The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37°C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G+C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carinii carinii isolated from infected rat lungs are haploid organisms.

Hintergrund: Ein sehr häufig in der nuklearmedizinischen Diagnostik genutztes Radionuklid ist 99mTc. Es emittiert Gammastrahlung mit einer relativ niedrigen Energie (140 keV) und hat eine kurze Halbwertszeit von 6 h. Zusätzlich zur Gammastrahlung entstehen bei jedem Zerfall von 99mTc Auger-Elektronen. Diese niederenergetischen Elektronen, sehr kurzer Reichweite verfügen über einen hohen LET und erzeugen somit eine ausreichende Energiedeposition, um direkte DSB zu erzeugen. Bei Untersuchungen zu Chemotoxizität und Radiotoxizität mit Zellexperimenten gilt es eine Vielzahl an verschiedenen Schutzmechanismen, Reparaturmechanismen und Signalkaskaden in Zellen zu beachten, welche häufig noch nicht vollständig erforscht sind. Um das schädigende Potential von unterschiedlichen Substanzen und Strahlenqualitäten auf die DNA zu untersuchen, wurde ein zellfreies System gewählt. Ziel dieser Arbeit war es, neben den Strahlenqualitäten der Alpha-, Beta, Gamma- und Röntgenstrahlung die Auger-Elektronen des 99mTc auf ihr Potential zur Induktion von DNA-Strangbrüchen zu untersuchen. Hierfür stand die Substanz 99mTc-HYNIC-DAPI zur Verfügung, welche 99mTc an das Plasmid binden und somit in direkte DNA-Nähe bringen kann. Material und Methode: Alle Versuche wurden mit dem Plasmid pUC 19, einem künstlich hergestellten, bakteriellen Plasmid mit 2686 Basenpaaren, welches als nackte DNA ohne Proteine vorliegt, durchgeführt. Der Vergleich zwischen bestrahltem Plasmid in Ab- und Anwesenheit des Radikalfängers DMSO gibt Hinweise darauf, ob Strangbrüche direkt induziert oder nach Radikalbildung indirekt erzeugt werden. Bei radikalvermittelter Wirkung verhindert DMSO DNA-Strangbrüche und die ungeschädigte Supercoiled-Plasmid-Konformation bleibt erhalten. Nach Bestrahlung des Plasmids erfolgte der Nachweis von Strangbrüchen mittels Agarose-Gelelektrophorese. Bekommt ein Plasmid Einzel- oder Doppelstrangbrüche, so verändert sich seine Konformation zu einem ringförmigen/open circle (ESB) oder einem linearen Plasmid (DSB). Durch veränderte Laufeigenschaften im Agarosegel sind die verschiedenen Konformationen voneinander trennbar. Nach Anfärben der DNA mit dem Fluoreszenzfarbstoff Ethidiumbromid konnte das fluoreszierende Plasmid fotografiert und die Intensität der Konformationsbanden quantifiziert werden. Ergebnisse: Zuerst wurde die Reproduzierbarkeit der Methodik überprüft und festgestellt, dass eine Korrelation zwischen Plasmidmasse und Fluoreszenzintensität besteht. Anschließend wurde in Vorversuchen gezeigt, dass die Inkubationstemperaturen, pH-Werte und der Radikalfänger DMSO keinen Einfluss auf die Plasmidintegrität haben. Bei Bestrahlung mit Röntgenstrahlung, dem Beta-Strahler 188Re und dem nicht DNA-gebundenen Gamma-Strahler und Auger-Emitter 99mTc konnte mit steigender Dosis eine Zunahme an ESB festgestellt werden. Vergleichsproben mit DMSO zeigten keinen Anstieg von ESB, was auf eine radikalvermittelte 67 DNA-Schädigung mittels Reaktiver Sauerstoffspezies (ROS) hinweist. Ab einer Energiedosis von ca. 80 Gy konnten nach Bestrahlung mit 188Re und 99mTc zusätzlich zu den ESB auch DSB nachgewiesen werden. DMSO konnte in den Vergleichsproben sowohl die ESB als auch die DSB erfolgreich verhindern. Bei einer sehr hohen Dosis ≥ 600 Gy zeigte DMSO Kapazitätsgrenzen und es konnten nicht mehr alle Strangbrüche verhindert werden. Die Bestrahlung mit dem Alpha-Strahler (hoher LET) 223Ra fügte, im Vergleich zu Strahlung mit niedrigem LET, dem Plasmid überproportional viele DSB zu. Einige dieser DSB konnten nicht durch DMSO verhindert werden, was auf einen direkten DNA-Schaden bzw. eine zu hohe Radikaldichte hinweist. Ein noch stärkerer direkter Effekt konnte beobachtet werden, wenn 99mTc über die Substanz 99mTc-HYNIC-DAPI an DNA gebunden wurde. Dabei konnten schon ab einer Energiedosis von 4 Gy DSB erzeugt werden, welche trotz Radikalfänger nicht verhindert werden konnten. Schlussfolgerung: Dieser bei 99mTc-HYNIC-DAPI beobachtete Effekt wird den Auger-Elektronen zugeschrieben. Aufgrund ihrer kurzen Reichweite und ihres hohen LET sind sie in der Lage direkte DSB zu erzeugen, wenn sie DNA-gebunden sind oder sich in geringem Abstand zur DNA befinden. Die Ergebnisse der Experimente weisen auf ein therapeutisches Potential von 99mTc hin. Weitere Untersuchungen müssen zeigen, ob eine Adressierung von 99mTc an die DNA im Zellkern einer intakten Zelle zu verwirklichen ist und ob DNA-gebundenes 99mTc durch die Energie der Auger-Elektronen den Zelltod herbeiführen kann. Im nächsten Schritt sollte die Erforschung von Trägersubstanzen erfolgen, welche es ermöglichen Auger-Emitter spezifisch an die DNA von Tumorzellen zu koppeln. / Introduction and aim of the study: A radionuclide commonly used in diagnostic nuclear medicine is 99mTc. It emits gamma rays with a relatively low energy (140 keV) and has a short half-time (6h). In addition to gamma rays, 99mTc radiates so called Auger-electrons with low energy, low range and high linear energy transfer. Due to the high-LET Auger-electrons have a sufficient energy deposition to induce direct double-strand breaks to the DNA. In these experiments we used plasmid DNA to evaluate damage induced to biological systems by different chemotoxical substances and radionuclides as well as external radiation. By using plasmids instead of cell cultures we avoid lots of unexplored signal pathways in cells and it is possible to quantify chemotoxical and radiation damage to the DNA. Materials and methods: The double-stranded plasmid pUC 19 with 2686 bp is used in all experiments. It is a synthetically produced bacterial plasmid without any proteins. To distinguish between directly and indirectly (radical induced) induced damage we used the radical scavenger DMSO. Indirectly induced damage via reactive oxygen species (ROS) can be prevented by DMSO. The quantification of supercoiled forms, single strand breaks (SSB) and double strand breaks (DSB) was measured by the method of agarose gel electrophoresis. After the electrophoresis, agarose gels are dyed in ethidium bromide and imaged with a ccd-camera using ultraviolet transillumination. The bands of the different plasmid forms were quantified through the FIJI computer program. Results: First of all a correlation between plasmid mass and fluorescence intensity was shown. In a pretrial no damaging effect to the plasmid from incubation temperature, pH-value and radical scavenger DMSO appeared. Afterwards we examined chemotoxical SnCl2, external x-rays, the alpha emitter 223Ra, the beta emitter 188Re, gamma- and Auger-emitter 99mTc and the DNA-bound 99mTc-HYNIC-DAPI. The radical scavenger DMSO was used to differentiate between indirect (radical induced) and direct DNA-damage. All different radiation qualities showed an increasing DNA-damage with increasing energy dose. For the low-LET radiation qualities like chemotoxical SnCl2, external x-rays, the beta emitter 188Re and not DNA-bound 99mTc, DMSO showed the quality to prevent the damage. After the deposition of an energy dose ≥ 600 Gy DMSO showed a limitation in his scavenger capacity. During radiation with high-LET beams like 223Ra or DNA-bound 99mTc-HYNIC-DAPI DMSO showed less or nearly no ability to prevent DNA-damage. A 4 Gy dose of 99mTc-HYNIC-DAPI was able to induce DSB into the plasmid. These DSB could not be prevented by DMSO. The lower ESB:DSB ratio for high-LET beams also displays that direct damage is more likely to create DSB than indirect damage. Conclusion: In conclusion we can say that DNA-bound 99mTc-HYNIC-DAPI was most appropriate to induce DSB via a direct effect. It was impossible to prevent this damage due to adding the 69 radical scavenger DMSO. We attribute this to low range, low-LET Auger-electrons and suppose that it may be possible to use DNA-bound 99mTc for therapeutic purpose. Further research has to show if 99mTc can be targeted to the DNA of intact cells and if suitable tracers can be found to safely target and kill tumor cells.

In patients with Parkinson's disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

The electromagnetic absorption properties of plasmonic nanostructures were utilized to develop mesoscopic sites for highly efficient photothermal generation steam, SERS biosensing, and light-triggered cellular delivery uptake. Plasmonic nanostructures embedded in common thermal solutions produces vapor without the requirement of heating the fluid volume. When particles are dispersed in water at ambient temperature, energy is directed primarily to vaporization of water into steam, with a much smaller fraction resulting in heating of the fluid. Solar illuminated aqueous nanoparticle solution can drive water-ethanol distillation, yielding fractions significantly richer in ethanol content than simple thermal distillation and also produced saturated steam destroying Geobacillus stearothermophilus bacteria in a compact solar powered autoclave. Subwavelength biosensing sites were developed using the plasmonic properties of gold nanoshells to investigate the properties of aptamer (DNA) target complexes. Nanoshells are tunable core-shell nanoparticles whose resonant absorption and scattering properties are dependent on core/shell thickness ratio. Nanoshells were used to develop a label free detection method using SERS to monitor conformational change induced by aptamer target binding. The conformational changes to the aptamers induced by target binding were probed by monitoring the aptamer SERS spectra reproducibility. Furthermore, nanoshells can serve as a nonviral light-controlled delivery vector for the precise temporal and spatial control of molecular delivery in vitro. The drug delivery concept using plasmonic vectors was shown using a monolayer of ds-DNA attached to the nanoshell surface and the small molecular “parcel” intercalated inside ds-DNA loops. DAPI, a fluorescent dye, was used as the molecular parcel to visualize the release process in living cells. Upon laser illumination at the absorption resonance the nanoshell converts photon energy into heat producing a local temperature gradient that induces DNA dehybridization, releasing the intercalated molecules.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CHAPTER II: Pequi, Caryocar brasiliense, is one of the species of highlight at the biome of the Brazilian savannah due to its utilization in culinary, popular medicine, industry in general, and iron and steel industry. It presents an elevated index of exploration, being capable of entering the list of the endangered species. In the region of São José do Xingu (MT), a tree of pequi without thorn at the mesocarp was found and this enables to improve pequi not only for consumption, taking advantage of the high appreciation it already has. To detect the existing genomic differences between the pequi with and without the thorn at the endocarp, RADP markers were utilized. The generated polymorphisms were cloned and sequenced in order to identify the sequences responsible for the fenotypical alteration. It was observed that the pequi without thorn is genetically isolated from the other populations of pequi with thorn at the endocarp, proving that this characteristic is related to the genetic divergence of the species. Analysis in BLASTn evidenced the similarity of the Dof1 genes of Zea mays, in the population with thorn, and in the population without thorn, with the gene of phosphinotricin acetyl transferase of Z. mays. In the analysis of BLASTx, the similarity was verified with the proteins responsible for the deficiency in ferric reductase 4, in the pequi without thorn and catalase, in the pequi without thorn. CHAPTER III:The pequi, Cayocar brasiliense Cambess. Is a feature of Brazilian cerrado plant suffering extractive action, mainly for food. It is a plant very appreciated in the regions of Minas Gerais, especially in the north of this state. In the region of Sao Jose do Xingu, Mato Grosso, was found in a plant which had no thorns in the core and this allowed further research to discover the lack of thorns as it is an unwanted feature of the plant. Thus, the cytogenetic study, using conventional Giemsa staining, NOR, banda C, DAPI and CMA3 were used for comparison between the pequi with and without spines in the putamen. It was found that these differences are not attributed to chromosomal differences between the two types of pequi. Both have the same chromosome number, the marking of NOR, C banding was not verified due to the size of chromosomes and differences in staining with DAPI and CMA3 could not be verified although related to these characteristics. CHAPTER IV: The pequi is a species that is gathered and today is seen as a source for the production of biodiesel. The objective of this study was to identify sequences from a cDNA library that are involved in the metabolic pathways for production of pequi fruit, to gain a better understanding of the species and its biological and genetic properties. To do so, a cDNA library was created and the clones were cloned and sequenced. These identified sequences were deposited in GenBank. Most of the sequences found are associated with protection against oxidative stress in plants, some are transcription factors and others provide structural and pathogenic resistance. / CAPÍTULO II: O pequi, Caryocar brasiliense, é uma das espécies de destaque no bioma do Cerrado devido a sua utilização na culinária, medicina popular, indústria e siderurgia. Apresenta índice de exploração elevado, podendo entrar na lista das espécies ameaçadas de extinção. Na região de São José do Xingu (MT) foi encontrada uma árvore produzindo pequi sem espinho no endocarpo, o que levantou a possibilidade de melhorar o pequi para consumo aproveitando a alta apreciação que já possui. Com o objetivo de analisar as diferenças genômicas entre o pequi com e o sem espinho no endocarpo, utilizou-se marcadores RADP (Random Amplified Polymorphism DNA). As bandas polimórficas geradas foram isoladas, clonadas e seqüenciadas, buscando identificar sequências responsáveis pela alteração fenotípica. Observou-se que o pequi sem espinho fica isolado geneticamente das demais populações de pequi com espinho no caroço, comprovando que essa característica está relacionada à divergência genética da espécie. Análises em BLASTn mostraram a similaridade aos genes Dof1 de Zea mays, na população com espinho e com o gene da Acetiltransferase Fosfinotricina de Z. mays, na população sem espinho. As análises em BLASTx revelaram similaridade com as proteínas responsáveis pela Deficiência em Redutase Férrica 4, no pequi sem espinho e com catalase, no pequi com espinho. CAPÍTULO III: O pequizeiro, Cayocar brasiliense Cambess., é uma planta característica do cerrado brasileiro que sofre ação extrativista, principalmente, para a alimentação. É uma planta bastante apreciada em regiões de Minas Gerais, especialmente no Norte desse estado. Na região de São José do Xingu, Mato Grosso, foi encontrada uma planta produzindo pequi sem espinhos no caroço. O estudo citogenético, por meio de coloração convencional de Giemsa, NOR, banda C, DAPI e CMA3 foi utilizado para a comparação entre plantas produtoras de pequi com e sem espinhos no caroço. Verificou-se que essas diferenças não podem ser atribuídas à diferenças cromossômicas entre os dois tipos de pequi. Ambas as plantas têm o mesmo número cromossômico, mesma marcação de NOR. Não foi verificado bandeamento C devido ao tamanho dos cromossomos. Diferenças de coloração com DAPI e CMA3 foram detectadas, porém não é possível afirmar que estejam relacionadas com a presença ou ausência de espinhos no fruto. CAPÍTULO IV: O pequizeiro (Caryocar brasiliense Cambess.) é uma espécie nativa do cerrado brasilieiro e que possui importância alimentar e social para a população que dela depende. É uma espécie que sofre ação extrativista e que, atualmente, é considerada fonte para a produção de biodiesel. Nesse trabalho o objetivo foi identificar, a partir de uma bilbioteca de cDNA, seqüências que estejam envolvidas em vias metabólicas para produção do fruto do pequi, para conhecimento da espécie, suas propriedades biológicas e genéticas. Dessa forma foi construída biblioteca de cDNA e os clones foram clonados e seqüenciados. As sequências identificadas foram depositadas no GenBank. Algumas das sequências encontradas estão relacionadas à proteção contra o estresse oxidativo em plantas, outras são fatores de transcrição e algumas sequências expressas têm funções estruturais e de resistência a patógenos. / Doutor em Genética e Bioquímica

In patients with Parkinson's disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

No description available.

This thesis is a biosystematic study focusing on the taxonomy and variability of selected taxa of the genus Sorbus, one of the most diverse and taxonomically complicated plant groups in Europe. Classical and modern biosystematic methods - comparative study, chromosome counting, analysis of nuclear microsatellite markers, flow cytometry, and traditional multivariate and outline morphometric analyses were used to assess the morphological, karyological and genetic variability of the genus. The final synthesis of these approaches led to the description of several new taxa (species, hybrids and a subgenus) and the correction of several taxonomic misinterpretations. To reveal ongoing evolutionary processes responsible for the generation of the observed variability, the reproductive modes of 42 Sorbus taxa were examined using flow-cytometric seed and pollen screens. Apart from revealing major trends, the study estimates the frequency of rare events and provides several novel conclusions that are relevant both specifically to Sorbus/Rosaceae and to apomixis in general.