Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the PDGFRA gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called Sequence Allocator, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.

The impact of CYP2A6 and CYP2B6 genetic variation on nicotine metabolism, tobacco use behaviours, and nicotine biomarkers was investigated in a group of Alaska Natives (n = 400). CYP2A6 and CYP2B6 allele frequencies were unique and associations of CYP2A6 genotype and CYP2A6 activity (plasma and urine trans 3’-hydroxycotinine/cotinine (3HC/COT) ratios) were robust. Notably, this population possessed a more rapid rate of CYP2A6 activity (higher plasma 3HC/COT) when compared to CYP2A6 wild-type individuals in other ethnic groups (ANOVA P < 0.001). Also demonstrated was a significant difference in urine total nicotine equivalents by CYP2A6 activity median split (t-test P < 0.01), the first evidence of nicotine titration by CYP2A6 activity within a light smoking population. Overall, this population possessed a distinctive pattern of CYP2A6 and CYP2B6 variant frequencies and a faster rate of nicotine metabolism, which may in part explain higher levels of tobacco use prevalence and tobacco-related disease risk.

The impact of CYP2A6 and CYP2B6 genetic variation on nicotine metabolism, tobacco use behaviours, and nicotine biomarkers was investigated in a group of Alaska Natives (n = 400). CYP2A6 and CYP2B6 allele frequencies were unique and associations of CYP2A6 genotype and CYP2A6 activity (plasma and urine trans 3’-hydroxycotinine/cotinine (3HC/COT) ratios) were robust. Notably, this population possessed a more rapid rate of CYP2A6 activity (higher plasma 3HC/COT) when compared to CYP2A6 wild-type individuals in other ethnic groups (ANOVA P < 0.001). Also demonstrated was a significant difference in urine total nicotine equivalents by CYP2A6 activity median split (t-test P < 0.01), the first evidence of nicotine titration by CYP2A6 activity within a light smoking population. Overall, this population possessed a distinctive pattern of CYP2A6 and CYP2B6 variant frequencies and a faster rate of nicotine metabolism, which may in part explain higher levels of tobacco use prevalence and tobacco-related disease risk.

In the present study, within and among breed genetic variability in seven native Turkish sheep breeds (Akkaraman, Dagli&ccedil / , G&ouml / k&ccedil / eada, ivesi, Karayaka, Kivircik and Morkaraman) were analyzed based on 20 microsatellite loci. For the analysis, various statistical methods such as Neighbor-Net, Factorial Correspondence Analysis (FCA) and Structure were used. High level of genetic variability within the Turkish breeds was observed. Gene pools of the breeds were visualized and found that they are highly overlapping with each other. As one of the reasons of this overlap, genetic exchange between the breeds was suggested. Dagli&ccedil / , claimed to be the ancestors of first domestic sheep in Anatolia, seemed to be the most admixed one. Yet Dagli&ccedil / , despite being the most introgressed one, still might be exhibiting its uniqueness. Observations implied that conservation practices concerning Dagli&ccedil / must be urgently revised. Results of the present study do not support previous observations about the genetic differentiation patterns of the breeds within Anatolia. Possible reasons of the discrepancies between the observations were discussed. Genetically extreme individuals can be identified by Structure, Assignment and FCA tests. These methods are found to be promising in establishing new relatively pure breeds or in saving the breeds from further genetic contamination. Genetically outlier individuals were shown not to exhibit any distinct morphological differences. Unknown band patterns were found by RFLP and SSCP of mtDNA Control Region and the individuals harboring those were sequenced. They were shown to belong to the common haplogroups A, B or C. No novel haplogroup was found.

The objective of this study is to elucidate the efficiency of enation-structure (at node) recognition method at pre-flowing stage and to understand the population genetic variation of the Mikania weeds in Taiwan. The plant materials collected by recognizing enation-structure symptom method from North, Central, South and East Taiwan and off-shore islands. Using PCR¡V sequencing marker techniques, the sequencing revealed that nrDNA ITS region could identify three Mikania weeds and the 97% similarity of phylogenetic relationship between M.cordata and M.micrantha are more closer than that between M.cordata and M.scandens, whose ITS sequence is obtained from GeneBank. However ,the sequences of chloroplast DNA of M.cordata and M.micrantha at trnL intron¡]436bp¡^or trnL-trnF IGS¡]345 bp¡^are almost the same and could not be used as molecular markers. The recognizing techniques of enation-structure was supported by the nrDNA ITS region and ISSR results at the end, thus the finding can be recommended to the Council of Agriculture in order to eliminate the weed and to reduce the impact on M.cordata, which is native in Taiwan. Moreover, the findings of ISSR analysis in the aspect of population genetic variation indicated that high genetic differentiation ¡]Gst>0.5¡^was found among the M. cordata and M.micrantha populations. Based on the Mantle test, there was no relationship between genetic distance and geographic distance in M.micrantha¡]r=0.0053,p=0.47¡^.This phenomenon revealed that populations of M.micrantha had complex population variability within the short-term invaded into Taiwan that might be resulted from the random dispersion of human activities. The population of M.micrantha was established by few individuals (founders) and grown rapidly in Taiwan, resulting in population differentiation via genetic drift. In contrast to M.micrantha ,there was relationship between genetic distance and geographic distance in M.cordata ¡]r=0.44,p=0.025¡¯¡^.It revealed that populations of M.cordata agreed to the concept of isolation by distance model, which might be evolved from the result of natural dispersion. In conclusion, the population structure of M.micrantha in Taiwan is stable , suggesting that control of Mikania population should be based on different populations where have large differentiation among them .

The two most common varieties of the grass Bouteloua curtipendula (Michx.)Torr. in North America use different strategies of clonal growth and have contrasting continental distributions. Variety caespitosa (phalanx form) ranges from central Texas westward to Arizona and California and var. curtipendula (guerrilla form) occurs in a more mesic region over the North American Great Plains. This study sought to determine whether the strategies had an ecological significance and investigated the possible relationship between changes in environmental factors and characteristics of each clonal growth strategy. Varieties showed to be morphologically variable, but the variability did not follow the pattern of the precipitational gradient. The abundance of var. curtipendula was related to soil depth and parent material (limestone types). Abundance of var. caespitosa could not be explained by any environmental factor separately. The performance of clones of the two growth forms in response to changes in resource availability (light and nutrients) and defoliation suggested similarities between the varieties in photosynthetic rate and only showed differences in water potential under extreme conditions. The major differences were related to the proportion of biomass allocated to structures related with seed production versus propagation by rhizomes. Intra-variety genetic variation for several life history traits was detected even with a very small sample size. The caespitose growth form showed more biomass and rate of tiller recruitment after defoliation on average, but responses between genotypes were dissimilar. Varieties also showed levels of plasticity in the allocation to reproductive structures in response to environmental factors. Responses to fire were compared between varieties by experimental burnings with increasing load of artificial fuel. Plants of the two varieties reached similar maximum temperatures although var. caespitosa suffered temperatures considered to be lethal for longer periods of time. Results from this study suggested that, although characteristic of the pattern of clonal growth were not distinctly associated to resource availability or defoliation, distribution of the varieties may be related to a combination of biotic and abiotic factors beyond the factors studied here.

Abiotic and biotic variation has been shown to be important in regulating nutrient cycling and belowground communities in natural systems. However, genetic variation in dominant plants as a driver of rates of nutrient cycling is still poorly understood and few studies have looked at genotype interactions across multiple environments. Using Populus angustifolia and a common garden approach, we hypothesized that all three factors: tree genetic variation, environmental conditions and genetic by environment (G x E) interactions would affect soil carbon (C) storage and nitrogen (N) cycling. Replicated copies of five different reciprocally planted Populus genotypes were studied in three separate 18-21 year old common gardens at different elevations (1300m, 1384m and 1587m) in northern Utah, to measure the genotype and environmental effects on pools of soil C and N as well as rates of soil net N nitrification and net mineralization. Our results indicate that genotypes influence pools of soil C, total N and C:N, but genotype did not influence net rates of nitrogen mineralization. Environmental variation significantly influenced pools of soil C, total N, soil C:N and rates of net nitrification and net N mineralization. As predicted, G x E interactions significantly influenced both pools and processes of soil C and N cycling. Overall, we found that genetic variation in plant traits (tree diameter and leaf/root chemistry) as well as soil texture across gardens were significant predictors of soil C and N pools and fluxes across seasons. These data help us understand the relative role of genotypic variation on above- and belowground interactions in different environments and the consequences of these interactions on ecosystem processes. The results from this study show that across an environmental gradient Populus angustifolia genotypes can influence nitrogen mineralization through feedbacks between environmental variation, tree phenotype and soils.

Intraspecific genetic diversity is an important attribute of natural populations and is deemed critical for their adaptive potential and persistence. However, we have limited empirical understanding of the impact of genetic diversity on population performance under different conditions. For my dissertation, I conducted long-term laboratory experiments with populations of the flour beetle Tribolium castaneum to test the consequences of genetic variation for population dynamic stability and niche evolution. In Chapter 1, I show that genetic variation prevented population extinction in a novel habitat. In addition, genetically diverse populations were more stable, both in a novel heterogeneous habitat and in their ancestral habitat. In the ancestral habitat, alleles from a single founding lineage dominated the dynamics, leading to increased stability of genetically diverse populations. However, such as selective effect was not observed in the novel heterogeneous habitat. Therefore, while genetic variation within populations increased their stability and persistence, the magnitude of the impact and its mechanism depended on the selective habitat. In Chapter 2, I ask whether genetic variation also facilitates resource niche expansion, i.e., use of a novel resource. Using stable carbon isotopes, I analyzed diets of beetles sampled from the above experiment and quantified the rate of change in resource use. Contrary to theoretical predictions, I found that genetic variation for resource use had no effect on the rate of niche evolution. Furthermore, behavioral niche expansion accounted for most of the adaptation to the novel resource, and the behavioral change hindered subsequent evolutionary change in resource use. It is thus apparent that in the short term, behavioral plasticity in niche use may impose far greater constraints on niche evolution than the amount of standing genetic variation. Mathematical models predict that intraspecific competition generates selection for niche evolution, and that genetic variation increases the response to selection. Therefore, I hypothesized that the impact of genetic variation on resource niche evolution may depend on the degree of intraspecific competition. In the final chapter of this thesis, I describe results of an experiment to test this hypothesis. I found that genetic variation and competition indeed interacted to increase the rate of niche expansion in T. castaneum, but that their impacts were temporally variable. Furthermore, the two factors acted on different components of niche evolution: while competition only affected the degree of niche expansion, genetic variation also promoted maintenance of individual variation in resource use. In summary, my thesis describes experiments to test for the ecological and evolutionary impacts of intraspecific genetic variation; and its interaction with behavioral plasticity, intraspecific competition, and resource availability. Genetic diversity and behavioral plasticity are common features of living organisms, and therefore it is vital to understand their combined consequences for population ecological and evolutionary dynamics. In addition, natural populations often face intense competition for limited resources. Hence the experimental results presented here can help us to better understand how populations overcome these resource constraints, given their specific genetic composition. Biologists are increasingly aware that the intricate connection between ecological and evolutionary dynamics is important to gain a more complete understanding of population biology. The work described here represents one of the few experiments providing such detailed mechanistic understanding of the interactions between- and consequences of - key ecological and evolutionary parameters. Finally, the results have important implications for conservation biology, because they show that the effects of genetic diversity can vary greatly depending on a number of population and environmental parameters. / text

Nicotine, the main addictive compound in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Substantial genetic variation in the CYP2A6 gene contributes to large variation in nicotine metabolism which alters numerous smoking behaviours. The goal of this study was to identify and characterize novel CYP2A6 variants. The CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced. Seven novel non-synonymous variants were identified: 468G>A (V68M), 1767C>G (I149M), 3515G>A (R265Q), 3524T>C (I268T), 4406C>T (T303I), 5661G>A (E390K), 6531T>C (L462P). They were introduced into a cDNA expression construct where they displayed lower protein expression, reduced nicotine metabolism to cotinine, and/or reduced stability as evaluated by western blotting and enzymatic activity. Genotyping assays were developed and assessed in 512 African Americans. Allelic frequencies ranged from 0.1-0.6% with a collective genotype frequency of 3.2%. Here we identified novel variants with reduced/loss of CYP2A6 activity, increasing our understanding of CYP2A6 variability.

Nicotine, the main addictive compound in tobacco, is metabolically inactivated to cotinine primarily by the hepatic enzyme CYP2A6. Substantial genetic variation in the CYP2A6 gene contributes to large variation in nicotine metabolism which alters numerous smoking behaviours. The goal of this study was to identify and characterize novel CYP2A6 variants. The CYP2A6 gene from African American phenotypically slow nicotine metabolizers was sequenced. Seven novel non-synonymous variants were identified: 468G>A (V68M), 1767C>G (I149M), 3515G>A (R265Q), 3524T>C (I268T), 4406C>T (T303I), 5661G>A (E390K), 6531T>C (L462P). They were introduced into a cDNA expression construct where they displayed lower protein expression, reduced nicotine metabolism to cotinine, and/or reduced stability as evaluated by western blotting and enzymatic activity. Genotyping assays were developed and assessed in 512 African Americans. Allelic frequencies ranged from 0.1-0.6% with a collective genotype frequency of 3.2%. Here we identified novel variants with reduced/loss of CYP2A6 activity, increasing our understanding of CYP2A6 variability.