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Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1978 K44 / Master of Science

LD2668 .T4 1962 R44

published_or_final_version / Pathology / Master / Master of Medical Sciences

Multicolor fluorescence in-situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Often, the intensity distributions between different images are found to be considerably different and the difference becomes the source of misclassifications of the pixels. Improved pixel classification accuracy is the most important task to ensure the success of the M-FISH technique. Along with a reliable pixel classification method, automation of the karyotyping process is another important goal. The automation requires segmentation of chromosomes, which not only involves object/background separation but also involves separating touching and overlapping chromosomes. While automating the segmentation of partially occluded chromosomes is an extremely challenging problem, a pixel classification method that satisfies both high accuracy and minimum human intervention has not been realized. The main contributions of this dissertation include development of a new feature normalization method for M-FISH images that reduces the difference in the feature distributions among different images, and development of a new decomposition method for clusters of overlapping and touching chromosomes. A significant improvement was achieved in pixel classification accuracy after the new feature normalization. The overall pixel classification accuracy improved by 40% after normalization. Given a cluster, a number of hypotheses was formed utilizing the geometry of a cluster, pixel classification results, and chromosome sizes, and a hypotheis that maximized the likelihood function was chosen as the correct decomposition. Superior decomposition results were obtained using the new method compared to the previous methods. Contributions also include development of a color compensation method for combinatorially stained FISH images (including M-FISH images) based on a new signal model for multicolor/multichannel FISH images. The true signal was recovered based on the signal model after color compensation. The resulting true signal does not have color spreading (channel crosstalk) among different color channels. Two new unsupervised nonparametric classification methods for M-FISH images are also introduced in this dissertation: a fuzzy logic classifier and a template matching method (a minimum distance classifier). While both methods produce an equivalent accuracy compared to a supervised classification method, their computation time is significantly less than a Bayes classifier. Highly sophisticated and practical algorithms have been developed through this research. Using the developed methods, the amount of human intervention required will be significantly reduced: chromosomes are reliably and accurately segmented from the background, pixels are accurately classified, and clusters of overlapping and touching chromosomes are automatically decomposed. / text

Symbioses between cool-season grasses (Subfamily Pooideae) and endophytic fungi in the genera Epichlo and Neotyphodium straddle a continuum of interactions from antagonistic to highly mutualistic. Although these two genera of endophytes are closely related, Neotyphodium endophytes are strictly seed-transmitted and provide many physiological and defensive benefits to their hosts, while Epichlo spp. have an obligately sexual contagious stage wherein host inflorescences are replaced by fungal sexual structures (stromata), effectively sterilizing the plant. Between these two extremes of interactions are Epichlo spp. with a mixed strategy, where some grass tillers are sterilized while others develop normally and yield healthy endophyte-infected seeds. These symbioses offer a unique opportunity to dissect evolutionary mechanisms that may drive movement along this continuum. The research presented characterizes distinct hybridization processes in endophytes and grasses that result in the generation of astounding genetic diversity for the symbiosis. Interspecific hybridization via hyphal anatomosis is a common feature of Neotyphodium endophytes, and may promote mutualism by combining suites of defensive alkaloid genes and ameliorating the adverse evolutionary effects of an asexual lifestyle. My results demonstrate that several genetically distinct hybrid endophytes infect grass species in tribe Poeae. Further, I show that a highly mutualistic asexual endophyte infecting tall fescue (=Festuca arundinaceum Schreb.), Neotyphodium coenophialum, also infects two closely related and interfertile relatives of this host. My findings suggest that this seed-borne endophyte may have been introgressed into these grasses through sexual grass hybridization events. These findings highlight interspecific hybridization as a means of generating tremendous genetic variability in both endophytes and their hosts, thus magnifying the adaptive evolutionary potential of these symbioses. Further, I establish a phylogenetic framework for grasses naturally harboring Epichlo and Neotyphodium endophytes. I show that patterns of genetic divergence among grass lineages are emulated by those of their fungal symbionts. These results suggest that endophytes have co-evolved with grasses in subfamily Pooideae, and may have played a critical role in the evolutionary success and radiation of this group of grasses.

Ce travail s’inscrit dans le cadre de la recherche relative au développement de biosenseurs à ADN électrochimiques. Des aspects fondamentaux, ainsi que des aspects d’application de la détection d’hybridation d’ADN sont envisagés. Dans un premier temps, le comportement interfacial et le processus d’hybridation d’oligonucléotides d’ADN linéaires et ADN hairpin (structure en épingle à cheveux) nonmarqués sont étudiés en formant des monocouches auto-assemblées mixtes de monobrins d’ADN (ssADN) thiolés et d’un hydroxyalcanethiol (4-mercaptobutan-1-ol) par coadsorption spontanée sur des électrodes d’or polycristallin. L’immobilisation de monocouches mixtes ssADN/MCB est caractérisée par voie électrochimique et par spectroscopie des photoélectrons X. Des mesures de chronocoulométrie, en présence de [Ru(NH3)6]3+ (RuHex), permettent de déterminer la quantité d’ADN dans la monocouche mixte formée. Les résultats montrent que l’excès superficiel d’ADN linéaire est plus important que l’excès superficiel d’ADN hairpin sous des conditions de formation identiques. La réaction de reconnaissance moléculaire d’hybridation est détectée par des mesures d’impédance en présence de [Fe(CN)6]3-/4-. L’hybridation se traduit dans le cas de l’ADN linéaire par une augmentation de la résistance au transfert d’électron Rct tandis que dans le cas de l’ADN hairpin, Rct diminue. Ces différences sont dues au plus faible recouvrement et au changement de conformation des molécules d’ADN hairpin lors de l’hybridation. Des mesures de réflectivité de neutrons nous ont permis de mettre en évidence l’augmentation de l’épaisseur du film d’ADN hairpin et de confirmer le changement conformationel ces sondes lors de la reconnaissance moléculaire. Dans la seconde partie, nous présentons une nouvelle méthode électrochimique de détection d’hybridation, basée sur les interactions électrostatiques entre le complexe cationique RuHex et les groupements phosphates de l’ADN. Afin d’améliorer la détection des molécules de PNA (peptide nucleic acid) ont été immobilisées comme sondes de reconnaissance moléculaire. Après hybridation des sondes PNA avec le brin complémentaire, RuHex s’adsorbe sur l’ADN hybridé et un signal de réduction de ces complexes redox, enregistré par voltampérométrie alternative, constitue une signature claire de l’hybridation d’ADN à l’interface modifiée. Les interactions RuHex/PNA-ADN ont été étudiées. La constante d’adsorption de RuHex sur l’électrode modifiée PNA/MCB après hybridation est évaluée à 2,9 (±0,3) 105 M-1 en milieu Tris-HCl 0,01M, selon une isotherme de Langmuir. Les performances analytiques de la méthode de détection (sensibilité, sélectivité et reproductibilité) ont été évaluées et optimisées pour la détection des séquences d’ADN du gène de l’ARNr 23S d’Helicobacter pylori. La méthode de détection électrochimique présentée est assez sélective pour permettre de discriminer les mutations ponctuelles A2143G et A2144C de la séquence de type sauvage. La diminution significative des signaux d’admittance enregistrés en présence des séquences mutées est attribuée à la capacité accrue de discrimination de mutations ponctuelles des molécules PNA. La réponse de détection est linéaire en fonction du logarithme de la concentration de la cible d’ADN sur plus de quatre ordres de grandeur (10-6 M à 10-10 M). La limite de détection de l’oligonucléotide d’ADN complémentaire de 80 pM est très bonne. La méthode a été appliquée avec succès à la détection de fragments PCR complémentaires de 100 et 400 paires de bases, amplifiés à partir de souches SS1 d’H.pylori.

Four species of the genus Parthenium were involved in a hybridization attempt. The species were: guayule (Parthenium argentatum Gray), Parthenium fruticosum Less, Parthenium bipinnatifidum (Ortega) Rollins, and Parthenium incanum H. B. K. Primary attention was given to the formation of hybrids between P. argentatum and P. fruticosum. Characteristics of the latter species such as size, growth rate, possible disease resistance, and wider geographical adaptation were desirable for transfer to P. argentatum. Reciprocal crosses were made between these two species and the production of hybrids was not difficult. However, in the case of selfing, backcross and sibcrosses, germination percent was 0.86 from a total of 3,471 achenes. Therefore, a search for the cause or causes of the negative results was initiated. The factors which were considered of primary interest were: incompatibility, genic and chromosomal sterility, pollination and planting techniques, and seed germination treatment. Incompatibility was considered the main limiting factor in the formation of a backcross population. Rubber analysis was performed in the interspecific hybrids. Mean rubber percent for hybrids between P. artentatum and P. fruticosum was 1.19; and for the reciprocal cross it was 0.39. Hybrids of the cross P. fruticosum x P. bipinnatifidum had a mean rubber percent of 0.19.

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.