Rapid microbiological detection method based on ultrasonic instrumentation

Los métodos de detección rápida de microorganismos se están convirtiendo en una herramienta esencial para el control de calidad en el área de la biotecnología, como es el caso de las industrias de alimentos y productos farmacéuticos y bioquímicos. En este escenario, el objetivo de esta tesis doctoral es desarrollar una técnica de inspección rápida de microoganismos basada en ultrasonidos. La hipótesis propuesta es que la combinación de un dispositivo ultrasónico de medida y un medio líquido diseñado específicamente para producir y atrapar burbujas, pueden constituir la base de un método sensible y rápido de detección de contaminaciones microbianas. La técnica presentada es efectiva para bacterias catalasa-positivas y se basa en la hidrólisis del peróxido de hidrógeno inducida por la catalasa. El resultado de esta reacción es un medio con una creciente concentración de burbujas. Tal medio ha sido estudiado y modelado desde el punto de vista de la propagación ultrasónica. Las propiedades deducidas a partir del análisis cinemático de la enzima se han utilizado para evaluar el método como técnica de inspección microbiana. En esta tesis, se han investigado aspectos teóricos y experimentales de la hidrólisis del peróxido de hidrógeno. Ello ha permitido describir cuantitativamente y comprender el fenómeno de la detección de microorganismos catalasa-positivos mediante la medida de parámetros ultrasónicos. Más concretamente, los experimentos realizados muestran cómo el oxígeno que aparece en forma de burbujas queda atrapado mediante el uso de un gel sobre base de agar. Este gel fue diseñado y preparado especialmente para esta aplicación. A lo largo del proceso de hidrólisis del peróxido de hidrógeno, se midió la atenuación de la onda y el “backscattering” producidos por las burbujas, utilizando una técnica de pulso-eco. Ha sido posible detectar una actividad de la catalasa de hasta 0.001 unidades/ml. Por otra parte, este estudio muestra que por medio del método propuesto, se puede lograr una detección microbiana para concentraciones de 105 células/ml en un periodo de tiempo corto, del orden de unos pocos minutos. Estos resultados suponen una mejora significativa de tres órdenes de magnitud en comparación con otros métodos de detección por ultrasonidos. Además, la sensibilidad es competitiva con modernos y rápidos métodos microbiológicos como la detección de ATP por bioluminiscencia. Pero sobre todo, este trabajo muestra una metodología para el desarrollo de nuevas técnicas de detección rápida de bacterias basadas en ultrasonidos. ABSTRACT In an industrial scenario where rapid microbiological methods are becoming essential tools for quality control in the biotechnological area such as food, pharmaceutical and biochemical; the objective of the work presented in this doctoral thesis is to develop a rapid microorganism inspection technique based on ultrasounds. It is proposed that the combination of an ultrasonic measuring device with a specially designed liquid medium, able to produce and trap bubbles could constitute the basis of a sensitive and rapid detection method for microbial contaminations. The proposed technique is effective on catalase positive microorganisms. Well-known catalase induced hydrogen peroxide hydrolysis is the fundamental of the developed method. The physical consequence of the catalase induced hydrogen peroxide hydrolysis is an increasingly bubbly liquid medium. Such medium has been studied and modeled from the point of view of ultrasonic propagation. Properties deduced from enzyme kinematics analysis have been extrapolated to investigate the method as a microbial inspection technique. In this thesis, theoretical and experimental aspects of the hydrogen peroxide hydrolysis were analyzed in order to quantitatively describe and understand the catalase positive microorganism detection by means of ultrasonic measurements. More concretely, experiments performed show how the produced oxygen in form of bubbles is trapped using the new gel medium based on agar, which was specially designed for this application. Ultrasonic attenuation and backscattering is measured in this medium using a pulse-echo technique along the hydrogen peroxide hydrolysis process. Catalase enzymatic activity was detected down to 0.001 units/ml. Moreover, this study shows that by means of the proposed method, microbial detection can be achieved down to 105 cells/ml in a short time period of the order of few minutes. These results suppose a significant improvement of three orders of magnitude compared to other ultrasonic detection methods for microorganisms. In addition, the sensitivity reached is competitive with modern rapid microbiological methods such as ATP detection by bioluminescence. But above all, this work points out a way to proceed for developing new rapid microbial detection techniques based on ultrasound.

Point diffraction interferometer with a liquid crystal monopixel

In this work a novel point diffraction interferometer based on a variable liquid crystal wave plate (LCWP) has been implemented. The LCWP consists of a 3x3 cm2 monopixel cell with parallel alignment. The monopixel cell was manufactured such that the electrode covers the entire surface except in a centered circular area of 50 μm of diameter. This circle acts as a point perturbation which diffracts the incident wave front giving rise to a spherical reference wave. By applying a voltage to the LCWP we can change the phase of the wave front that passes through the monopixel, except at the center. Phase shifting techniques are used in order to calculate the amplitude and phase distribution of the object wave front. The system allows a digital hologram to be obtained, and by using the Fresnel diffraction integral it is possible to digitally reconstruct the different planes that constitute the three dimensional object.

Multi-Camera very wide baseline feature matching based on view-adaptive junction detection

This paper presents a strategy for solving the feature matching problem in calibrated very wide-baseline camera settings. In this kind of settings, perspective distortion, depth discontinuities and occlusion represent enormous challenges. The proposed strategy addresses them by using geometrical information, specifically by exploiting epipolar-constraints. As a result it provides a sparse number of reliable feature points for which 3D position is accurately recovered. Special features known as junctions are used for robust matching. In particular, a strategy for refinement of junction end-point matching is proposed which enhances usual junction-based approaches. This allows to compute cross-correlation between perfectly aligned plane patches in both images, thus yielding better matching results. Evaluation of experimental results proves the effectiveness of the proposed algorithm in very wide-baseline environments.

Anomaly Detection Using Remote Sensing for the Archaeological Heritage Registration

The aim of this work is an approach using multisensor remote sensing techniques to recognize the potential remains and recreate the original landscape of three archaeological sites. We investigate the spectral characteristics of the reflectance parameter and emissivity in the pattern recognition of archaeological materials in several hyperspectral scenes of the prehispanic site in Palmar Sur (Costa Rica), the Jarama Valley site and the celtiberian city of Segeda in Spain. Spectral ranges of the visible-near infrared (VNIR), shortwave infrared (SWIR) and thermal infrared (TIR) from hyperspectral data cubes of HyMAP, AHS, MASTER and ATM have been used. Several experiments on natural scenarios of Costa Rica and Spain of different complexity, have been designed. Spectral patterns and thermal anomalies have been calculated as evidences of buried remains and change detection. First results, land cover change analyses and their consequences in the digital heritage registration are discussed.

Negative density dependence and environmental heterogeneity effects on tree ferns across succession in a tropical montane forest.

Although tree ferns are an important component of temperate and tropical forests, very little is known about their ecology. Their peculiar biology (e.g., dispersal by spores and two-phase life cycle) makes it difficult to extrapolate current knowledge on the ecology of other tree species to tree ferns. In this paper, we studied the effects of negative density dependence (NDD) and environmental heterogeneity on populations of two abundant tree fern species, Cyathea caracasana and Alsophila engelii, and how these effects change across a successional gradient. Species patterns harbor information on processes such as competition that can be easily revealed using point pattern analysis techniques. However, its detection may be difficult due to the confounded effects of habitat heterogeneity. Here, we mapped three forest plots along a successional gradient in the montane forests of Southern Ecuador. We employed homogeneous and inhomogeneous K and pair correlation functions to quantify the change in the spatial pattern of different size classes and a case-control design to study associations between juvenile and adult tree ferns. Using spatial estimates of the biomass of four functional tree types (short- and long-lived pioneer, shade- and partial shade-tolerant) as covariates, we fitted heterogeneous Poisson models to the point pattern of juvenile and adult tree ferns and explored the existence of habitat dependencies on these patterns. Our study revealed NDD effects for C. caracasana and strong environmental filtering underlying the pattern of A. engelii. We found that adult and juvenile populations of both species responded differently to habitat heterogeneity and in most cases this heterogeneity was associated with the spatial distribution of biomass of the four functional tree types. These findings show the effectiveness of factoring out environmental heterogeneity to avoid confounding factors when studying NDD and demonstrate the usefulness of covariate maps derived from mapped communities.

Impacts of global change on iberian forest ecosystems inferred from the spanish national forest inventory = Impactos del cambio global en ecosistemas forestales ibéricos a partir del inventario forestal nacional

Los efectos del cambio global sobre los bosques son una de las grandes preocupaciones de la sociedad del siglo XXI. Algunas de sus posibles consecuencias como son los efectos en la producción, la sostenibilidad, la pérdida de biodiversidad o cambios en la distribución y ensamblaje de especies forestales pueden tener grandes repercusiones sociales, ecológicas y económicas. La detección y seguimiento de estos efectos constituyen uno de los retos a los que se enfrentan en la actualidad científicos y gestores forestales. En base a la comparación de series históricas del Inventario Forestal Nacional Español (IFN), esta tesis trata de arrojar luz sobre algunos de los impactos que los cambios socioeconómicos y ambientales de las últimas décadas han generado sobre nuestros bosques. En primer lugar, esta tesis presenta una innovadora metodología con base geoestadística que permite la comparación de diferentes ciclos de inventario sin importar los diferentes métodos de muestreo empleados en cada uno de ellos (Capítulo 3). Esta metodología permite analizar cambios en la dinámica y distribución espacial de especies forestales en diferentes gradientes geográficos. Mediante su aplicación, se constatarán y cuantificarán algunas de las primeras evidencias de cambio en la distribución altitudinal y latitudinal de diferentes especies forestales ibéricas, que junto al estudio de su dinámica poblacional y tasas demográficas, ayudarán a testar algunas hipótesis biogeográficas en un escenario de cambio global en zonas de especial vulnerabilidad (Capítulos 3, 4 y 5). Por último, mediante la comparación de ciclos de parcelas permanentes del IFN se ahondará en el conocimiento de la evolución en las últimas décadas de especies invasoras en los ecosistemas forestales del cuadrante noroccidental ibérico, uno de los más afectados por la invasión de esta flora (Capítulo 6). ABSTRACT The effects of global change on forests are one of the major concerns of the XXI century. Some of the potential impacts of global change on forest growth, productivity, biodiversity or changes in species assembly and spatial distribution may have great ecological and economic consequences. The detection and monitoring of these effects are some of the major challenges that scientists and forest managers face nowadays. Based on the comparison of historical series of the Spanish National Forest Inventory (NFI), this thesis tries to shed some light on some of the impacts driven by recent socio-economic and environmental changes on our forest ecosystems. Firstly, this thesis presents an innovative methodology based on geostatistical techniques that allows the comparison of different NFI cycles regardless of the different sampling methods used in each of them (Chapter 3). This methodology, in conjunction with other statistical techniques, allows to analyze changes in the spatial distribution and population dynamics of forest species along different geographic gradients. By its application, this thesis presents some of the first evidences of changes in species distribution along different geographical gradients in the Iberian Peninsula. The analysis of these findings, of species population dynamics and demographic rates will help to test some biogeographical hypothesis on forests under climate change scenarios in areas of particular vulnerability (Chapters 3, 4 and 5). Finally, by comparing NFI cycles with permanent plots, this thesis increases our knowledge about the patterns and processes associated with the recent evolution of invasive species in the forest ecosystems of North-western Iberia, one of the areas most affected by the invasion of allien species at national scale (Chapter 6).

Deciduous tree reconstruction algorithm based on cylinder fitting from mobile terrestrial laser scanned point clouds

Vector reconstruction of objects from an unstructured point cloud obtained with a LiDAR-based system (light detection and ranging) is one of the most promising methods to build three dimensional models of orchards. The cylinder fitting method for woody structure reconstruction of leafless trees from point clouds obtained with a mobile terrestrial laser scanner (MTLS) has been analysed. The advantage of this method is that it performs reconstruction in a single step. The most time consuming part of the algorithm is generation of the cylinder direction, which must be recalculated at the inclusion of each point in the cylinder. The tree skeleton is obtained at the same time as the cluster of cylinders is formed. The method does not guarantee a unique convergence and the reconstruction parameter values must be carefully chosen. A balanced processing of clusters has also been defined which has proven to be very efficient in terms of processing time by following the hierarchy of branches, predecessors and successors. The algorithm was applied to simulated MTLS of virtual orchard models and to MTLS data of real orchards. The constraints applied in the method have been reviewed to ensure better convergence and simpler use of parameters. The results obtained show a correct reconstruction of the woody structure of the trees and the algorithm runs in linear logarithmic time

Optimization of Dengue immunoassay by label-free interferometric optical detection method

In this communication we report a direct immunoassay for detecting dengue virus by means of a label-free interferometric optical detection method. We also demonstrate how we can optimize this sensing response by adding a blocking step able to significantly enhance the optical sensing response. The blocking reagent used for this optimization is a dry milk diluted in phosphate buffered saline. The recognition curve of dengue virus over the proposed surface sensor demonstrates the capacity of this method to be applied in Point of Care technology.

What role does climate change play in agricultural market uncertainty? An integrated assessment taking into account market-driven adjustments

Recent studies point to climate change being one of the long-term drivers of agricultural market uncertainty. To advance in the understanding of the influence of climate change on future agricultural market developments, we compare a reference scenario for 2030 with alternative simulation scenarios that differ regarding: (1) emission scenarios; (2) climate projections; and (3) the consideration of carbon fertilization effects. For each simulation scenario, the CAPRI model provides global and EU-wide impacts of climate change on agricultural markets. Results show that climate change would considerably affect agrifood markets up to 2030. Nevertheless, market-driven adaptation strategies (production intensification, trade adjustments) would soften the impact of yield shocks on supply and demand. As a result, regional changes in production would be lower than foreseen by other studies focused on supply effects.

Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe’s average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 μg unspecific DNA without post-PCR probe manipulations could be achieved with different primer/probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Phosphorylation of Tyrosine 992, 1068, and 1086 Is Required for Conformational Change of the Human Epidermal Growth Factor Receptor C-Terminal Tail

We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the β-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr→Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2–binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation.

Paleoproterozoic snowball Earth: Extreme climatic and geochemical global change and its biological consequences

Geological, geophysical, and geochemical data support a theory that Earth experienced several intervals of intense, global glaciation (“snowball Earth” conditions) during Precambrian time. This snowball model predicts that postglacial, greenhouse-induced warming would lead to the deposition of banded iron formations and cap carbonates. Although global glaciation would have drastically curtailed biological productivity, melting of the oceanic ice would also have induced a cyanobacterial bloom, leading to an oxygen spike in the euphotic zone and to the oxidative precipitation of iron and manganese. A Paleoproterozoic snowball Earth at 2.4 Giga-annum before present (Ga) immediately precedes the Kalahari Manganese Field in southern Africa, suggesting that this rapid and massive change in global climate was responsible for its deposition. As large quantities of O2 are needed to precipitate this Mn, photosystem II and oxygen radical protection mechanisms must have evolved before 2.4 Ga. This geochemical event may have triggered a compensatory evolutionary branching in the Fe/Mn superoxide dismutase enzyme, providing a Paleoproterozoic calibration point for studies of molecular evolution.

Point of care testing: randomised controlled trial of clinical outcome

Objectives: To describe the proportion of patients attending an accident and emergency department for whom blood analysis at the point of care brought about a change in management; to measure the extent to which point of care testing resulted in differences in clinical outcome for these patients when compared with patients whose samples were tested by the hospital laboratory.

Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. donovani (TUBA5) that is deficient in LdNT1 transport and consequently resistant to tubercidin. In TUBA5 cells, the LdNT1.2 genes had the same sequence as wild-type cells. However, because LdNT1.2 mRNA is not detectable in either wild-type or TUBA5 promastigotes, LdNT1.2 does not contribute to nucleoside transport in this stage of the life cycle. In contrast, the TUBA5 cells were compound heterozygotes at the LdNT1.1 locus containing two mutant alleles that encompassed distinct point mutations, each of which impaired transport function. One of the mutant LdNT1.1 alleles encoded a G183D substitution in predicted TM 5, and the other allele contained a C337Y change in predicted TM 7. Whereas G183D and C337Y mutants had only slightly elevated adenosine Km values, the severe impairment in transport resulted from drastically (≈20-fold) reduced Vmax values. Because these transporters were correctly targeted to the plasma membrane, the reduction in Vmax apparently resulted from a defect in translocation. Strikingly, G183 was essential for pyrimidine nucleoside but not adenosine transport. A mutant transporter with a G183A substitution had an altered substrate specificity, exhibiting robust adenosine transport but undetectable uridine uptake. These results suggest that TM 5 is likely to form part of the nucleoside translocation pathway in LdNT1.1

Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060.

The 1,25-dihydroxyvitamin D3 [1,25-(OH)2vitamin D3] analog KH1060 exerts very potent effects on cell proliferation and cell differentiation via the vitamin D receptor (VDR). However, the activities of KH1060 are not associated with an increased affinity for the VDR. We now show that increased stabilization of the VDR-KH1060 complex could be an explanation for its high potencies. VDR half-life studies performed with cycloheximide-translational blocked rat osteoblast-like ROS 17/2.8 cells demonstrated that, in the absence of ligand, VDR levels rapidly decreased. After 2 hr, less than 10% of the initial VDR level could be measured. In the presence of 1,25-(OH)2vitamin D3, the VDR half-life was 15 hr. After 24 hr. less than 20% of the initial VDR content was detectable, whereas, at this time-point, when the cells were incubated with KH1060 80% of the VDR was still present. Differences in 1,25-(OH)2vitamin D3- and KH1060-induced conformational changes of the VDR could underlie the increased VDR stability. As assessed by limited proteolytic digestion analysis, both 1,25-(OH)2vitamin D3 and KH1060 caused a specific conformational change of the VDR. Compared with 1,25-(OH)2vitamin D3, KH1060 induced a conformational change that led to a far more dramatic protection of the VDR against proteolytic degradation. In conclusion, the altered VDR stability and the possibly underlying change in VDR conformation caused by KH1060 could be an explanation for its enhanced bioactivity.