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Modèles prédictifs utilisant des données moléculaires de haute dimension pour une médecine de précision en oncologie / Predictive models using high dimensional molecular data for precision medicine in oncologyFerte, Charles 17 December 2013 (has links)
Le niveau médiocre des taux de réponses et des améliorations de survie lorsque des stratégies conventionnelles sont appliquées souligne la nécessité de développer des outils prédictifs performants, robustes et applicables en clinique. La démocratisation des technologies d’analyses à haut-débit est le substrat de la médecine de précision permettant le développement de modèles prédictifs capables d’orienter les stratégies thérapeutiques et la définition d’une nouvelle taxonomie des cancers par l’intégration de données moléculaires de haute dimension. A travers cette thèse, nous avons d’abord analysé des données publiques d’expression génique de cancer bronchique non à petites cellules dans le but de prédire la probabilité de survie à trois ans. Le fort pouvoir prédictif de la TNM seule et la faible taille des cohortes de validation ont malheureusement limité la possibilité de traduire nos résultats en clinique. Nous avons ensuite développé un prédicteur du phénotype « KRAS muté » spécifique du cancer colorectal, permettant d’identifier de nouveaux traits moléculaires responsables de ce phénotype et d’améliorer la prédiction de la réponse au cetuximab chez les patients KRAS sauvage. Enfin, nous avons combiné les données moléculaires des panels de lignées cellulaires CCLE et Sanger avec les données des cohortes du TCGA pour produire des prédicteurs performants de la sensibilité aux drogues. Ces modèles sont concordants avec des screens produits par interférence RNA et permettent d’expliquer la réponse extrême de patients sectionnés dans le programme de screening moléculaire MOSCATO.Les défis spécifiques posés par les données moléculaires de haute dimension dans le développement d’outils prédictifs applicables en clinique sont discutés dans cette thèse. / The mediocre level of the rates of answers and the improvements of survival when conventional strategies are applied underlines the necessity of developing successful, strong and applicable predictive tools in private hospital. The democratization of the technologies of analyses with top-debit(-flow) is the substratum of the medicine of precision allowing the development of predictive models capable of directing the therapeutic strategies and the definition of a new taxonomy of cancers by the integration of molecular data of high dimension(size).Through this thesis(theory), we analyzed at first public data of genic expression of bronchial cancer not in small cells(units) with the aim of predicting the probability of survival in three years. The strong predictive power of the only TNM and
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Sistemática de Barnadesioideae (Asteraceae) com ênfase em Dasyphyllum / Systematics of Barnadesioideae (Asteraceae) with emphasis on DasyphyllumPaola de Lima Ferreira 30 April 2015 (has links)
Barnadesioideae é uma subfamília monofilética subordinada a família Asteraceae, que consiste em 9 gêneros e 85 espécies endêmicos da América do Sul. O interesse em Barnadesioideae vem aumentando consideravelmente desde que resultados em biologia molecular revelaram que constituem o grupo irmão para o restante das Asteraceae. Hipóteses filogenéticas para Barnadesioideae vêm sendo propostas nas últimas duas décadas com resultados incongruentes, especialmente em relação à monofilia de Dasyphyllum e sua classificação infragenérica. O intuito desde projeto é propor hipóteses filogenéticas baseadas em dados moleculares a fim de testar a monofilia de Dasyphyllum, de sua classificação infragenérica e as relações filogenéticas de D. diacanthoides, D. excelsum, D. hystrix (incertae sedis) e D. capixaba. As análises filogenéticas foram baseadas em duas regiões não codificantes do cpDNA, trnL-trnF e psbA-trnH, e uma região nuclear, ITS. As regiões plastidiais e nuclear foram analisadas separadamente e combinadas, sob os critérios de parcimônia e análise Bayesiana. Os resultados obtidos demonstram que Dasyphyllum não é um grupo monofilético pelo posicionamento de D. diacanthoides e D. excelsum em um clado como grupo irmão de Fulcaldea e Arnaldoa e distante filogeneticamente das outras espécies do gênero. Além disso, a monofilia de Dasyphyllum seção Macrocephala e Dasyphyllum seção Dasyphyllum em sua circunscrição atual foram rejeitadas. Visando a monofilia de Dasyphyllum, Dasyphyllum subgênero Archidasyphyllum é elevado ao status de gênero, Archidasyphyllum, com duas novas combinações, Archidasyphyllum diacanthoides e Archidasyphyllum excelsum. / Barnadesioideae is a monophyletic subfamily of Asteraceae, which includes 9 genera and 85 species endemic to South America. The interest in Barnadesioideae increased considerably since results with molecular data revealed this subfamily is the sister group to the other Asteraceae. Phylogenetic hypotheses for Barnadesioideae have been proposed in the last two decades with inconsistent results, especially regarding the monophyly of Dasyphyllum and its infrageneric classification. The objectives of this study were to propose phylogenetic hypotheses based on molecular data to test the monophyly of Dasyphyllum, its infrageneric classification, besides the phylogenetic relationships of D. diacanthoides, D. excelsum, D. hystrix (incertae sedis) and D. capixaba. The phylogenetic analyzes were based on two non-coding regions of cpDNA, trnL-trnF and psbA-trnH, and a nuclear region, ITS. The nuclear and plastid regions were analyzed separately and combined under the parsimony criterion and with Bayesian analysis. The results have shown that Dasyphyllum is not a monophyletic group by the positioning of D. diacanthoides and D. excelsum in a clade as sister group of Fulcaldea and Arnaldoa and phylogenetically distant from the other species of the genus. In addition, the monophyly of Dasyphyllum section Macrocephala and Dasyphyllum section Dasyphyllum in its current circumscription were rejected. In order to achieve the monophyly of Dasyphyllum, Dasyphyllum subgenus Archidasyphyllum is elevated to the status of genus, Archidasyphyllum, with two new combinations, Archidasyphyllum diacanthoides and Archidasyphyllum excelsum.
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Étude théorique de collisions d’intérêt interstellaire / Theoretical study of collisions in the interstellar mediumOuerfelli, Ghofrane 12 November 2016 (has links)
Le satellite Herschel Space Observatory a permit de détecter la lumière dans l'infrarouge lointain, ce qui correspond aux fréquences auxquelles émettent les molécules légères lors de leurs transitions rotationnelles. ALMA, un interféromètre situé sur un plateau du désert chilien a pris le relais d'Herschel et va permettre de nouvelles observations des nuages moléculaires froids avec une bonne précision angulaire. ALMA observe dans la gamme du millimétrique et du sub-millimétrique ce qui le rend complémentaire du satellite Herschel (infra lointain). Ces avancées considérables concernant les interactions entre particules au niveau microscopique, permettant de produire et piéger des diatomiques dans des états internes bien précis, ouvrent de nouvelles perspectives dans le domaine de la physique des collisions et de la chimie théorique. L'observation des molécules interstellaires a bénéficié des avancées de la radioastronomie, permettant d'identifier les transitions rotationnelles et vibrationnelles des molécules. De plus, les informations spectroscopiques nous procurent des données importantes sur l’état du milieu interstellaire : ionisé ou neutre.Le cation méthylidène CH+ a été observé dans le milieu diffus par sa transition électronique X1Σ+ -A1Π.Il joue un rôle important dans les différentes étapes du réseau chimique complexe grâce à des processus et réactions moléculaires qui se produisent dans les régions interstellaires et circumstellaires. Donc CH+lance une vaste chaine chimique de processus qui peut évoluer vers la formation d'espèces plus complexes.La transition de structure fine de C+; est la plus forte raie en émission de la voie lactée. L’ion C+ est un traceur de la densité et de la température dans les nuages diffus et les régions dominées par les photons (PDR). La raie de C+ constitue un important outil pour sonder le contenu en gaz et les processus de formation des étoiles dans la Voie lactée et dans d'autres galaxies.Les collisions de C+ avec H2 peuvent mener à la formation de CH+. Cette réaction a été très étudiée théoriquement et expérimentalement, cependant, étant endothermique par 3211cm-1 et, pour les températures typiques du MIS et pour H2 dans son niveau fondamental, la réaction ne se produit pas. Le seul processus est alors le processus d'excitation de spin-orbite de C+.La relaxation spin orbite C+(2P1/2) + H2(v; j) = C+(2P3/2) + H2(v0; j0) qui a été tout d'abord étudiée dans cette thèse contribue au refroidissement du gaz constituant les nuages interstellaires.L'excitation vibrationnelle de H2 (v > 0), qui a lieu lors des collisions avec C+ possède une influence notable sur les abondances de CH+. CH+ est un ion très réactif, il se détruit à partir de la réaction d'abstraction de l'hydrogène qui a été prise en considération dans ce travail. Il est donc intéressant d'évaluer précisément l'efficacité réelle de ce chemin de destruction. Le dilemme est que cet ion est également abondamment trouvé dans le milieu neutre et froid.Ce travail de thèse a comporté des études de collisions inélastiques et réactive d'intérêt interstellaire.Nous avons utilisé les calculs ab initio hautement corrélés. De plus, la dynamique nucléaire des systèmes considérés a été étudiée à l'aide d'un formalisme quantique et indépendant du temps, basé sur les coordonnées hypersphériques : soit inélastique dans le cas de l’excitation spin-orbite de C+(2P) + ortho-H2, et para-H2 et l’excitation rotationnelle de (CD+) + He, ou réactif dans le cas de l’abstraction d’un hydrogène dans H + CH+.Notre soucis a été de donner une base compréhensible des mécanismes et de fournir une quantification des sections efficaces de relaxation spin-orbite et des taux de réaction permettant de remonter aux observations spectroscopiques. Les nouvelles constantes de vitesse que nous avons obtenues devraient aider à mieux interpréter les observations de l'émission de C+ obtenues par les télescopes actuels et futurs. (...) / The Herschel Space Observatory satellite has permitted to detect light in the far infrared, corresponding to frequencies at which molecules emit light through rotational transitions. ALMA, an interferomete rlocated on the Chilean desert of Acatama took over Herschel and will allow new observations of cold molecular clouds with an accurate angular accuracy. ALMA observes in the range of millimeter andsub-millimeter which makes it complementary to the Satellite Herschel (far infrared). These significant advances in observing particle interactions at the microscopic level, to produce and trap diatomic molecules in specific internal states, open new perspectives in the field of collision physics and theoretical chemistry.Observation of interstellar molecules has benefited from advances in astronomy, to identify vibrational rotational ransitions of molecules. Furthermore, spectroscopic data provide us with important information on the state of the interstellar medium: ionized or neutral.The cation methylidene CH + was observed in the diffuse medium through its X1Σ + -A1Π electronictransition. It plays an important role in the different stages of the complex chemical behaviour through processes and molecular reactions that occur in interstellar and circumstellar regions. So CH+ launches large chain chemical processes that can progress to the formation of more complex species.The fine structure transition of C +; is the strongest emission line of the Milky Way. The C + ion is atracer of density and temperature in diffuse clouds and regions dominated by photons (PDR). The C +line is an important tool to probe the gas content and star formation processes in the Milky Way andother galaxies.C+ + H2 collisions can lead to the formation of CH +. This reaction has been extensively studied theoretically and experimentally, however, it is endothermic by 3211cm-1 and at the typical temperatures for MIS and H2 in its ground vibrational state, the reaction does not occur. The only process is then the C + spin-orbit excitation process.Spin orbit relaxation C + (2P1 / 2) + H 2 (v j) = C + (2P3 / 2) + H 2 (v0; j0) which was first studied inthis thesis contributes to the cooling of the gas constituting the interstellar clouds.The vibrational excitation of H2 (v> 0), which takes place during collisions with C + has a significant influence on the abundance of CH +. CH + is a highly reactive ion, it is destroyed by the abstractionreaction of hydrogen that has been considered in this work. It is therefore interesting to accurately assess the effectiveness of this path of destruction. The dilemma is that this ion is also abundantly found in the neutral and cold environment.This thesis focuses on the inelastic and reactive collisions studies of interstellar interest. We used ab initio highly correlated methods to tackle the electronic structure parts. Moreover, the nuclear dynamics of the systems was studied using a time independent quantum formalism, based on the Jacobi coordinates in the case of the spin-orbit excitation of C + (2P) + ortho H2, and para-H2 and rotational excitation of (+ CD) + He, or the hyper spherical coordinates for the reactive process in the case of the abstraction of a hydrogen in H + CH +.Our concern was to give a comprehensive basis of the mechanisms and provide a quantification of the effective spin-orbit relaxation cross sections and reaction rates to confront with spectroscopic observations. The new rate constants we obtained should help to better interpret the observations of C+ radiation emissions obtained by current and future telescopes.
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Vanadium Oxide in the Spectra of Mira VariablesCastelaz, Michael W., Luttermoser, Donald G., Piontek, Robert A. 20 July 2000 (has links)
As a preliminary step in deducing Teff and log (g) of Mira variables as a function of phase, a comparison is made between spectra synthesized from LTE stellar atmosphere models and observed spectra. The observed spectra show obvious vanadium oxide (VO) absorption bands. However, the molecular line list used to produce the synthetic spectra does not include the bound-bound VO opacities. The wavenumbers, line oscillator strengths, and lowest energy levels are needed to calculate these opacities. The equations, constants, and experimentally determined factors required to calculate the line oscillator strengths and lowest energy levels from experimentally determined wavenumbers are presented. The effect of including the wavenumbers, line oscillator strengths, and lowest energy levels of the VO BX (0, 0) band are calculated and show the expected absorption features observed in the spectra of Mira variables. In the VO B-X (0, 0) band the line oscillator strengths range from about 0.05 to 3.
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Multi-Platform Molecular Data Integration and Disease Outcome AnalysisYoussef, Ibrahim Mohamed 06 December 2016 (has links)
One of the most common measures of clinical outcomes is the survival time. Accurately linking cancer molecular profiling with survival outcome advances clinical management of cancer. However, existing survival analysis relies intensively on statistical evidence from a single level of data, without paying much attention to the integration of interacting multi-level data and the underlying biology. Advances in genomic techniques provide unprecedented power of characterizing the cancer tissue in a more complete manner than before, opening the opportunity of designing biologically informed and integrative approaches for survival analysis. Many cancer tissues have been profiled for gene expression levels and genomic variants (such as copy number alterations, sequence mutations, DNA methylation, and histone modification). However, it is not clear how to integrate the gene expression and genetic variants to achieve a better prediction and understanding of the cancer survival.
To address this challenge, we propose two approaches for data integration in order to both biologically and statistically boost the features selection process for proper detection of the true predictive players of survival. The first approach is data-driven yet biologically informed. Consistent with the biological hierarchy from DNA to RNA, we prioritize each survival-relevant feature with two separate scores, predictive and mechanistic. With mRNA expression levels in concern, predictive features are those mRNAs whose variation in expression levels are associated with the survival outcome, and mechanistic features are those mRNAs whose variation in expression levels are associated with genomic variants (copy number alterations (CNAs) in this study). Further, we propose simultaneously integrating information from both the predictive model and the mechanistic model through our new approach GEMPS (Gene Expression as a Mediator for Predicting Survival). Applied on two cancer types (ovarian and glioblastoma multiforme), our method achieved better prediction power than peer methods. Gene set enrichment analysis confirms that the genes utilized for the final survival analysis are biologically important and relevant.
The second approach is a generic mathematical framework to biologically regularize the Cox's proportional hazards model that is widely used in survival analysis. We propose a penalty function that both links the mechanistic model to the clinical model and reflects the biological downstream regulatory effect of the genomic variants on the mRNA expression levels of the target genes. Fast and efficient optimization principles like the coordinate descent and majorization-minimization are adopted in the inference process of the coefficients of the Cox model predictors. Through this model, we develop the regulator-target gene relationship to a new one: regulator-target-outcome relationship of a disease. Assessed via a simulation study and analysis of two real cancer data sets, the proposed method showed better performance in terms of selecting the true predictors and achieving better survival prediction. The proposed method gives insightful and meaningful interpretability to the selected model due to the biological linking of the mechanistic model and the clinical model.
Other important forms of clinical outcomes are monitoring angiogenesis (formation of new blood vessels necessary for tumor to nourish itself and sustain its existence) and assessing therapeutic response. This can be done through dynamic imaging, in which a series of images at different time instances are acquired for a specific tumor site after injection of a contrast agent. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a noninvasive tool to examine tumor vasculature patterns based on accumulation and washout of the contrast agent. DCE-MRI gives indication about tumor vasculature permeability, which in turn indicates the tumor angiogenic activity. Observing this activity over time can reflect the tumor drug responsiveness and efficacy of the treatment plan. However, due to the limited resolution of the imaging scanners, a partial-volume effect (PVE) problem occurs, which is the result of signals from two or more tissues combining together to produce a single image concentration value within a pixel, with the effect of inaccurate estimation to the values of the pharmacokinetic parameters. A multi-tissue compartmental modeling (CM) technique supported by convex analysis of mixtures is used to mitigate the PVE by clustering pixels and constructing a simplex whose vertices are of a single compartment type. CAM uses the identified pure-volume pixels to estimate the kinetics of the tissues under investigation. We propose an enhanced version of CAM-CM to identify pure-volume pixels more accurately. This includes the consideration of the neighborhood effect on each pixel and the use of a barycentric coordinate system to identify more pure-volume pixels and to test those identified by CAM-CM. Tested on simulated DCE-MRI data, the enhanced CAM-CM achieved better performance in terms of accuracy and reproducibility. / Ph. D.
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Properties and evolution of dense structures in the interstellar medium / Propriétés et évolution des structures denses en milieu interstellaireParikka, Anna 28 September 2015 (has links)
Mon travail de thèse présente deux types de structures denses : des sources froides compactes détectées par Planck et des condensations denses dans une région de photodissociation (PDR), à savoir la Barre d’Orion. Les deux types de structures sont étroitement liées à la formation des étoiles. Les sources froides sont étudiées comme objets potentiellement gravitationnellement liés, c’est-à-dire comme objets préstellaires. La Barre d’Orion est intéressante en tant que PDR à fort champ UV (G0 ∼104) prototypique, avec plusieurs disques protoplanétaires connus, éclairés par les jeunes étoiles du Trapèze.D’abord, je présente un article publié dans A&A: The Physical state of selected cold clumps. Dans cet article, nous avons comparé les observations du continuum de la poussière par Herschel provenant de l’open time key program Galactic Cold Cores (Herschel) aux observations de raies moléculaires par le radiotélescope de 20-m de l’Onsala Space Observatory en Suède. Les objets ont été sélectionnés en fonction de leur luminosités et faibles températures de couleur des poussières (T∼10−15 K). Nous avons calculé les masses du viriel et de Bonnor-Ebert et les avons comparées aux masses déduites à partir des observations. Les résultats indiquent que la plupart des objets froids observés ne sont pas nécessairement préstellaires.Dans mon étude de la Barre d’Orion, j’utilise des observations de l’instrumentPACS d’Herschel du programme Unveiling the origin and excitation mechanisms of the warm CO, OH and CH+ . Je présente des cartes de 110” ×110” du cation méthylidyne (CH+ J=3-2), des doublets de OH à 84 μm, et des raies de CO á hauts J (J=19-18). C’est la première fois que ces traceurs des PDR ont présentés avec une telle résolution spatiale et un tel rapport signal-sur-bruit.La répartition spatiale de CH+ et OH montre la même structure de la Barre qui a été vue dans d’autres observations. La morphologie du CH+ et H2 confirme que la formation et l’excitation de CH+ est fortement dépendante du H2 excité vibrationnellement. Le maximum d’émission de OH84 μm correspond à un objet brillant jeune, identifié comme le disque protoplanetaire 244-440.Je présente également des transitions rotationnelles de CO de moyenne (∼20 K) et haute (∼1000 K) énergie. La morphologie de l’émission du CO rotationnellement excité est corrélée avec la présence des petites structures denses irradiées. Nous établissons le lien entre le coeur de ces structures, tracé en CS J=2-1 par Lee et al. (2013) et H 13 CN par Lis and Schilke (2003) et le bord de la PDR, tracé en CO J=19-18 et le H 2 vibrationnellement excitée. Nous montrons également que le CO est principalement excité par le chauffage UV. / In this thesis I present a study of two kinds of dense ISM structures: compact cold sources detected by Planck and dense condensations in a photodissociation region (PDR), namely the Orion Bar detected by ground-based and Herschel telescopes. Both kinds of structures are closely related to star formation. The cold sources are investigated as potentially gravitationally bound, prestellar, objects. The Orion Bar is a highly FUV-illuminated (G0=104) prototypical PDR, with several known protoplanetary disks, illuminated by the young Trapezium stars.First I introduce a paper published in A&A: The Physical state of selected cold clumps. In this paper we compared the Herschel dust continuum observations from the open time key program Galactic Cold Cores to ground based molecular line observations from the 20-m radio telescope of the Onsala Space Observatory in Sweden. The clumps were selected based on their brightness and low dust color temperatures (T=10-15 K). We calculated the virial and Bonnor-Ebert masses and compared them to the masses calculated from the observations. The results indicate that most of the observed cold clumps are not necessarily prestellar.Then I move on to the warm and dense condensations of the ISM. In my study of the Orion Bar, I use observations from PACS instrument on board Herschel from the open time program Unveiling the origin and excitation mechanisms of the warm CO, OH and CH+. I present maps of 110”x110” of the methylidyne cation (CH+ J=3-2), OH doublets at 84 µm, and high-J CO (J=19-18). This is the first time that these PDR tracers are presented in such a high spatial resolution and high signal-to-noise ratio. The CH+ and OH have critical densities (1010 cm-3) and upper level energy temperatures (250 K). In addition the endothermicity of the CH+ + H2 reaction (4300 K) that forms CH+ is comparable to the activation barrier of the O + H2 reaction (4800 K) forming OH. Given these similarities it is interesting to compare their emission. The spatial distribution of CH+ and OH shows the same clumpy structure of the Bar that has been seen in other observations. The morphology of CH+ and H2 confirms that CH+ formation and excitation is strongly dependent on the vibrationally excited H2, while OH is not. The peak in the OH 84 µm emission corresponds to a bright young object, identified as the externally illuminated protoplanetary disk 244-440.Finally, I study the high-J CO in the Orion Bar. I also introduce low- and mid-J CO observations of the area. The high-J CO morphology shows a clumpy structure in the Bar and we establish a link between the dense core of the clumps, traced in CS J=2-1 by Lee et al. (2013) and in H13CN by Lis and Schilke (2003). We also show that the high-J CO is mainly excited by the UV heating.
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Contributions to the systematics and biocultural value of Aloe L. (Asphodelaceae)Grace, Olwen Megan 13 July 2009 (has links)
Aloe L. (Asphodelaceae) is a monocotyledonous group of considerable popularity among succulent plant collectors and with a long history of medicinal use. It comprises ca. 500 species occurring throughout Africa, the Arabian Peninsula and western Indian Ocean islands. The first comprehensive ethnobotanical study of Aloe (excluding the cultivated A. vera) was undertaken using the literature as a surrogate for data gathered by interview methods. Over 1400 use records representing 173 species were collated, the majority (74%) of which described medicinal uses, including species used for natural products. In southern Africa, 53% of approximately 120 Aloe species in the region are used for health and wellbeing. Consensus ratios indicated that the uses of Aloe spp. for medicine and pest control are of the greatest biocultural importance. Utility has contributed to the recognition of diversity, taxonomic complexity, and conservation concerns, in Aloe. A systematic evaluation of the problematic maculate (spotted) species complex, section Pictae, was undertaken. New sequences were acquired of the nuclear ribosomal internal transcribed spacer (ITS), chloroplast trnL intron, trnL–F spacer and matK gene in 29 maculate species of Aloe. A well supported monophyletic (holophyletic) maculate group was recovered in phylogenetic trees of comparable topology generated by parsimony analysis and Bayesian inference. A representative of the related section Paniculatae, A. striata, was recovered in the maculate group, whereas doubtful maculate species with unusual floral morphology (A. leptosiphon and A. suffulta) comprised a sister group. Analogous patterns were identified in chemosystematic and comparative morphological studies of 34 and 36 maculate species, respectively, and insights were gained into interspecific relationships. The flavonoids isoorientin and isovitexin, and a new C-glycosylanthrone, 6′-malonylnataloin, were characterised using hyphenated chromatographic techniques and nuclear magnetic resonance (NMR) spectroscopy. Leaf surface sculpturing, stomata and lobes surrounding the epistomatal pore observed under a scanning electron microscope (SEM) are of potential taxonomic significance. Available evidence indicates that floral characters, namely a basally swollen perianth with constriction above the ovary, are of greater significance than maculate leaves as synapomorphies for section Pictae. An evolutionary hypothesis for section Pictae excludes marginal maculate species with unusual flowers. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted
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Linienformation in M Klasse Sternen / Line Formation in M-type StarsWende, Sebastian 28 October 2010 (has links)
No description available.
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