased steadily throughout the first three years of the study, surpassing the coverage of macroalgae before the end of the second year, much to the detriment of coral transplants and many coral recruits. By the end of the study, over 75% of the transplants were overgrown by D. anchorata, and density of new coral recruits on the Pads treatment modules was lower than the other treatments and controls. Coral recruitment was dominated by Porites astreoides on all treatments and controls, and the number of corals increased steadily throughout the study. The controls had consistently greater numbers of corals than the treatments, as well as lower percent coverage of macroalgae and sponges. Total abundance and species richness of reef fishes was generally unaffected by the treatments. However, at the family and species level, several differences were detected, particularly for the substrate pads treatment and to a lesser extent for the coral transplants treatment. For future restoration or mitigation efforts utilizing similar or identical treatments to artificial substrates, this study suggests that, in the absence of routine maintenance, greater success may be achieved after waiting several years post-deployment for the initial wave of unchecked growth by benthic organisms (i.e., macroalgae and sponges) to reach a balance point before a large investment of resources is devoted to coral transplanting. Further recommendations include routine monthly or quarterly on-site maintenance to enhance transplant survival, as well as a longer monitoring window to assess community development in response to experimental treatments. The results of this study suggest that the experimental treatments did indeed have an effect on the biota, but whether or not the effect was beneficial largely depends upon perspective. The Pads treatment in particular had the greatest effect on both reef fish and benthic community development, however, it was not beneficial for stony coral recruitment. Additional research is needed to fully understand the long-term performance and effects of the padding material on biotic assemblage development for future restoration or mitigation projects.

Floodplain lakes are strongly regulated by river connectivity because floodwaters exert strong influence on the water balance, the physical, chemical and biological limnological conditions, and the influx of contaminants. The Peace-Athabasca Delta (PAD) in northern Alberta (Canada) is a hydrologically complex landscape and is an important node in the upper Mackenzie River Drainage Basin. The ecological integrity of the PAD is potentially threatened by multiple environmental stressors, yet our understanding of the hydroecology of this large floodplain remains underdeveloped. Indeed, ever since the planning and construction of the WAC Bennett Dam (1960s), concerns have grown over the effects of upstream human activities on the lakes of the PAD. More recently, concerns over the health of the PAD have intensified and come to the fore of national and international dialogue due to water abstraction and mining and processing activities by the rapidly expanding oil sands industry centred in Fort McMurray Alberta. Currently, widespread perception is that upstream human activities have reduced water levels and frequency of flooding at the PAD, which have lowered nutrient availability and productivity of perched basin lakes, and have increased supply of pollutants from oil sands. However, these perceptions remain based on insufficient knowledge of pre-impact conditions and natural variability. Current and past relations between hydrology and limnology of PAD lakes are mostly undocumented, particularly during the important spring freshet period when the effects of river flood waters are strongest. Similarly, knowledge of the deposition of oil-sands- related contaminants in the PAD remains insufficient to determine whether anthropogenic activities have increased the deposition of important oil-sands-related contaminants such as polycyclic aromatic compounds (PACs) relative to natural processes. Such knowledge gaps must be filled to achieve effective monitoring, policy and governance concerning impacts of industrial development and the protection of human and environmental health within the PAD and Mackenzie drainage basin. This thesis examines the effects of river flooding (and the lack of) on water clarity, nutrients, chemistry, phytoplankton abundance, epiphyton community composition and the deposition of polycyclic aromatic compounds (PACs) in lakes of the Peace-Athabasca Delta. To determine the role of flooding on contemporary epiphytic diatom communities (an abundant and diverse guild of primary producers in PAD lakes), a field experiment was conducted examining the community composition and abundance of epiphytic diatoms in four PAD lakes. Two of these four lakes had received floodwaters that spring and two had not. Epiphytic diatom communities in each lake were sampled during the peak macrophyte biomass period (summer) from two macrophyte taxa (Potamogeton zosteriformis, P. perfoliatus var. richardsonii) and from polypropylene artificial substrates previously deployed that spring. A two-way analysis of similarity (ANOSIM) test identified that epiphytic diatom community composition differed between lakes that flooded and those that did not flood. From the use of similarity percentage (SIMPER) analysis, diatom taxa were identified that discriminate between flooded and non-flooded lakes. The relative abundance of ‘strong flood indicator taxa’ was used to construct an event-scale flood record spanning the past ~180 years using analyses of sedimentary diatom assemblages from a closed-drainage lake (PAD 5). Results were verified by close agreement with an independent paleo-flood record from a nearby flood-prone oxbow lake (PAD 54) and historical records. Comparison of epiphytic diatoms in flooded and non-flooded lakes in this study provides a promising approach to detect changes in flood frequency, and may have applications for reconstructing other pulse-type disturbances such as hurricanes and pollutant spills. Additionally, this study demonstrates that artificial substrates can provide an effective bio-monitoring tool for lakes of the PAD and elsewhere. To improve our understanding of the hydrolimnological responses of lake in the PAD to flooding, repeated measurements over three years (2003-05) were made on a series of lakes along a hydrological gradient. This allowed the role of river flooding to be characterized on limnological conditions of lakes and to identify the patterns and timescales of limnological change after flooding. River floodwaters elevate lake water concentrations of suspended sediment, total phosphorus (TP), SO4 and dissolved Si (DSi), and reduce concentrations of total Kjeldahl nitrogen (TKN), DOC and most ions. River flooding increases limnological homogeneity among lakes, because post-flood conditions are strongly affected by the river water properties. After floodwaters recede, limnological conditions become more heterogeneous among lakes in response to diversity of local basin influences (geology, slope, vegetation, depth, fetch, and biological communities and processes), and limnological changes occur at two distinct timescales. In the weeks to months after flooding, water clarity increases as suspended sediments and TP settle out of the water column. In the absence of flooding for many years to decades, evaporative concentration leads to an increase in most nutrients (TKN, inorganic N, and dissolved P), DOC and ions. Contrary to a prevailing paradigm, these results suggest that regular flooding is not required to maintain high nutrient concentrations. In light of anticipated declines in river discharge, limnological conditions in the southern Athabasca sector will become increasingly less dominated by the short-term effects of flooding, and resemble nutrient- and solute-rich lakes in the northern Peace sector that are infrequently flooded. To determine the roles of the Athabasca River and atmospheric transport as vectors for the deposition of PACs in the PAD, sediment cores spanning the last ~200 years were collected from three lakes within the delta. A closed-drainage basin elevated well above the floodplain (PAD 18) was selected to determine temporal patterns of change in PAC concentration due to atmospheric deposition and within-basin production of PACs. Known patterns of paleohydrological changes at the other two lakes (PAD 23 and 31) were used to assess the role of the Athabasca River in delivering PACs to the Athabasca Delta during the ~200 year. Well- dated sediment core samples were analysed for 52 alkylated and non-alkylated PACs (method EPA 3540/8270-GC/MS). Sediments deposited in the non-flood prone lake (PAD 18) contained lower concentrations of total PACs compared to sediments deposited during flood-prone periods in the other study lakes, and were dominated by PACs of a pyrogenic rather than bitumen origin. Multivariate analysis of similarity tests identified that the composition of PACs differs between sediments deposited during not flood-prone and flood-prone periods. Subsequent Similarities Percentage (SIMPER) analysis was used and identified seven PACs that are preferentially deposited during flood-prone periods. These seven PACs are bitumen-associated, river-transported and account for 51% of the total PACs found in oil-sands sediment. At PAD 31, which has been flood-prone both before and since onset of Athabasca oil sands development, identified no measureable differences in both the proportion and concentration of the river-transported indicator PACs in sediments deposited pre-1940s versus post-1982. Our findings suggest that natural erosion of exposed bitumen along the banks of the Athabasca River and its tributaries is the main process delivering PACs to the Athabasca Delta, and that the spring freshet is a key period for contaminant mobilization and transport. Such key baseline environmental information is essential for informed management of natural resources and human-health concerns by provincial and federal regulatory agencies and industry, and for designing effective long-term monitoring and surveillance programs for the lower Athabasca River watershed in the face of future oil sands development. Further monitoring activities and additional paleolimnological studies of the depositional history of PACs and other oil-sands- and non-oil-sands-related contaminants is strongly recommended. Overall, results of this research identify that river flooding exerts strong control on physical, chemical and biological conditions of lakes within the PAD. However, contrary to prevailing paradigms, the PAD is not a landscape that has been adversely and permanently affected by regulation of the Peace River and industrial development of the oil sands along the Athabasca River. Instead, data from contemporary and paleolimnological studies identify that natural processes continue to dominate the delivery of water and contaminants to the delta. Regular and frequent flooding is not essential to maintain the supply of nutrients and productivity of delta lakes, which has been a widespread paradigm that developed in the absence of objective scientific data. Instead, nutrient concentrations rise over years to decades after flooding and lake productivity increases. During the thesis research, novel approaches were developed and demonstrated to be effective. Namely, new artificial substrate samplers were designed for aquatic biomonitoring that accrue periphyton and can identify the occurrence of flood events. Also, paleolimnological methods were employed to characterize the composition and concentration of PACs supplied by natural processes prior to oil sands industrial activity, which serves as an important benchmark for assessing industrial impacts. These are effective methods that can be employed to improve monitoring programs and scientific understanding of the factors affecting this world-renowned landscape, as well as floodplains elsewhere.

Indiana University-Purdue University Indianapolis (IUPUI) / It is increasingly recognized that mechanical properties of substrates play a pivotal role in the regulation of cellular fate and function. However, the underlying mechanisms of cellular mechanosensing still remain a topic of open debate. Traditionally, advancements in this field have been made using polymeric substrates of adjustable stiffness with immobilized linkers. While such substrates are well suited to examine cell adhesion and migration in an extracellular matrix environment, they are limited in their ability to replicate the rich dynamics found at cell-cell interfaces. To address this challenge, we recently introduced a linker-functionalized polymer-tethered multi-bilayer stack, in which substrate stiffness can be altered by the degree of bilayer stacking, thus allowing the analysis of cellular mechanosensitivity. Here, we apply this novel biomembrane-mimicking cell substrate design to explore the mechanosensitivity of C2C12 myoblasts in the presence of cell-cell-mimicking N-cadherin linkers. Experiments are presented, which demonstrate a relationship between the degree of bilayer stacking and mechanoresponse of plated cells, such as morphology, cytoskeletal organization, cellular traction forces, and migration speed. Furthermore, we illustrate the dynamic assembly of bilayer-bound N-cadherin linkers underneath cellular adherens junctions. In addition, properties of individual and clustered N-cadherins are examined in the polymer-tethered bilayer system in the absence of plated cells. Alternatively, substrate stiffness can be adjusted by the concentration of lipopolymers in a single polymer-tethered lipid bilayer. On the basis of this alternative cell substrate concept, we also discuss recent results on a linker-functionalized single polymer-tethered bilayer substrate with a lateral gradient in lipopolymer concentration (substrate viscoelasticity). Specifically, we show that the lipopolymer gradient has a notable impact on spreading, cytoskeletal organization, and motility of 3T3 fibroblasts. Two cases are discussed: 1. polymer-tethered bilayers with a sharp boundary between low and high lipopolymer concentration regions and 2. polymer-tethered bilayers with a gradual gradient in lipopolymer concentration.