Laser-mediated, site-specific inactivation of RNA transcripts

The biological function of specific gene products often is determined experimentally by blocking their expression in an organism and observing the resulting phenotype. Chromophore-assisted laser inactivation using malachite green (MG)-tagged antibodies makes it possible to inactivate target proteins in a highly restricted manner, probing their temporally and spatially resolved functions. In this report, we describe the isolation and in vitro characterization of a MG-binding RNA motif that may enable the same high-resolution analysis of gene function specifically at the RNA level (RNA-chromophore-assisted laser inactivation). A well-defined asymmetric internal bulge within an RNA duplex allows high affinity and high specificity binding by MG. Laser irradiation in the presence of low concentrations of MG induces destruction of the MG-binding RNA but not of coincubated control RNA. Laser-induced hydrolysis of the MG-binding RNA is restricted predominantly to a single nucleotide within the bulge. By appropriately incorporating this motif into a target gene, transcripts generated by the gene may be effectively tagged for laser-mediated destruction.

A natural experiment on plant acclimation: lifetime stomatal frequency response of an individual tree to annual atmospheric CO2 increase.

Carbon dioxide (CO2) has been increasing in atmospheric concentration since the Industrial Revolution. A decreasing number of stomata on leaves of land plants still provides the only morphological evidence that this man-made increase has already affected the biosphere. The current rate of CO2 responsiveness in individual long-lived species cannot be accurately determined from field studies or by controlled-environment experiments. However, the required long-term data sets can be obtained from continuous records of buried leaves from living trees in wetland ecosystems. Fine-resolution analysis of the lifetime leaf record of an individual birch (Betula pendula) indicates a gradual reduction of stomatal frequency as a phenotypic acclimation to CO2 increase. During the past four decades, CO2 increments of 1 part per million by volume resulted in a stomatal density decline of approximately 0.6%. It may be hypothesized that this plastic stomatal frequency response of deciduous tree species has evolved in conjunction with the overall Cenozoic reduction of atmospheric CO2 concentrations.

In vivo protein-DNA interactions at human DNA replication origin.

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basic-helix-loop-helix family, nuclear respiratory factor 1, transcription factor Sp1, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation.

(Table 1) Age model of sediment core MD03-2705

A reconstruction of northwest African summer monsoon strength during the cold marine isotopic stage (MIS) 6 indicates a link to the seasonal migration of the Intertropical Convergence Zone (ITCZ). High-resolution studies of eolian dust supply and sea surface temperature recorded in marine core MD03-2705, on the Mauritanian margin, provide a better understanding about the penultimate glacial history of northwestern African aridity/humidity and upwelling coastal activity. Today, site MD03-2705 experiences increased upwelling and dust flux during the winter months, when the ITCZ is in a southerly position. Analyses of foraminifera isotopic composition suggest that during MIS 6.5 (180-168 ka) the average position of the ITCZ migrated north, marked by an increase in the strength of the summer monsoon, which decreased eolian dust transport and the coastal upwelling activity. The northward migration is in phase with a specific orbital combination of a low precessional index with a high obliquity signal. High-resolution analysis of stable isotopes (d18O and d13C) and microscale resolution geochemical (Ti/Al and quartz grain counts) determinations reveal that the transition between monsoonal humid (MIS 6.5) and dry (MIS 6.4) conditions has occurred in less than 1.3 ka. Such rapid changes suggest a nonlinear link between the African monsoonal rainfall system and environmental changes over the continent. This study provides new insights about the influence of vegetation and oceanic temperature feedbacks on the onset of African summer monsoon and demonstrates that, during the penultimate glacial period, changes in tropical dynamics had regional and global impacts.

From cardinal spline wavelet bases to highly coherent dictionaries

Wavelet families arise by scaling and translations of a prototype function, called the mother wavelet. The construction of wavelet bases for cardinal spline spaces is generally carried out within the multi-resolution analysis scheme. Thus, the usual way of increasing the dimension of the multi-resolution subspaces is by augmenting the scaling factor. We show here that, when working on a compact interval, the identical effect can be achieved without changing the wavelet scale but reducing the translation parameter. By such a procedure we generate a redundant frame, called a dictionary, spanning the same spaces as a wavelet basis but with wavelets of broader support. We characterize the correlation of the dictionary elements by measuring their 'coherence' and produce examples illustrating the relevance of highly coherent dictionaries to problems of sparse signal representation.

(Table 1) TOC, d13C, concentrations of C37 alkenones, crenarchaeol and GDGTs of turbidites

One of the primary prerequisites for the application of organic proxies is that they should not be substantially affected by diagenesis. However, studies have shown that oxic degradation of biomarker lipids can affect their relative distribution. We tested the diagenetic stability of the UK'37 and TEX86 palaeothermometers upon long term oxygen exposure. For this purpose, we studied the distributions of alkenones and glycerol dialkyl glycerol tetraethers (GDGTs) in different sections of turbidites at the Madeira Abyssal Plain (MAP) that experienced different degrees of oxygen exposure. Sediments were deposited anoxically on the shelf and then transported by turbidity currents to the MAP, which has oxic bottom water. This resulted in partial degradation of the turbidite organic matter as a result of long term exposure to oxic bottom water. Concentrations of GDGTs and alkenones were reduced by one to two orders of magnitude in the oxidized parts of the turbidites compared to the unoxidized parts, indicating substantial degradation. High-resolution analysis of the Pleistocene F-turbidite showed that the UK'37 index of long chain alkenones increased only slightly (0.01, corresponding to <0.5 °C) in the oxidized part of the turbidite, suggesting minor preferential degradation of the C37:3 alkenone, in agreement with previous studies. TEX86 values showed a small increase (0.02, corresponding to ~2 °C) in the F-turbidite, like UK'37 , while for other Pliocene/Miocene turbidites it either remained unchanged or decreased substantially (up to 0.06, corresponding to ~6 °C). Previous observations showed that the BIT index, a proxy for the contribution of soil organic matter to total organic carbon, was always substantially higher in the oxidized part in all the turbidites, as a result of preferential degradation of marine-derived GDGTs. This relative increase in soil-derived GDGTs affects TEX86, as the isoprenoid GDGT distribution on the continent can be quite different from that in the marine environment. Our results indicate that the organic proxies are affected by long term oxic degradation to different extents; this should be taken into account when applying these proxies in palaeoceanographic studies of sediments which have been exposed to prolonged oxic degradation.

From the traditional verification approach to the spatial methods: a study of applicability of the SAL metric in the framework of the WMO project MesoVICT

Increasing in resolution of numerical weather prediction models has allowed more and more realistic forecasts of atmospheric parameters. Due to the growing variability into predicted fields the traditional verification methods are not always able to describe the model ability because they are based on a grid-point-by-grid-point matching between observation and prediction. Recently, new spatial verification methods have been developed with the aim of show the benefit associated to the high resolution forecast. Nested in among of the MesoVICT international project, the initially aim of this work is to compare the newly tecniques remarking advantages and disadvantages. First of all, the MesoVICT basic examples, represented by synthetic precipitation fields, have been examined. Giving an error evaluation in terms of structure, amplitude and localization of the precipitation fields, the SAL method has been studied more thoroughly respect to the others approaches with its implementation in the core cases of the project. The verification procedure has concerned precipitation fields over central Europe: comparisons between the forecasts performed by the 00z COSMO-2 model and the VERA (Vienna Enhanced Resolution Analysis) have been done. The study of these cases has shown some weaknesses of the methodology examined; in particular has been highlighted the presence of a correlation between the optimal domain size and the extention of the precipitation systems. In order to increase ability of SAL, a subdivision of the original domain in three subdomains has been done and the method has been applied again. Some limits have been found in cases in which at least one of the two domains does not show precipitation. The overall results for the subdomains have been summarized on scatter plots. With the aim to identify systematic errors of the model the variability of the three parameters has been studied for each subdomain.

Toward an understanding of the atmospheric forcing uncertainties in the Mediterranean Sea

The thesis has extensively investigated for the first time the statistical distributions of atmospheric surface variables and heat fluxes for the Mediterranean Sea. After retrieving a 30-year atmospheric analysis dataset, we have captured the spatial patterns of the probability distribution of the relevant atmospheric variables for ocean atmospheric forcing: wind components (U,V), wind amplitude, air temperature (T2M), dewpoint temperature (D2M) and mean sea-level pressure (MSL-P). The study reveals that a two-parameter PDF is not a good fit for T2M, D2M, MSL-P and wind components (U,V) and a three parameter skew-normal PDF is better suited. Such distribution captures properly the data asymmetric tails (skewness). After removing the large seasonal cycle, we show the quality of the fit and the geographic structure of the PDF parameters. It is found that the PDF parameters vary between different regions, in particular the shape (connected to the asymmetric tails) and the scale (connected to the spread of the distribution) parameters cluster around two or more values, probably connected to the different dynamics that produces the surface atmospheric fields in the Mediterranean basin. Moreover, using the atmospheric variables, we have computed the air-sea heat fluxes for a 20-years period and estimated the net heat budget over the Mediterranean Sea. Interestingly, the higher resolution analysis dataset provides a negative heat budget of –3 W/m2 which is within the acceptable range for the Mediterranean Sea heat budget closure. The lower resolution atmospheric reanalysis dataset(ERA5) does not satisfy the heat budget closure problem pointing out that a minimal resolution of the atmospheric forcing is crucial for the Mediterranean Sea dynamics. The PDF framework developed in this thesis will be the basis for a future ensemble forecasting system that will use the statistical distributions to create perturbations of the atmospheric ocean forcing.

Texture analysis of high resolution MRI allows discrimination between febrile and afebrile initial precipitating injury in mesial temporal sclerosis

A computational pipeline combining texture analysis and pattern classification algorithms was developed for investigating associations between high-resolution MRI features and histological data. This methodology was tested in the study of dentate gyrus images of sclerotic hippocampi resected from refractory epilepsy patients. Images were acquired using a simple surface coil in a 3.0T MRI scanner. All specimens were subsequently submitted to histological semiquantitative evaluation. The computational pipeline was applied for classifying pixels according to: a) dentate gyrus histological parameters and b) patients' febrile or afebrile initial precipitating insult history. The pipeline results for febrile and afebrile patients achieved 70% classification accuracy, with 78% sensitivity and 80% specificity [area under the reader observer characteristics (ROC) curve: 0.89]. The analysis of the histological data alone was not sufficient to achieve significant power to separate febrile and afebrile groups. Interesting enough, the results from our approach did not show significant correlation with histological parameters (which per se were not enough to classify patient groups). These results showed the potential of adding computational texture analysis together with classification methods for detecting subtle MRI signal differences, a method sufficient to provide good clinical classification. A wide range of applications of this pipeline can also be used in other areas of medical imaging. Magn Reson Med, 2012. (c) 2012 Wiley Periodicals, Inc.

Analysis of the E-J Curve of HTS Tapes Under DC and AC Magnetic Fields at 77 K

The evaluation of the electrical characteristics of technical HTS tapes are of the key importance in determining the design and operational features of superconducting power apparatuses as well as to understand the external factors which affect the superconducting performance. In this work we report the systematic measurements of the electric field versus current density, E-J relation of short samples for three commercial HTS tapes (BSCCO-2223 tapes, with and without steel reinforcement, and YBCO-coated conductor) at 77 K. In order to get sensitive and noiseless voltage signals the measurements were carried out with DC transport current and subjecting the broad surface tape to DC (0-300 mT) and AC (0-62 mT, 60 Hz) magnetic fields. The voltage is measured by a sensitive nanovoltmeter and the applied magnetic field is monitored by a Hall sensor placed on the tape broad surface. The comparison between the results obtained from the three tapes was done by fitting a power-law equation for currents in the vicinity of the critical current. For the current regime below the critical one a linear correlation of the electric field against the current density is observed. The BSCCO samples presented the same behavior, i.e., a decreasing of n-index with the increasing DC and AC magnetic field strength. Under AC field the decreasing slope of n-index is steeper as compared to DC field. The n-index curve for the YBCO tape showed similar behavior for AC field, however under DC field in the 0-390 mT range exhibited a slight decreasing of the n-index.

Analysis of Detergent-Insoluble and Whole Cell Lysate Fractions of Resting Neutrophils Using High-Resolution Mass Spectrometry

Neutrophilic granulocytes play a major role in the initiation and resolution of the inflammatory response, and demonstrate significant transcriptional and translational activity. Although much was known about neutrophils prior to the introduction of proteomics, the use of MS-based methodologies has provided an unprecedented tool to confirm and extend previous findings. In the present study, we performed a Gel-LC-MS/MS analysis of neutrophil detergent insoluble and whole cell lysate fractions of resting neutrophils. We achieved a set of identifications through the use of high-resolution mass spectrometry and validation of its data. We identified a total of 1249 proteins with a wide range of intensities from both detergent-insoluble and whole cell lysate fractions, allowing a mapping of proteins such as those involved in intracellular transport (Rab and Sec family proteins) and cell signaling (S100 proteins). These results represent the most comprehensive proteomic characterization of resting human neutrophils to date, and provide important information relevant for further studies of the immune system in health and disease. The methods applied here can be employed to help us understand how neutrophils respond to various physiologic and pathophysiologic conditions and could be extended to protein quantitation after cell activation.

Analysis of PCBs in soils and sediments by microwave-assisted extraction, headspace-SPME and high resolution gas chromatography with ion-trap tandem mass spectrometry

A procedure for the determination of seven indicator PCBs in soils and sediments using microwave-assisted extraction (MAE) and headspace solid-phase microextraction (HS-SPME) prior to GC-MS/MS is described. Optimization of the HS-SPME was carried out for the most important parameters such as extraction time, sample volume and temperature. The adopted methodology has reduced consumption of organic solvents and analysis runtime. Under the optimized conditions, the method detection limit ranged from 0.6 to 1 ng/g when 5 g of sample was extracted, the precision on real samples ranged from 4 to 21% and the recovery from 69 to 104%. The proposed method, which included the analysis of a certified reference material in its validation procedure, can be extended to several other PCBs and used in the monitoring of soil or sediments for the presence of PCBs.

Multi-elemental analysis of ready-to-eat “baby leaf” vegetables using microwave digestion and high-resolution continuum source atomic absorption spectrometry

The mineral content (phosphorous (P), potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn) and copper (Cu)) of eight ready-to-eat baby leaf vegetables was determined. The samples were subjected to microwave-assisted digestion and the minerals were quantified by High-Resolution Continuum Source Atomic Absorption Spectrometry (HR-CS-AAS) with flame and electrothermal atomisation. The methods were optimised and validated producing low LOQs, good repeatability and linearity, and recoveries, ranging from 91% to 110% for the minerals analysed. Phosphorous was determined by a standard colorimetric method. The accuracy of the method was checked by analysing a certified reference material; results were in agreement with the quantified value. The samples had a high content of potassium and calcium, but the principal mineral was iron. The mineral content was stable during storage and baby leaf vegetables could represent a good source of minerals in a balanced diet. A linear discriminant analysis was performed to compare the mineral profile obtained and showed, as expected, that the mineral content was similar between samples from the same family. The Linear Discriminant Analysis was able to discriminate different samples based on their mineral profile.

Principal component analysis of the yield curve

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and Economics