Fortschritte in der molekulargenetischen Charakterisierung von Tumorerkrankungen haben in den letzten Jahren die klinische Praxis zunehmend beeinflusst. Das Ziel dieser Arbeit war die Untersuchung von astrozytären Tumoren der WHO-Grade II bis IV und ihre Subtypisierung anhand der gefundenen chromosomalen Aberrationen. Ferner sollte der Einfluss der gefundenen Aberrationen auf klinische Parameter wie das Gesamtüberleben oder die rezidivfreie Zeit untersucht werden. Dazu wurden paraffinfixierte Proben von insgesamt 184 primären astrozytären Tumoren (28 low-grade Astrozytome, 6 low-grade Oligoastrozytome, 50 anaplastische Astrozytome, 4 anaplastische Oligoastrozytome, 96 Glioblastoma multiforme) mit der Comparativen Genomischen Hybridisierung (CGH) untersucht. Häufige Aberrationen in allen Malignitätsgruppen stellten chromosomale Zugewinne auf Chromosom 7 sowie Verluste von Chromosom 10 und 9p dar. High-grade Astrozytome zeigten ferner häufig Zugewinne von Chromosom 19 und 20 sowie Verluste von 13q, 14q und 15q. WHO-Grad-II-Astrozytome wiesen häufig Zugewinne auf Chromosom 8 sowie Verluste von Chromosom 4q und 6q auf. Eine kürzeres Gesamtüberleben zeigten high-grade Gliome mit Verlusten von Chromosom 10q und Zugewinnen auf 7p. In Glioblastomen verursachten zusätzlich Zugewinne auf 7q sowie Verluste von 14q, in anaplastischen Astrozytomen zusätzlich Verluste von 10p ein verringertes Gesamtüberleben. WHO-Grad-II-Astrozytome zeigten bei Verlusten von 3p ein schlechteres Gesamtüberleben. Chromosomale Aberrationen, die zu einem verlängerten Gesamtüberleben führten, waren Verluste von 1p und Zugewinne von 10p in WHO-Grad-III-Tumoren. Die rezidivfreie Zeit wurde in high-grade Gliomen durch Zugewinne auf 7p und Verluste von 10p verringert. Eine Verkürzung der rezidivfreien Zeit in Glioblastomen zeigten außerdem Tumoren mit Verlusten von 7q, 10q und 14q. In anaplastischen Astrozytomen führten Verluste von 1p und 19q sowie Zugewinne auf 8q und 10p, in WHO-Grad-II-Astrozytomen Verluste von Chromosom 6 zu einer verlängerten Zeit bis zum Rezidiv. Die Anfertigung onkogenetischer Baummodelle stellte verschiedene genetische Wege der Tumorgenese dar. Ein Cluster war gekennzeichnet durch einen Verlust von 6q, ein weiterer wurde initialisiert durch den Verlust von 13q, der dritte durch den Verlust von 9p. Der vierte Cluster wurde charakterisiert durch Zugewinne auf Chromosom 7 und Verluste von Chromosom 10, während der fünfte Cluster Zugewinne auf 8q sowie Verluste von 4q aufwies.

The methods presented in this study are aimed at the identification of subpopulations (clones) of genetically similar cells within tissue samples through measurement of loci-specific Fluorescence in-situ hybridization (FISH) spot signals for each nucleus and analyzing cell spatial distributions by way of Voronoi tessellation and Delaunay triangulation to robustly define cell neighbourhoods. The motivation for the system is to examine lung cancer patient for subpopulations of Non-Small Cell Lung Cancer (NSCLC) cells with biologically meaningful gene copy-number profiles: patterns of genetic alterations statistically associated with resistance to cis-platinum/vinorelbine doublet chemotherapy treatment. Current technologies for gene-copy number profiling rely on large amount of cellular material, which is not always available and suffers from limited sensitivity to only the most dominant clone in often heterogeneous samples. Thus, through the use of FISH, the detection of gene copy-numbers is possible in unprocessed tissues, allowing identification of specific tumour clones with biologically relevant patterns of genetic aberrations. The tissue-wide characterization of multiplexed loci-specific FISH signals, described herein, is achieved through a fully automated, multicolour fluorescence imaging microscope and object segmentation algorithms to identify cell nuclei and FISH spots within. Related tumour clones are identified through analysis of robustly defined cell neighbourhoods and cell-to-cell connections for regions of cells with homogenous and highly interconnected FISH spot signal characteristics. This study presents experiments which demonstrate the system’s ability to accurately quantify FISH spot signals in various tumour tissues and in up to 5 colours simultaneously or more through multiple rounds of FISH staining. Furthermore, the system’s FISH-based cell classification performance is evaluated at a sensitivity of 84% and specificity 81% and clonal identification algorithm results are determined to be comparable to clone delineation by a human-observer. Additionally, guidelines and procedures to perform anticipated, routine analysis experiments are established.

Rampant lateral gene transfer (LGT) among prokaryotes, hybridization in plants and other reticulate evolutionary processes invalidate typical phylogenetic tree models by violating the assumption that organisms only inherit genetic information from a single parent species. Comparing the different evolutionary histories of multiple genes is necessary to identify and assess these processes. In this work I develop efficient approximation and fixed-parameter algorithms for computing rooted maximum agreement forests (MAFs) and maximum acyclic agreement forests (MAAFs) of pairs of phylogenetic trees. Their sizes correspond to the subtree-prune-and-regraft (SPR) distance and the hybridization number of these pairs of trees, which are important measures of the dissimilarity of phylogenies used in studying reticulate evolution. Although these MAFs and MAAFs are NP-hard to compute, my fixed-parameter algorithms are practical because they scale exponentially with the computed distance rather than the size of the trees. I contribute efficient fixed-parameter algorithms for computing MAFs and MAAFs of two binary rooted trees and give the first efficient fixed-parameter and approximation algorithms for computing MAFs of two multifurcating rooted trees. My open-source implementation of the MAF algorithms is orders of magnitude faster than previous approaches, reducing the time required to compute SPR distances of 46 between trees of 144 species to fractions of a second whereas previous approaches required hours to compute SPR distances of 25. These fast MAF-based distance metrics enable the construction of supertrees to reconcile a collection of gene trees and rapid inference of LGT. Simulations demonstrate that supertrees minimizing the SPR distance are more accurate than other supertree methods under plausible rates of LGT. I constructed an SPR supertree from a phylogenomic dataset of 40,631 gene trees covering 244 genomes from several major bacterial phyla and inferred "highways" of gene transfer between these bacterial classes and genera; a small number of these highways connect distantly related genera and can highlight specific genes implicated in long-distance LGT. These fast MAF algorithms are thus practical and enable new analyses of reticulate evolution.

No description available.

The propagation of ultrasound through two- and three-dimensional strongly scattering media, with either random or ordered internal structures, has been investigated through experiments and finite element simulations. All media investigated have strong scattering resonances, leading to novel transport behaviour. The two-dimensional samples consist of nylon rods immersed in water. When the nylon rods are arranged in a triangular lattice to form two-dimensional phononic crystals, very unusual dispersion properties are observed when the lattice constant is adjusted so that Bragg and hybridization gaps overlap in frequency. This behaviour is attributed to the competition between two co-existing propagating modes, leading to a new method for tuning bandgap properties and adjusting the transmission by orders of magnitude. The scattering resonance of the nylon rods also leads to unusual Dirac cone properties at the K point of the triangular lattice. The three-dimensional media were fabricated by brazing aluminum beads together to form a disordered porous solid network, with either vacuum or air in the pores, depending on the experiment. This system is of particular interest because it has been shown to exhibit Anderson localization of ultrasound. Two experimental approaches were developed to investigate previously unstudied properties of this system. By directly counting the modes in the frequency domain, the density of states was measured. At intermediate frequencies, the density of states was found to be approximately independent of frequency, while at higher frequencies, the frequency dependence was consistent with traditional density-of-states models. The level statistics of the modes was also investigated to determine the conditions under which level repulsion occurs. By using a laser interferometer to measure the ultrasonic displacements on the surface of a large slab-shaped sample, sub-diffusive behaviour was observed, demonstrating the feasibility of using such measurements to investigate the transition to Anderson localization in these samples.

Early diagnosis of endometrial cancer (EC) is uncertain and women undergo preventive hysterectomy in cases where a non-invasive treatment can be used instead. To contribute to solving this challenge we investigated if early changes in the nuclear 3D telomere architecture during carcinoma development can be detected prior to the first morphological evidence of precancerous lesions. We utilized Pten heterozygous mice that develop progressive carcinoma in the endometrial tissue similar to EC development in women. We used telomere fluorescence in situ hybridization (FISH), 3D molecular imaging and analysis techniques on interphase nuclei of endometrial glandular epithelial cells to identify alterations in the 3D-telomere profile. We found that telomere dysfunction in Pten heterozygous mice is present already in endometrial simple hyperplasia lesions prior to detectable loss of PTEN protein expression and that the 3D telomere architecture has a specific signature that indicates early telomere dysfunction predictive for endometrial malignant transformation.

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

Early diagnosis of endometrial cancer (EC) is uncertain and women undergo preventive hysterectomy in cases where a non-invasive treatment can be used instead. To contribute to solving this challenge we investigated if early changes in the nuclear 3D telomere architecture during carcinoma development can be detected prior to the first morphological evidence of precancerous lesions. We utilized Pten heterozygous mice that develop progressive carcinoma in the endometrial tissue similar to EC development in women. We used telomere fluorescence in situ hybridization (FISH), 3D molecular imaging and analysis techniques on interphase nuclei of endometrial glandular epithelial cells to identify alterations in the 3D-telomere profile. We found that telomere dysfunction in Pten heterozygous mice is present already in endometrial simple hyperplasia lesions prior to detectable loss of PTEN protein expression and that the 3D telomere architecture has a specific signature that indicates early telomere dysfunction predictive for endometrial malignant transformation.

A photolabile terpolymer containing 6-nitroveratyloxycarbonyl (NVOC) protected amine, epoxy and trimethoxysilyl functionality in 1:3:2 monomer ratio was synthesized to template glass surfaces for specific site directed coupling of non-probe oligomers and probe oligonucleotides. Non-probe oligomers were introduced to the surface to control the environment of the probes by reducing probe-to-probe and probe-to-surface interactions. The trimethoxysilyl group served as the anchoring site for the terpolymer to be covalently bound to glass and silicon wafers. Amine terminated non-probe oligomers were coupled to the epoxy sites and thiolated 19-mer SMN1 probes were directed to the deprotected amine sites via the heterobifunctional linker, sulfosuccinimidyl-4-[maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Characterization of the terpolymer was done using 1H NMR, 13C NMR, MALDI-ToF and elemental analysis. NVOC deprotection was monitored by UV absorption, and surface characterization of the bound terpolymer on silicon wafers was investigated with XPS, ToF-SIMS, ellipsometry and static contact angle. Neutral polyethylene glycol (PEG), negatively charged methacrylic acid (MAA) oligomer and dC20 oligonucleotides were used as non-probe oligomers. The probe density on the surface was estimated to be 2.2 ± 0.3 x 10^12 molecules/cm2 and the presence of the oligomers on the surface did not significantly affect probe immobilization efficiency. The mixed films were functional for target hybridization and its selectivity towards partially-mismatched targets was investigated at different solution pH, ionic strength and temperature. It was demonstrated that pH can be tuned to ameliorate non-specific adsorption and ionic strength governed the selectivity of the surfaces. Improved selectivity was achieved at high salt concentration (1 M NaCl) on PEG and dC20 mixed films at room temperature. The MAA surface did not show significant improvements in selectivity. This indicated that charge of the oligomers does not dominate control of selectivity. The results suggested that the terpolymer construct played a role in depression of the melting temperature of the hybridized duplex to within 5 to 10 oC of room temperature. With the melting temperature shifted closer to room temperature, it is possible to improve selectivity for room temperature detections of single nucleotide polymorphism.

Recent genetic studies have confirmed that there was admixture between African early modern humans and archaic populations throughout the Old World. In this dissertation, I examine European early modern human dental morphology to assess the evidence for Neandertal-human admixture. The focus of this study is not on the question of taxonomic designations of Late Pleistocene Homo, but rather on the interactions of these populations in Europe. This focus on gene flow itself redefines the Neandertal question. Rather than asking if Neandertals are a different species from H. sapiens, I focus on the nature of the interactions between archaic and modern populations, which is essential to understanding the history of modern H. sapiens regardless of species definitions. I recorded dental metric measurements and morphology observations on 85 fossil Neandertals and early modern humans and a recent modern human comparative sample of 330 Native Americans and Spaniards. I examined each trait distribution individually and through the use of Mahalanobis D2, mean measure of divergence, principle components analysis, discriminant function analysis, k-means cluster analysis, and a population genetics program, structure. Through these methods, I found evidence of admixture in the dental trait distributions of European early modern humans. However, it is not evident in traditional distance measures or cluster analyses. The earliest European modern humans do not follow the trend of dental reduction found throughout the Pleistocene and into the Holocene and do not uniformly classify with any fossil population in discriminant function analysis of metric traits. The non-metric trait sample size is too small to make any definitive conclusions, but a mosaic pattern of trait frequencies also suggests admixture. The recent modern human sample shows that while increased variation and a mosaic of non-metric traits persist through many generations after admixture has ceased, traditional methods of distance analysis cannot detect low levels of admixture within 200 years. The program structure is effective in finding patterns of variation within and among populations using morphological data. It will be useful for future analyses of dental traits and other fossil data, given the ability to use it with an incomplete data set.