Variabilidade genética em cultivares de soja determinada com marcadores microssatélites em gel de agarose

O objetivo deste trabalho foi avaliar a variabilidade genética de cultivares de soja com marcadores microssatélites selecionados e caracterizados quanto à informatividade para uso em gel de agarose. O DNA de 23 cultivares de soja foi amplificado com 283 marcadores microssatélites em gel de agarose a 3%. Posteriormente, 53 marcadores que apresentaram polimorfismo facilmente detectável nos géis de agarose foram utilizados na caracterização de 53 cultivares. Nessas cultivares foram detectados 124 alelos, com média de 2,34 alelos por loco, e os valores de conteúdo de informação polimórfica variaram entre 0,16 e 0,66, com média de 0,47. As frequências alélicas variaram de 0,02 a 0,91, com média de 0,43. A distância genética calculada variou de 0,02 a 0,73, com média de 0,47. A menor distância observada foi entre as cultivares CD201 e CD208, e a maior distância entre CD210 e BRSMT Uirapuru. Os marcadores utilizados possibilitaram a identificação das 53 cultivares avaliadas. Os locos microssatélites de soja, avaliados em gel de agarose, apresentam elevada informatividade. É possível detectar variabilidade significativa no germoplasma brasileiro de soja avaliado, mesmo entre cultivares elite, quando se usa marcadores moleculares microssatélites selecionados por sua informatividade.

Sedimentation of macroscopic rigid knots and its relation to gel electrophoretic mobility of DNA knots.

We address the general question of the extent to which the hydrodynamic behaviour of microscopic freely fluctuating objects can be reproduced by macrosopic rigid objects. In particular, we compare the sedimentation speeds of knotted DNA molecules undergoing gel electrophoresis to the sedimentation speeds of rigid stereolithographic models of ideal knots in both water and silicon oil. We find that the sedimentation speeds grow roughly linearly with the average crossing number of the ideal knot configurations, and that the correlation is stronger within classes of knots. This is consistent with previous observations with DNA knots in gel electrophoresis.

Mechanisms of interaction of therapeutic nanoparticles with cells

Nanoparticles (NPs) have gained a lot of interest in recent years due to their huge potential for applications in industry and medicine. Their unique properties offer a large number of attractive possibilities in the biomedical field, providing innovative tools for diagnosis of diseases and for novel therapies. Nevertheless, a deep understanding of their interactions with living tissues and the knowledge about their possible effects in the human body are necessary for the safe use of nanoparticulate formulations. The aim of this PhD project was to study in detail the interactions of therapeutic NPs with living cells, including cellular uptake and release, cellular localization and transport across the cell layers. Moreover, the effects of NPs on the cellular metabolic processes were determined using adapted in vitro assays. We evaluated the biological effect of several NPs potentially used in the biomedical field, including titanium dioxide (Ti02) NPs, 2-sized fluorescent silica NPs, ultrasmall superparamagnetic iron oxide (USPIO) NPs, either uncoated or coated with oleic acid or with polyvinylamine (aminoPVA) and poly(lactic-co-glycolic acid) - polyethylene-oxide (PLGA-PEO) NPs. We have found that the NPs were internalized by the cells, depending on their size, chemical composition, surface coating and also depending on the cell line considered. The uptake of aminoPVA-coated USPIO NPs by endothelial cells was enhanced in the presence of an external magnetic field. None of the tested USPIO NPs and silica NPs was transported across confluent kidney cell layers or brain endothelial cell layers, even in the presence of a magnetic field. However, in an original endothelium-glioblastoma barrier model which was developed, uncoated USPIO NPs were directly transferred from endothelial cells to glioblastoma cells. Following uptake, Ti02 NPs and uncoated USPIO NPs were released by the kidney cells, but not by the endothelial cells. Furthermore, these NPs induced an oxidative stress and autophagy in brain endothelial cells, possibly associated with their enhanced agglomeration in cell medium. A significant DNA damage was found in brain endothelial cells after their exposure to TiO2NPs. Altogether these results extend the existing knowledge about the effects of NPs on living cells with regard to their physicochemical characteristics and provide interesting tools for further investigation. The development of the in vitro toxicological assays with a special consideration for risk evaluation aims to reduce the use of animal experiments. -Les nanoparticules (NPs) présentent beaucoup d'intérêt dans le domaine biomédical et industriel. Leurs propriétés uniques offrent un grand nombre de possibilités de solutions innovantes pour le diagnostique et la thérapie. Cependant, pour un usage sûr des NPs il est nécessaire d'acquérir une connaissance approfondie des mécanismes d'interactions des NPs avec les tissus vivants et de leur effets sur le corps humain. Le but de ce projet de thèse était d'étudier en détail les mécanismes d'interactions de NPs thérapeutiques avec des cellules vivantes, en particulier les mécanismes d'internalisation cellulaire et leur subséquente sécrétion par les cellules, leur localisation cellulaire, leur transport à travers des couches cellulaires, et l'évaluation des effets de NPs sur le métabolisme cellulaire, en adaptant les méthodes existante d'évaluation cyto-toxico logique s in vitro. Pour ces expériences, les effets biologiques de nanoparticules d'intérêt thérapeutique, telles que des NPs d'oxyde de titane (TiO2), des NPs fluorescents de silicate de 2 tailles différentes, des NPs, d'oxyde de fer super-para-magnétiques ultra-petites (USPIO), soit non- enrobées soit enrobées d'acide oléique ou de polyvinylamine (aminoPVA), et des NPs d'acide poly(lactique-co-glycolique)-polyethylene-oxide (PLGA-PEO) ont été évalués. Les résultats ont démontré que les NPs sont internalisées par les cellules en fonction de leur taille, composition chimique, enrobage de surface, et également du type de cellules utilisées. L'internalisation cellulaire des USPIO NPs a été augmentée en présence d'un aimant externe. Aucune des NPs de fer et de silicate n'a été transportée à travers des couches de cellules épithéliales du rein ou endothéliales du cerveau, même en présence d'un aimant. Cependant, en développant un modèle original de barrière endothélium-glioblastome, un transfert direct de NPs d'oxyde de fer de cellule endothéliale à cellule de glioblastome a été démontré. A la suite de leur internalisation les NPs d'oxyde de fer et de titane sont relâchées par des cellules épithéliales du rein, mais pas des cellules endothéliales du cerveau. Dans les cellules endothéliales du cerveau ces NPs induisent en fonction de leur état d'agglomération un stress oxydatif et des mécanismes d'autophagie, ainsi que des dommages à l'ADN des cellules exposées aux NPs d'oxyde de titane. En conclusion, les résultats obtenus élargissent les connaissances sur les effets exercés par des NPs sur des cellules vivantes et ont permis de développer les outils expérimentaux pour étudier ces effets in vitro, réduisant ainsi le recours à des expériences sur animaux.

Alterações fisiológicas e bioquímicas em sementes de café secas em sílica gel e soluções salinas saturadas

O objetivo deste trabalho foi avaliar alterações fisiológicas e bioquímicas em sementes de café submetidas à secagem rápida, em sílica gel, e à secagem lenta, em soluções salinas saturadas. As sementes foram secas até que atingissem os seguintes teores de água: 40, 30, 20, 15, 10 e 5% (base úmida). Após a secagem, uma parte das sementes foi imediatamente avaliada quanto ao desempenho fisiológico e ao perfil de enzimas do processo oxidativo, e outra parte foi avaliada após armazenagem em condição hermética, em câmara fria e seca, por quatro meses. A velocidade de secagem e o teor final de água tiveram efeito significativo sobre a qualidade fisiológica das sementes. Após a secagem rápida em sílica gel, as sementes toleraram teores finais de água mais baixos. No entanto, após a secagem lenta, as sementes com teores finais de água mais elevados apresentaram maior qualidade. O período de armazenamento não afetou a germinação, mas prejudicou o vigor das sementes. A secagem rápida apresenta maior potencial de dano ao endosperma do que aos embriões. O perfil enzimático das sementes de café é afetado pelo teor final de água e pela velocidade de secagem.

Simulation of Boundary Layers and Heat Transfer in the Entrance Region of Pipes

The study of fluid flow in pipes is one of the main topic of interest for engineers in industries. In this thesis, an effort is made to study the boundary layers formed near the wall of the pipe and how it behaves as a resistance to heat transfer. Before few decades, the scientists used to derive the analytical and empirical results by hand as there were limited means available to solve the complex fluid flow phenomena. Due to the increase in technology, now it has been practically possible to understand and analyze the actual fluid flow in any type of geometry. Several methodologies have been used in the past to analyze the boundary layer equations and to derive the expression for heat transfer. An integral relation approach is used for the analytical solution of the boundary layer equations and is compared with the FLUENT simulations for the laminar case. Law of the wall approach is used to derive the empirical correlation between dimensionless numbers and is then compared with the results from FLUENT for the turbulent case. In this thesis, different approaches like analytical, empirical and numerical are compared for the same set of fluid flow equations.

Neurotrophic activity of human adipose stem cells isolated from deep and superficial layers of abdominal fat.

New approaches to the clinical treatment of traumatic nerve injuries may one day utilize stem cells to enhance nerve regeneration. Adipose-derived stem cells (ASC) are found in abundant quantities and can be harvested by minimally invasive procedures that should facilitate their use in such regenerative applications. We have analyzed the properties of human ASC isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures. Cells from the superficial layer proliferate significantly faster than those from the deep layer. In both the deep and superficial layers, ASC express the pluripotent stem cell markers oct4 and nanog and also the stro-1 cell surface antigen. Superficial layer ASC induce the significantly enhanced outgrowth of neurite-like processes from neuronal cell lines when compared with that of deep layer cells. However, analysis by reverse transcription with the polymerase chain reaction and by enzyme-linked immunosorbent assay has revealed that ASC isolated from both layers express similar levels of the following neurotrophic factors: nerve growth factor, brain-derived neurotrophic factor and glial-derived neurotrophic factor. Thus, human ASC show promising potential for the treatment of traumatic nerve injuries. In particular, superficial layer ASC warrant further analysis of their neurotrophic molecules.

MOCVD growth andproperties of GaN and AlGaN layers

In this work GaN and AlGaN layers were grown by metal-organic chemical vapor deposition (MOCVD) on sapphire substrates. The research was carried out at Micro and Nanoscience Laboratory of Helsinki University of Technology. The objective of this thesis is the study of MOCVD technique for the growth of GaN and AlGaN films and optimization of growth parameters in purpose to improve crystal quality of the films. The widely used two-step and the new multistep methods have been used for GaN, AlGaN MOCVD growth on c-plane sapphire. Properties of the GaN and AlGaN layers were studied using in-situ reflectance monitoring during MOCVD growth, atomic force microscopy and x-ray diffraction. Compared to the two step method, the multistep method has produced even better qualities of the GaN and AlGaN layers and significant reduction of threading dislocation density.

Investigations of a thermosensitive gel to temporarily occlude crural arteries in femoro-distal bypass surgery.

OBJECTIVES: Long occlusions in calcified crural arteries are a major cause of endovascular technical failure in patients with critical limb ischaemia. Therefore, distal bypasses are mainly performed in patients with heavily calcified arteries and with consequently delicate clamping. A new reverse thermosensitive polymer (RTP) is an alternative option to occlude target vessels. The aim of the study is to report our technical experience with RTP and to assess its safety and efficiency to temporarily occlude small calcified arteries during anastomosis time. METHODS: Between July 2010 and December 2011, we used RTP to occlude crural arteries in 20 consecutive patients with 20 venous distal bypasses. We recorded several operative parameters, such as volume of injected RTP, duration of occlusion and anastomotic time. Quality of occlusion was subjectively evaluated. Routine on-table angiography was performed to search for plug emboli. Primary patency, limb salvage and survival rates were reported at 6 months. RESULTS: In all patients, crural artery occlusion was achieved with the RTP without the use of an adjunct occlusion device. Mean volume of RTP used was 0.3 ml proximally and 0.25 ml distally. Mean duration of occlusion was 14.4 ± 4.5 min, while completion of the distal anastomosis lasted 13.4 ± 4.3 min. Quality of occlusion was judged as excellent in eight cases and good in 12 cases. Residual plugs were observed in two patients and removed with an embolectomy catheter, before we amended the technique for dissolution of RTP. At 6 months, primary patency rate was 75% but limb salvage rate was 87.5%. The 30-day mortality rate was 10%. CONCLUSIONS: This study shows that RTP is safe when properly dissolved and effective to occlude small calcified arteries for completion of distal anastomosis.

Biogeochemical processes controlling oxygen and carbon isotopes of diatom silica in Late Glacial to Holocene lacustrine rhythmites

Biogeochemical cycles and sedimentary records in lakes are related to climate controls on hydrology and catchment processes. Changes in the isotopic imposition of the diatom frustules (δ 18 O diatom and δ 13 C diatom ) in lacustrine sediments can be used to reconstruct palaeoclimatic and palaeoenvironmental changes. The Lago Chungará (Andean Altiplano, 18°15 ′ S, 69°10 ′ W, 4520 masl) diatomaceous laminated sediments are made up of white and green multiannual rhythmites. White laminae were formed during short-term diatom super-blooms, and are composed almost exclusively of large-sized Cyclostephanos andinus.These diatoms bloom during mixing events when recycled nutrients from the bottom waters are brought to the surface and/or when nutrients are introduced from the catchment during periods of strong runoff. Conversely, the green laminae are thought to have been deposited over several years and are composed of a mixture of diatoms (mainly smaller valves of C. andinus and Discostella stelligera ) and organic matter. These green laminae reflect the lake's hydrological recovery from a status favouring the diatom super-blooms (white laminae) towards baseline conditions. δ 18 O diatom and δ 13 C diatom from 11,990 to 11,530 cal years BP allow us to reconstruct shifts in the precipitation/evaporation ratio and changes in the lake water dissolved carbon concentration, respectively. δ 18 O diatom values indicate that white laminae formation occurred mainly during low lake level stages, whereas green laminae formation generally occurred during high lake level stages. The isotope and chronostratigraphical data together suggest that white laminae deposition is caused by extraordinary environmental events. El Niño-Southern Oscillation and changes in solar activity are the most likely climate forcing mechanisms that could trigger such events, favouring hydrological changes at interannual-to-decadal scale. This study demonstrates the potential for laminated lake sediments to document extreme pluriannual events.

Tailoring the surface density of silicon nanocrystals embedded in SiOx single layers

In this article, we explore the possibility of modifying the silicon nanocrystal areal density in SiOx single layers, while keeping constant their size. For this purpose, a set of SiOx monolayers with controlled thickness between two thick SiO2 layers has been fabricated, for four different compositions (x=1, 1.25, 1.5, or 1.75). The structural properties of the SiO x single layers have been analyzed by transmission electron microscopy (TEM) in planar view geometry. Energy-filtered TEM images revealed an almost constant Si-cluster size and a slight increase in the cluster areal density as the silicon content increases in the layers, while high resolution TEM images show that the size of the Si crystalline precipitates largely decreases as the SiO x stoichiometry approaches that of SiO2. The crystalline fraction was evaluated by combining the results from both techniques, finding a crystallinity reduction from 75% to 40%, for x = 1 and 1.75, respectively. Complementary photoluminescence measurements corroborate the precipitation of Si-nanocrystals with excellent emission properties for layers with the largest amount of excess silicon. The integrated emission from the nanoaggregates perfectly scales with their crystalline state, with no detectable emission for crystalline fractions below 40%. The combination of the structural and luminescence observations suggests that small Si precipitates are submitted to a higher compressive local stress applied by the SiO2 matrix that could inhibit the phase separation and, in turn, promotes the creation of nonradiative paths.