Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Oceanografia Física, Química e Geológica, Instituto de Oceanografia, 2006. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2013-02-18T14:26:38Z No. of bitstreams: 1 2007_ana_renata.pdf: 1874697 bytes, checksum: bf17d3113d513b65a1b3794a25a72638 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2013-06-12T01:26:50Z (GMT) No. of bitstreams: 1 2007_ana_renata.pdf: 1874697 bytes, checksum: bf17d3113d513b65a1b3794a25a72638 (MD5) / Made available in DSpace on 2013-06-12T01:26:50Z (GMT). No. of bitstreams: 1 2007_ana_renata.pdf: 1874697 bytes, checksum: bf17d3113d513b65a1b3794a25a72638 (MD5) Previous issue date: 2006 / Em estuários, o carbono e o nitrogênio orgânicos particulados (COP e NOP) estão entre os principais constituintes do material em suspensão (MS). O carbono orgânico pode provir de fontes naturais em muitas áreas costeiras, porém tem-se observado processos de eutrofização originados de intensos aportes antrópicos de COP e NOP. Pesquisas relativas à fração orgânica do MS no estuário da Lagoa dos Patos são escassas para o carbono e nitrogênio. Em função de sua importância o presente trabalho estuda este aspecto, sendo parte do grupo temático “Qualidade Ambiental e Biodiversidade”, que se enquadrada no projeto “Uso e Apropriação dos Recursos Costeiros (RECOS)”, do Instituto do Milênio. O presente estudo tem como objetivo avaliar a distribuição de COP e NOP nas enseadas Saco da Mangueira (impactada) e Saco do Arraial (menos impactada), através do desenvolvimento do método de análise elementar CHNS, para avaliar o estado de contaminação orgânica das referidas enseadas e compará-lo com outros ambientes aquáticos. O presente estudo ocorreu de outubro/03 a junho/04, nas duas enseadas, com coletas bimensais da água de superfície em 4 locais de cada uma. As amostras foram filtradas, após os filtros foram secos a 60°C por 24 horas, descarbonatados com vapor de HCl concentrado e novamente secos a 60°C por 24 horas. As amostras foram analisadas para carbono e nitrogênio no equipamento Perkin Elmer CHN/S modelo 2400. Os resultados revelaram variações nas concentrações de COP e NOP na água de superfície, com maiores valores encontrados em dezembro/03 (primavera) e fevereiro/04 (verão). A enseada do Saco do Arraial mostrou características aproximadamente constantes nos níveis de COP (344 a 2354 μg L-1) e NOP (130 a 836 μg L-1), quando comparado com o Saco da Mangueira, sendo que as concentrações mais elevadas estão associadas a contribuições autóctones de produtores primários, destacando-se o fitoplâncton. Entretanto, a enseada do Saco da Mangueira apresentou influência provável de fatores antrópicos nos níveis de COP (193 a 2794 μg L-1) e NOP (72 a 2386 μg L-1) sendo mais expressivos no final da primavera e verão. Em ambas regiões, a participação média do fitoplâncton ao COP menor que 30%, associada aos baixos valores de relação C/N (4–5), confirmam a participação das bactérias juntamente com o fitoplâncton nos níveis de COP e NOP. Os valores mínimos e máximos de COP foram semelhantes entre os dois locais pesquisados, mas a concentração máxima de NOP esteve aproximadamente três vezes maior no Saco da Mangueira, sendo o parâmetro que melhor caracterizou sua eutrofização. Os elementos particulados apresentaram concentrações mínimas e máximas maiores que os valores obtidos nos demais estuários que participaram do Programa RECOS, e semelhantes aos níveis encontrados em outros ambientes aquáticos. Exceção foi encontrada para o valor máximo de NOP no Saco da Mangueira que ficou com concentrações acima dos demais estuários, devido à possibilidade do nitrogênio constituinte do MS ressuspendido ter uma origem não apenas orgânica, mas também inorgânica. Isso se deve ao longo histórico de efeitos humanos no Saco da Mangueira, com elevados aportes de nutrientes e materiais particulados provenientes da área urbana e industrial da cidade de Rio Grande. / In estuaries, the particulate organic carbon and nitrogen (POC and PON) are one of the main constituents of the material in suspension (MS). The organic carbon can come from natural sources in many coastal areas, however it has observed eutrofization processes originated of intense antropic input of POC and PON. Research about the organic fraction of the MS in the estuary of the Patos Lagoon is scarce for carbon and nitrogen. In function of its importance the present work study this subject, being part of the thematic group "Quality Ambient and Biodiversity", that belongs in the project "Use and Appropriation of Recursos Costeiros (RECOS)", of the Institute of the Milênio. The present study has as objective to evaluate the distribution of POC and PON in the Saco da Mangueira (impacted) and Saco do Arraial (less impacted), through the development of the method of elementary analysis CHNS, to evaluate the state of organic contamination of the mentioned embayments and to compare it with other aquatic environments. The present study occurred from outubro/03 to junho/04, in both embayments, with semimonthly collections of surface water in 4 places of each one. The samples were filtered, then the filters were dry at 60°C for 24 hours, exposed to a vapor of HCl and again dried at 60°C for 24 hours. The samples were analyzed for carbon and nitrogen in the equipment Perkin Elmer CHN/S model 2400. The results showed variations in the concentrations of POC and PON in the surface water, with higher values found in dezembro/03 (spring) and fevereiro/04 (summer). The Saco do Arraial showed approximate3ly constant characteristics in the levels of POC (344 the 2354 μg L-1) and PON (130 the 836 μg L-1), when compared with the Saco da Mangueira, being high concentrations associated with autoctones contributions of primary producers, distinguishing the phytoplankton. However, the Saco da Mangueira presented likely influence of antropic factors in the levels of POC (193 the 2794 μg L-1) and PON (72 the 2386 μg L-1) being expressive at the end of the spring and summer. In both regions, the average participation of phytoplankton to the POC lesser that 30%, associated to the low values of relation C/N (4-5), confirm the participation of the bacteria with phytoplankton in the levels of POC and PON. The minimum and maximum values of POC were similar between the two study places, but the maximum concentration of PON was approximately three times bigger in the Saco da Mangueira, being the parameter that better characterized its eutrofization. These particulate elements presented minimum and maximum concentrations greater that values of other estuaries, which participated of Program RECOS, and similar to the levels found in other aquatic environments. Exception was found for the maximum value of PON in the Saco da Mangueira that had concentrations higher than other estuaries, due to the nitrogen constituent of the ressuspended MS to have not only an organic origin, but also inorganic. This should be due to the long historic of human effect in the Saco da Mangueira, with high inputs of nutrients and particulate materials proceeding from urban and industrial areas of the city of Rio Grande.

Arctic rivers transport 31-42 Tg organic carbon (OC) each year to the Arctic Ocean, which is equal to 10% of the global riverine OC discharge. Since the Arctic Ocean only holds approximately 1% of the global ocean volume, the influence of terrestrially derived organic carbon (OCter) in the Arctic Ocean is relatively high. Despite the global importance of this region the behavior of the, by far largest fraction of the OCter, the dissolved organic carbon (DOC) in Arctic and sub-arctic estuaries is still a matter of debate. This thesis describes data originating from field cruises in Arctic and sub-arctic estuaries and coastal areas with the aim to improve the understanding of the fate of OCter in these areas, with specific focus on DOC. All presented studies indicate that DOCter and terrestrially derived particulate organic carbon (POCter) are subjected to substantial degradation in high-latitude estuaries, as shown by the non-conservative behavior of DOC in the East Siberian Arctic Shelf Seas (ESAS) (paper I) and the even more rapid degradation of POC in the same region (paper II). The removals of OCter in Arctic shelf seas were further supported by multiple isotope studies (paper III and IV), which showed that a use of 13C/12C in both OC and DIC, together with 34S/32S is a powerful tool to describe the sources and fate of OCter in estuaries and coastal seas. High-latitude estuaries play a key role in the coupling between terrestrial and marine carbon pools. In contrast to the general perception, this thesis shows that they are not only transportation areas for DOCter from rivers to the ocean, but are also active sites for transformation, degradation and sedimentation of DOCter, as well as for POCter. In a rapidly changing climate, the importance of these areas for the coupling between inorganic and organic carbon pools cannot be underestimated. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: In press. Paper 2: Submitted. Paper 4: Manuscript.</p>

The work described in this dissertation focuses on developing simple yet effective assays integrating nucleic acid (NA) aptamers with rolling circle amplification (RCA) for the detection of non-NA biomarkers. The first project, a comprehensive literature review, highlights the current state of the art in functional NA-based RCA applications, and identifies shortcomings in the detection of non-NA targets with RCA. Biosensor design is critically evaluated from four key perspectives: regulation, efficiency, and detection of RCA, and the integration of all three components for point of care (POC) applications. The second project investigates how target-binding to a linear aptamer can be utilized to regulate RCA in a simple and inexpensive format. Phi29 DNA polymerase (DP) exhibits difficulty processing DNA strands that are bound to non-NA materials such as proteins. The work uses this restriction of phi29 DP as a feature by utilizing protein-binding aptamers as primer strands (aptaprimers) for RCA. The simplicity is showcased by adapting the method to a cellulose paper-based device for the real-time detection and quantification of PDGF or thrombin within minutes. As the second project is a turn-off sensor, the third project exploits the inherent 3’-exonuclease activity of phi29 DP to generate a simple turn-on assay instead. As target-bound aptamers were shown to be resistant to exonuclease activity, the phi29 DP preferentially digests target-free aptaprimers instead of target-bound aptaprimers. The target-bound aptaprimer could be liberated by a circular template (CT) by incorporating toehold-mediated strand displacement (TMSD), and used for RCA. Sensitivity was improved relative to project two, though the dynamic range was narrow owing to difficulty liberating target-bound aptaprimer at high target concentrations. Project four instead used RecJ, which has 5’-exonuclease activity, to modulate aptaprimer availability. Similarly to project three, target-binding conferred protection on the aptaprimer from 5’-exonuclease digestion by RecJ. By including a free 3’ terminus on the aptaprimer, inhibition of RCA due to target binding was avoided and CT-mediated TMSD was not needed, simplifying the assay. As well, this approach was generalizable as it was demonstrated using both a protein (thrombin) and a small molecule (ochratoxin A) target. This turn-on method further improved the assay compared to project three with a 100-fold enhancement in sensitivity and a restoration of the dynamic range. In sum, this work contributed multiple simple and sensitive approaches for the real-time fluorescent detection of proteins and small molecules with the RCA of linear aptamers. / Thesis / Doctor of Science (PhD)

Preterm birth (PTB) results in over 15 million early births annually and is the leading cause of neonatal deaths. There are no clinical methods currently available to evaluate risk of PTB at early stages in pregnancy; thus, a rapid diagnostic to analyze PTB risk would be beneficial. Microfluidic immunoaffinity extraction is a promising platform for preparing complex samples, such as maternal serum with PTB risk biomarkers. 3D printed microfluidic devices have advantages over conventional microfluidic systems including simple fabrication and potential for iterative optimization to improve designs. In this work, I developed immunoaffinity monoliths in 3D printed microfluidic devices modified with antibodies to enrich PTB biomarkers from human blood serum. I retained and eluted a peptide PTB biomarker in both buffer and blood serum using an immunoaffinity column. An additional three PTB biomarkers were also successfully extracted either from buffer or blood serum on single-antibody columns. Both polyclonal and monoclonal antibodies to PTB biomarkers were characterized by dot blots, biolayer interferometry, and surface plasmon resonance to determine their specificity and dissociation constants. I created multiplexed immunoaffinity columns to simultaneously enrich three PTB biomarkers from depleted human blood serum in a single extraction. This is the first demonstration of multiplexed immunoaffinity columns for PTB biomarkers in a 3D printed microfluidic device. My work is a key step towards the future development of 3D printed microfluidic devices for rapid PTB testing.

No description available.

No description available.

Ce travail de thèse porte sur l’étude des mécanismes et des bilans de transfert des pesticides vers les cours d’eau sur deux bassins versants agricoles emboités, de tailles différentes en Gascogne (Sud Ouest de la France) : la Save à Larra (1110 km2).et le Montoussé (3,28 km2) à Auradé. Dans cette région, les pratiques d’une agriculture intensive conduisent à des risques importants pour les ressources en eau, notamment pendant les périodes de crue. C’est pour cela que nous avons porté une attention particulière dans cette étude à ces événements au cours desquels une grande quantité de contaminants sont transportés par les cours d’eau. Quatorze molécules de pesticides (herbicides et fongicides) largement utilisées sur ces bassins versants ont été étudiées grâce à un échantillonnage intensif, notamment en périodes de crue, durant deux années hydrologiques (2007/2008 et 2008/2009). La majorité des molécules étudiées présentent des concentrations qui dépassent les limites autorisées par l’Union Européenne pour l’eau potable (0.1 μg.L-1 pour chaque molécule et 0.5 μg.L-1 pour l’ensemble des molécules), notamment en périodes de crue. L’analyse des hystérésis mises en évidence sur les relations entre les concentrations (pesticides, MES, COD, POC) et les débits du cours d’eau permet de mieux comprendre les mécanismes de transfert des pesticides et de leurs paramètres de contrôle. Ces hystérésis montrent pour un même débit des concentrations différentes en montée et en descente de crue ; elles peuvent être dextres (sens des aiguilles d’une montre) ou senestres (sens inverse) suivant l’origine des molécules et l’écoulement responsable de leur transfert des sols vers les eaux. C’est pourquoi nous avons procédé dans cette étude à une séparation des différentes composantes (ruissellement superficiel, écoulement hypodermique, écoulement de nappe) de l’écoulement fluvial pour mieux comprendre la dynamique de transferts des pesticides et de leurs paramètres de contrôle. On peut ainsi mettre en évidence des relations positives entre MES, COD, COP ou certaines molécules de pesticides et les débits des écoulements de surface (ruissellement superficiel ou écoulement hypodermique suivant les caractéristiques physico-chimiques des molécules). Les calculs de flux des différents pesticides exportés par les cours d’eau montrent que 60 à 90% (suivant les molécules) des transports annuels se font durant les épisodes de crue. Les flux spécifiques (masse exportée par unité de surface) calculés sont plus importants sur le bassin versant de la Save que sur celui du Montoussé et les relations flux spécifiques-débits mettent en évidence des concentrations plus élévées sur la Save où l’utilisation des pesticides est globalement plus importante que sur le Montoussé où les pratiques sont raisonnées. L’analyse des pesticides dans l’eau filtrée et non filtrée nous a permis d’estimer la distribution de chaque molécule entre les phases particulaires et dissoutes (Kd). De plus, les valeurs de flux calculés pour chaque crue nous ont permis d’estimer un Kd moyen pour chaque molécule. Ces valeurs de Kd présentent une très bonne relation avec les valeurs de Kow (coefficient de partage octanol-eau) extraites de la littérature. De même, les pourcentages calculés de chaque pesticide exporté sous forme particulaire sont aussi très bien corrélés au Kow de chaque molécule. / The mechanisms of pesticides transport to stream flow were studied in two agricultural nested catchments of different size in Gascogne region (South West of France): the Save river basin at Larra (1110 km2) and the Montoussé experimental watershed at Auradé (3.28 km2). The intensive agricultural practices used in this region lead to an important risk for water resources by pesticides, especially during storm events. This is why we have paid special attention on storm events when a large quantity of contaminant was transported during hydrological periods. Fourteen molecules of pesticides (herbicides and fungicides) were investigated during the study period. Both of these groups are widely used for agricultural purposed in these catchments. The results achieved over the two years monitoring (2007-2009) enable us to emphasize the principal processes, implied in pesticide transfer on these agricultural catchments. The majority of compounds are detected during storm runoff events. And, the average concentrations of some pesticides are exceeded at the authorization limit of the European Union for pesticide concentrations in drinking water (0.1 µg.L-1 for individual pesticides and 0.5 µg.L-1 for total pesticides). To better understand the mechanisms of pesticide transport hysteresis, patterns on the concentration-discharge relationship (result of different concentration of pesticides in rising and falling limb of storm) were studied. However, clockwise or anticlockwise hysteresis patterns could be observed for some molecules of pesticide and their controlling factors such as dissolved organic carbon (DOC), particulate organic carbon (POC) and total suspended matters (TSM) according to their transfer dynamic in the catchment. We proceeded with hydrograph separation of the main stormflow components (surface runoff, subsurface flow and groundwater) so that the main pesticide routing could be traced for its soil-river transfers. We also came to the conclusion that there is a positive relationship between riverine TSM, DOC and pesticide, concentrations and the discharges of surface or subsurface runoffs according to pesticide properties. Pesticide flux calculation shows between 60 to 90% of the molecule transport takes place during storm periods. Specific flux calculation also demonstrated the higher flux value in Save catchment than in Aurade with higher pesticide concentration for a given specific discharge. The latter result may be due to the more consumption of pesticide in Save catchment. The analyses of pesticides both in filtered and unfiltered water enabled us to estimate the distribution of pesticides into particulate and dissolved phases. Moreover, the pesticide flux values allow calculating average partition coefficients kd between dissolved and particulate fractions which present good relationship with Kow values (octanol-water) extracted from literature. The percentage of each pesticide transported as particulate forms is also well correlated to Kow.

Existing terrestrial carbon accounting models have mainly investigated atmosphere-vegetationsoil stocks and fluxes but have largely ignored the hydrological flux of organic carbon. It is generally assumed that biomass and soil carbon are the only relevant pools in a landscape ecosystem. However, recent findings have suggested that significant amounts of organic carbon can dissolve (dissolved organic carbon or DOC) or particulate (particulate organic carbon or POC) in water and enter the hydrological flux at the catchment scale. A significant quantity of total organic carbon (TOC) sequestered through photosynthesis may be exported from the landscape through the hydrological flux and stored in downstream stocks.¶ This thesis presents a catchment-scale case study investigation into the export of organic carbon through a river system in comparison with carbon that is produced by vegetation through photosynthesis. The Cotter River Catchment was selected as the case study. It is a forested catchment that experienced a major wildfire event in January 2003. The approach is based on an integration of a number of models. The main input data were time series of in-stream carbon measurements and remotely sensed vegetation greenness. The application of models to investigate diffuse chemical substances has dramatically increased in the past few years because of the significant role of hydrology in controlling ecosystem exchange. The research firstly discusses the use of a hydrological simulation model (IHACRES) to analyse organic carbon samples from stream and tributaries in the Cotter River Catchment case study. The IHACRES rainfall-runoff model and a regionalization method are used to estimate stream-flow for the 75 sub-catchments. The simulated streamflow data were used to calculate organic carbon loads from concentrations sampled at five locations in the catchment.¶ The gross primary productivity (GPP) of the vegetation cover in the catchment was estimated using a radiation use efficiency (RUE) model driven by MODIS TERRA data on vegetation greenness and modeled surface irradiance (RS). The relationship between total organic carbon discharged in-stream and total carbon uptake by plants was assessed using a cross-correlation analysis.¶ The IHACRES rainfall-runoff model was successfully calibrated at three gauged sites and performed well. The results of the calibration procedure were used in the regionalization method that enabled streamflow to be estimated at ungauged locations including the seven sampling sites and the 75 sub-catchment areas. The IHACRES modelling approach was found appropriate for investigating a wide range of issues related to the hydrological export of organic carbon at the catchment scale. A weekly sampling program was implemented to provide estimates of TOC, DOC and POC concentrations in the Cotter River Catchment between July 2003 and June 2004. The organic carbon load was estimated using an averaging method.¶ The rate of photosynthesis by vegetation (GPP) was successfully estimated using the radiation use efficiency model to discern general patterns of vegetation productivity at sub-catchment scales. This analysis required detailed spatial resolution of the GPP across the entire catchment area (comprising 75 sub-catchment areas) in addition to the sampling locations. Important factors that varied at the catchment scale during the sampling period July 2003 – June 2004, particularly the wildfire impacts, were also considered in this assessment. ¶ The results of the hydrologic modelling approach and terrestrial GPP outcome were compared using cross correlation and regression analysis. This comparison revealed the likely proportion of catchment GPP that contributes to in-stream hydrological flux of organic carbon. TOC Load was 0.45% of GPP and 22.5 - 25% of litter layer. As a result of this investigation and giving due consideration to the uncertainties in the approach, it can be concluded that the hydrological flux of organic carbon in a forested catchment is a function of gross primary productivity.

Medical devices are used widely at every stage of disease diagnosis and treatment. To eradicate certain infectious diseases, the development of highly sensitive diagnostic tools and techniques is essential. The work reported in this thesis presents a novel approach, which can be used for the diagnosis of various diseases in the field of clinical cytology. The central theme of this approach was to develop a simple, holistic and completely automated system for point-of-care (POC) diagnostics. This is realized through the Development of an Absorption Flow-Cytometer with Synergistic Integration of Microfluidic, Optics and simple Electronics. Quantitative diagnosis of malaria has been taken as test case for the characterization and validation of the developed technology. Malaria is a life-threatening disease widely prevalent in developing countries. Approximately half the world population undergoes a test of malaria and it kills close to half a million people every year. Early detection and treatment will reduce the number of fatalities and also decrease its transmission rate. In the recent past, several diagnostic tools have been developed to detect malaria but there are varied demands on diagnostic instruments in healthcare settings and endemic contexts. The objective of this thesis is to develop an instrument capable of identifying malaria-infected red blood cells (i-RBCs) from a given few micro-liters of whole blood. The optical absorption properties of blood cells were measured at a single-cell level to diagnose malaria. The proof-of-concept for the instrument was established in four stages, after which a prototype was also developed and validated. In the first stage, a system capable of simultaneously imaging cells and also measuring their optical absorbance properties was developed. The developed system was employed to characterize absorption properties of red blood cells (malaria-infected and healthy ones) on blood-smear. A custom-made bright-field transmission microscope in combination with a pair of laser diode and photo-detector was used to simultaneously image and measure transmittance of infected and uninfected RBCs. In the second stage, the technique was extended to enable high-throughput measurements with the use of microfluidic sample handling and synchronous data acquisition. Using this technique, the optical absorbance and morphology of infected and healthy RBCs have been characterized in statistically significant numbers. The correlation between cell morphology (from images) and single-cell optical absorbance level helped to establish the thresholds for differentiating healthy and infected cells. In the third stage, a portable prototype capable of assessing optical absorbance levels of single cells was fabricated. The developed prototype is capable of assessing cells at throughputs of about 1800 cells/ second. It was initially validated with sample suspensions containing infected and healthy RBCs obtained from malaria cultures. For the device to be usable at the field-level, it has to function in the presence of all other cellular components of whole blood. The optical absorbance of other cellular components of blood like white blood cells and platelets, were characterized. The device was finally tested with blood samples spiked with malaria-infected RBCs validating the overall proof-of-concept and the developed prototype. The deployment of such cost-effective, automated POC system would enable malaria diagnosis at remote locations and play a crucial role in the ongoing efforts to eradicate malaria. In future, the presented technology can be extended to develop POC diagnostic tool for other diseases as well. As it enables quantitative estimation of malaria, the present optical absorption flow analyzer would also find application in disease prognosis monitoring, anti-malarial drug development and other studies requiring measurements on a single-cell basis. The hyper-imaging system can be used to characterize and validate the threshold information, and can be incorporated in the prototype. Thus, it is a continuous process to characterization and implementation in the prototype. The optofluidic absorption flow analyzer will help enable affordable clinical diagnostic testing in resource limited settings. This approach will be extended to diagnose other diseases, using differences in optical absorption as criteria for differentiating healthy and infected cells.

No description available.