Simplified sample handing in mass spectrometry based protein research - focus on protein phosphorylation

The human genome comprises roughly 20 000 protein coding genes. Proteins are the building material for cells and tissues, and proteins are functional compounds having an important role in many cellular responses, such as cell signalling. In multicellular organisms such as humans, cells need to communicate with each other in order to maintain a normal function of the tissues within the body. This complex signalling between and within cells is transferred by proteins and their post-translational modifications, one of the most important being phosphorylation. The work presented here concerns the development and use of tools for phosphorylation analysis. Mass spectrometers have become essential tools to study proteins and proteomes. In mass spectrometry oriented proteomics, proteins can be identified and their post-translational modifications can be studied. In this Ph.D. thesis the objectives were to improve the robustness of sample handling methods prior to mass spectrometry analysis for peptides and their phosphorylation status. The focus was to develop strategies that enable acquisition of more MS measurements per sample, higher quality MS spectra and simplified and rapid enrichment procedures for phosphopeptides. Furthermore, an objective was to apply these methods to characterize phosphorylation sites of phosphopeptides. In these studies a new MALDI matrix was developed which allowed more homogenous, intense and durable signals to be acquired when compared to traditional CHCA matrix. This new matrix along with other matrices was subsequently used to develop a new method that combines multiple spectra from different matrises from identical peptides. With this approach it was possible to identify more phosphopeptides than with conventional LC/ESI-MS/MS methods, and to use 5 times less sample. Also, phosphopeptide affinity MALDI target was prepared to capture and immobilise phosphopeptides from a standard peptide mixture while maintaining their spatial orientation. In addition a new protocol utilizing commercially available conductive glass slides was developed that enabled fast and sensitive phosphopeptide purification. This protocol was applied to characterize the in vivo phosphorylation of a signalling protein, NFATc1. Evidence for 12 phosphorylation sites were found, and many of those were found in multiply phosphorylated peptides

Anchor or Accelerate – A Study on Cancer Cell Adhesion and Motility

Cell migration and adhesion to the extracellular matrix (ECM) are crucial in many biological and pathological processes such as morphogenesis, tissue repair, inflammatory responses, survival, and cancer. Cell-matrix adhesion is mediated by the integrin family of transmembrane receptors, which not only anchor cells to their surroundings, but also transmit bidirectional signalling at the cell surface and couple the ECM to the cytoskeleton. Another group of adhesion receptors are the syndecan proteoglycans, which engage the ECM and possess signalling activity in response to a variety of ligands. Cell migration is a complex process that requires spatial and temporal coordination of adhesion, cell contractility, intracellular traffic of integrins, and matrix turnover by matrix metalloproteinases (MMPs). Thus, integrins and syndecans, as well as MMPs, play essential roles in cancer cell migration and invasion. The understanding of the cooperation of syndecans and integrins was broadened in this thesis study. The results reveal that syndecan-1 functions in concert with 21 integrin in cell adhesion to collagen, whereas syndecan-4 is essential in 21 integrin-mediated matrix contraction. Finally, oncogenic K-Ras was shown to regulate 21 integrin, membrane-type 1 MMP, and syndecan-1 and -4 expression and their cooperation in cell invasion. Epithelial-mesenchymal transition (EMT) is fundamental during embryogenesis and organ development. Activation of EMT processes, including the upregulation of mesenchymal intermediate filament protein vimentin, has also been implicated in the acquisition of a malignant phenotype by epithelial cancer cells. Members of the protein kinase C (PKC) superfamily are involved in cell migration and various integrindependent cellular functions. One aim of this work was to shed light on the role of vimentin in the regulation of integrin traffic and cell motility. In addition, the mechanism by which vimentin participates in EMT was investigated. The results show that integrin recycling and motility are dependent on the PKC–mediated phosphorylation of vimentin. In addition, vimentin was found to be a positive regulator of EMT and regulate the expression of several migratory genes. Specifically, vimentin governs the expression of receptor tyrosine kinase Axl, which is implicated in tumour growth and metastasis. Taken together, the findings described in this thesis reveal novel aspects of the complex interplay between distinct cellular components: integrins, syndecans, and the vimentin cytoskeleton, which all contribute to the regulation of human cancer cell adhesion, migration, and invasion.

Potential impact of the global climate changes on the spatial distribution of areas of risk for the occurrence of eucalyptus rust in Brazil

Rust, caused by Puccinia psidii, is one of the most important diseases affecting eucalyptus in Brazil. This pathogen causes disease in mini-clonal garden and in young plants in the field, especially in leaves and juvenile shoots. Favorable climate conditions for infection by this pathogen in eucalyptus include temperature between 18 and 25 ºC, together with at least 6-hour leaf wetness periods, for 5 to 7 consecutive days. Considering the interaction between the environment and the pathogen, this study aimed to evaluate the potential impact of global climate changes on the spatial distribution of areas of risk for the occurrence of eucalyptus rust in Brazil. Thus, monthly maps of the areas of risk for the occurrence of this disease were elaborated, considering the current climate conditions, based on a historic series between 1961 and 1990, and the future scenarios A2 and B2, predicted by IPCC. The climate conditions were classified into three categories, according to the potential risk for the disease occurrence, considering temperature (T) and air relative humidity (RH): i) high risk (18 < T < 25 ºC and RH > 90%); ii) medium risk (18 < T < 25 ºC and RH < 90%; T< 18 or T > 25 ºC and RH > 90%); and iii) low risk (T < 18 or T > 25 ºC and RH < 90%). Data about the future climate scenarios were supplied by GCM Change Fields. In this study, the simulation model Hadley Centers for Climate Prediction and Research (HadCm3) was adopted, using the software Idrisi 32. The obtained results led to the conclusion that there will be a reduction in the area favorable to eucalyptus rust occurrence, and such a reduction will be gradual for the decades of 2020, 2050 and 2080 but more marked in scenario A2 than in B2. However, it is important to point out that extensive areas will still be favorable to the disease development, especially in the coldest months of the year, i.e., June and July. Therefore, the zoning of areas and periods of higher occurrence risk, considering the global climate changes, becomes important knowledge for the elaboration of predicting models and an alert for the integrated management of this disease.

Matrix- based logic for avoiding paradoxes and its paraconsistent alternative

The present article shows that there are consistent and decidable many- valued systems of propositional logic which satisfy two or all the three criteria for non- trivial inconsistent theories by da Costa (1974). The weaker one of these paraconsistent system is also able to avoid a series of paradoxes which come up when classical logic is applied to empirical sciences. These paraconsistent systems are based on a 6- valued system of propositional logic for avoiding difficulties in several domains of empirical science (Weingartner (2009)).

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

POPULATION STRUCTURE AND SPATIAL DISTRIBUTION OF Bauhinia cheilantha (Bong.) Steud. IN TWO FRAGMENTS AT DIFFERENT REGENERATION STAGES IN THE CAATINGA, IN SERGIPE, BRAZIL

ABSTRACTThis study aimed to analyze the vertical and diameter structure and the spatial distribution pattern of Bauhinia cheilantha in two Caatinga fragments in Sergipe, Brazil, at different regeneration stages. Thirty plots were demarcated in area I (Canindé de São Francisco and Poço Redondo), which has vegetation regeneration, and 25 plots in area II (Porto da Folha) with preserved vegetation, both having 400 m2. All B. cheilanthaindividuals had their height and circumference (circumference at breast height > 6 cm) measured. Possible differences in height and diameter at breast height were tested in the two populations by using Student’s T-test. The distribution pattern of species was calculated through Payandeh’s index. We sampled 154 B. cheilantha individuals, equivalent to 33.3% of the plots in area I and in 1,027 individuals in area II, totaling 100% frequency. Height and the diameter of the two populations were statistically different, where AI achieved all values lower than AII. The spatial distribution pattern of B. cheilantha found in both areas was aggregate, with values of 11.85 and 9.00, respectively. Thus, it became clear that the population in AII is at a more advanced successional status than AI, due to its longer conservation time.

Spatial variability of leaf wetness duration in a 'Niagara Rosada' vineyard

Despite considerable efforts to develop accurate electronic sensors to measure leaf wetness duration (LWD), little attention has been given to studies about how is LWD variability in different positions of the crop canopy. In order to evaluate the influence of 'Niagara Rosada' (Vitis labrusca) grapevine structure on the spatial variability of LWD, the objective of this study was to determine the canopy position of the ‘Niagara Rosada’ table grape with longer LWD and its correlation with measured standard LWD over turfgrass. LWD was measured in four different canopy positions of the vineyard (sensors deployed at 45º with the horizontal): at the top of the plants, with sensors facing southwest and northeast (Top-SW and Top-NE), and at the grape bunches height, with sensors facing southwest and northeast (Bottom-SW and Bottom-NE). No significant difference was observed between the top (1.6 m) and the bottom (1.0 m) of the canopy and also between the southwest and northeast face of the plants. The relationship between standard LWD over turfgrass and crop LWD in different positions of the grape canopy showed a define correlation, with R² ranging from 0.86 to 0.89 for all period, from 0.72 to 0.77 for days without rain, and from 0.89 to 0.91 for days with rain.

Determining the concrete stiffness matrix through ultrasonic testing

The determination of the modulus tangent (Eci ) and of the modulus secant (Ecs) of the concrete can be done using compression test but, to be simpler, it is used relations with characteristic strength (f ck). Relations are also used to determine the transversal modulus (Gc) and, in the case of the Poisson's ratio (ν), a fixed value 0.20 is established. The objective of this research was to evaluate the use of the ultrasonic propagation waves to determine these properties. For the tests were used specimens with f ck varying from 10 to 35 MPa. For the ultrasonic tests were used cylindrical and cubic specimens. The modulus of deformation obtained by ultrasound was statistically equivalent to the obtained by compression tests. The results of modules obtained using the relations with f ck was far away from those obtained by ultrasound or by compression tests. The Poisson's ratio obtained by ultrasound was superior to the fixed value. We can conclude that the concrete characterization by ultrasound is consistent and, to this characterization the cylindrical specimen, normally used to determine f ck, can be used.

Terrain forms and spatial variability of soil properties in an area cultivatedwith citrus

The technique of precision agriculture and soil-landscape allows delimiting areas for localized management, allowing a localized application of agricultural inputs and thereby may contribute to preservation of natural resources. Therefore, the objective of this work was to characterize the spatial variability of chemical properties and clay content in the context of soil-landscape relationship in a Latosol (Oxisol) under cultivation of citrus. Soil samples were collected at a depth of 0.0-0.2 m in an area of 83.5 ha planted with citrus, as a 50-m intervals grid, with 129 points in concave terrain and 206 points in flat terrain, totaling 335 points. Values for the variables that express the chemical characteristics and clay content of soil properties were analyzed with descriptive statistics and geostatistical modeling of semivariograms for making maps of kriging. The values of range and kriging maps indicated higher variability in the shape of concave topography (top segment) compared with the shape of flat topography (slope and hillside segments below). The identification of different forms of terrain proved to be efficient in understanding the spatial variability of chemical properties and clay content of soil under cultivation of citrus.

GPS receivers for georeferencing of spatial variability of soil attributes

The characterization of the spatial variability of soil attributes is essential to support agricultural practices in a sustainable manner. The use of geostatistics to characterize spatial variability of these attributes, such as soil resistance to penetration (RP) and gravimetric soil moisture (GM) is now usual practice in precision agriculture. The result of geostatistical analysis is dependent on the sample density and other factors according to the georeferencing methodology used. Thus, this study aimed to compare two methods of georeferencing to characterize the spatial variability of RP and GM as well as the spatial correlation of these variables. Sampling grid of 60 points spaced 20 m was used. For RP measurements, an electronic penetrometer was used and to determine the GM, a Dutch auger (0.0-0.1 m depth) was used. The samples were georeferenced using a GPS navigation receiver, Simple Point Positioning (SPP) with navigation GPS receiver, and Semi-Kinematic Relative Positioning (SKRP) with an L1 geodetic GPS receiver. The results indicated that the georeferencing conducted by PPS did not affect the characterization of spatial variability of RP or GM, neither the spatial structure relationship of these attributes.

Spatial variability of soil physical properties in two management systems in sugarcane crop

The aim of this study was to characterize the spatial variability of soil bulk density (Bd), soil moisture content (θ) and total porosity (Tp) in two management systems of sugarcane harvesting, with or without burning, in a Haplustox soil, in the 0-0.20 m layer. The study area is located in Rio Brilhante, state of Mato Grosso do Sul, Brazil, in Eldorado Sugar Mill. The plots have presented 180 m length, and 145.6 m width, totaling 90 points distributed in the form of a grid of nine rows by ten columns, with points spaced 20 m from its neighbor. Soil samples were collected at 0-0.20 m layer in 2007/2008 and 2008/2009 crops. The harvest with burning system had a higher density compared to mechanized harvest, in the two study periods. The moisture content as well as the porosity increased proportionally with the decrease of the density of the harvest burning system compared to the mechanized.

Spatial variability of the rainfall erosive potential in the State of Mato Grosso do Sul, Brazil

Information about rainfall erosivity is important during soil and water conservation planning. Thus, the spatial variability of rainfall erosivity of the state Mato Grosso do Sul was analyzed using ordinary kriging interpolation. For this, three pluviograph stations were used to obtain the regression equations between the erosivity index and the rainfall coefficient EI30. The equations obtained were applied to 109 pluviometric stations, resulting in EI30 values. These values were analyzed from geostatistical technique, which can be divided into: descriptive statistics, adjust to semivariogram, cross-validation process and implementation of ordinary kriging to generate the erosivity map.Highest erosivity values were found in central and northeast regions of the State, while the lowest values were observed in the southern region. In addition, high annual precipitation values not necessarily produce higher erosivity values.

Spatial variability of noise level in agricultural machines

The knowledge of the spatial variability of noise levels and the build of kriging maps can help the evaluation of the salubrity of environments occupied by agricultural workers. Therefore, the objective of this research was to characterize the spatial variability of the noise level generated by four agricultural machines, using geostatistics, and to verify if the values are within the limits of human comfort. The evaluated machines were: harvester, chainsaw, brushcutter and tractor. The data were collected at the height of the operator's ear and at different distances. Through the results, it was possible to verify that the use of geostatistics, by kriging technique, made it possible to define areas with different levels for the data collected. With exception of the harvester, all of machines presented noise levels above than 85 dB (A) near to the operator, demanding the use of hearing protection.

Spatial variability of yield and other parameters associated with pear trees

In the last few years, precision agriculture has become commonly used with many crops, particularly cereals, and there is also interest in precision horticulture. Pear is a seasonal fruit and well appreciated by Brazilian people, although it is mostly imported. Brazilian farmers are nowadays trying to increase pear production. Thus, this research aimed at mapping the yield of pear trees in order to study the spatial variability of yield as well as its comparison with spatial variability of soil and plant attributes. The experimental field had 146 pear trees, variety 'Pêra d'água', distributed on a 1.24 ha. Four harvests were performed according to the fruit ripening and from each tree; only the ripe fruits were harvested. In each harvest, all the fruits were weighed and the total yield was obtained based on the sum of each harvest. The soil attributes analyzed were P, K, Ca, Mg, pH in CaCl2, C, Cu, Zn, Fe, Mn and base saturation, and the plant attributes were fruit length, diameter and yield. Yield had low correlation with soil and plant attributes. An index of spatial variability was suggested in this study and helped in classifying levels of spatial dependence of the various soil and plant attributes: very low (fruit length); low (P, fruit diameter), medium (Mg, pH, Cu, Zn, Fe), high (Ca, K, base saturation and yield), and very high (Mn and C).