<p>Development of serological assays for the diagnosis of Baylisascaris larva migrans in birds and non-human primates</p>

Once thought to be an evolutionary dead-end, hybridization is now being detected in genomes across kingdoms, perhaps even playing an integral role in evolution. In chapter 1, I investigated the potential influences of hybridization on the evolution of a group of vagrant, asexual species in the Rhizoplaca melanophthalma species group. I sequenced the mitochondrial and nuclear genomes of 55 specimens and found well-supported nuclear phylogenies of both datasets. There were, however, multiple instances of discordance between the mitochondrial and nuclear trees, which can be caused by hybridization. PhyloNet and ABBA-BABA also detected widespread hybridization among this group. In chapter 2, I shifted to the Holarctic clade of lichen-forming fungi in Xanthoparmelia to characterize gene tree conflict and investigate the potential for hybrization. Here, I used three different tests for hybridization that account for incomplete lineage sorting "ABBA-BABA, PhyloNet, and SplitsTree" as well as PhyParts to characterize gene tree conflict. Like the Rhizoplaca species group, widespread hybridization was detected in the Holarctic clade despite robust phylogenies. My research underscores the value of investigating hybridization when studying species boundaries and evolutionary history.

The genus Chenopodium contains many economically important species in the New World, but is relatively understudied and poorly understood, especially in terms of evolutionary relationships. A better understanding of the structure of this genus could significantly help in breeding efforts on its cultivated members, notably the tetraploid C. quinoa and also certain varieties of C. berlandieri, also tetraploid. Of special concern is determining which diploid weed species are the most likely ancestors for C. quinoa, C. berlandieri, and the other tetraploid members of subsection Cellulata. The phylogeny can be understood in part by examining the ribosomal RNA loci and observing how many copies of the 5S and 45S loci each New World species contains. In this work, the 5S and 45S ribosomal RNA loci are characterized by means of fluorescence in situ hybridization in 23 Chenopodium species collected in the New World, with the 5S locus labeled red and the 45S locus labeled green. Based on these results, the pool of most likely candidate ancestor species for C. quinoa and C. berlandieri includes C. fremontii, C. incanum, C. neomexicanum, and C. watsonii.

Detection of single nucleotide substitutions (SNS) in DNA and RNA has a growing importance in biology and medicine. One traditional approach for recognition of SNS takes advantage of hybridization probes that bind target nucleic acids followed by measuring ?Tm, the difference in melting temperatures of matched and mismatched hybrids. The approach enables SNS differentiation at elevated temperatures (usually 40-65oC) often only in a narrow range of < 10oC and requires high-resolution melting devices. Here we demonstrate that a specially designed DNA probe (X sensor) can broaden ?Tm from ~10oC to ~16oC and distinguish SNS in the interval of ~5-40oC. Therefore, there is no need for heating or measuring Tm for accurate SNS differentiation. Our data indicate that this wide differentiation range is in part due to the non-equilibrium hybridization conditions. Further we explored the idea that it is possible to improve the performance of an X sensor operable in close to equilibrium conditions by shifting its operability to non-equilibrium conditions. One way to achieve this is to introduce as many as possible structured ligands in analyte's dissociated state. Here we show that by introducing the maximum possible conformational constraints in X probe it is possible to shift its operation to non-equilibrium conditions and to improve its selectivity at temperatures < 15oC. Thus, this work points towards a new strategy for the design of highly selective hybridization sensors which operate in non-equilibrium conditions at close to room temperature. The X sensors could be utilized in qPCR, microarrays, as well as RNA analysis in living cells and for ambient temperature point-of-care diagnostics. In the last part of this work, X sensors were used in real time detection of PCR products. The sensors were optimized to operate in PCR buffer with optimal Mg2+ concentration. They were able to detect the target amplicon together with nonspecific products. The results presented here suggest that X sensors might be adopted for real time PCR format.

Molecular and morphologic studies were used to determine the evolution, classification and differentiation of Nymphaea odorata. Molecular analyses of the nuclear internal transcribed spacer (ITS) region, the chloroplast trnL-F region, and inter-simple sequence repeat (ISSR) markers determined the variation present between and within two species of Nymphaea. The ITS region resulted in a phylogeny depicting strong separation between species (N. mexicana and N. odorata) and some separation between N. odorata's subspecies. The ITS region contained polymorphisms, which upon SAHN clustering and principle coordinate (PCOA) and minimum spanning tree (MST) analyses produced groups similar to the clades in the ITS phylogeny. Sixteen accessions were chosen for trnL-F analysis, where a subspecies-specific molecular marker was found. In most accessions the marker confirmed the original subspecies classification. Molecular analyses using ISSRs characterized among population variation in N. odorata and N. mexicana using five primers. ISSR markers among populations were highly variable within a species and were used in UPGMA, PCOA and MST analysis, which resulted in separation between the subspecies. Both univariate and multivariate analyses were performed on quantitative and qualitative morphological characters. An analysis of variance resulted in six morphological characteristics that were statistically significant (P< 0.05), the majority being leaf blade characteristics. Multivariate statistics of principle component analysis and discriminate analysis resulted in groups for each subspecies, both emphasized the importance of quantitative leaf blade characteristics. Overall, both morphology and molecular characteristics supported the classification of subspecies for ssp. odorata and ssp. tuberosa, due a lack of strong segregation of characteristics. / Master of Science

No description available.

No description available.

No description available.

No description available.

No description available.