Evolution is an ubiquitous feature of living systems. The genetic composition of a population changes in response to the primary evolutionary forces: mutation, selection and genetic drift. Organisms undergoing rapid adaptation acquire multiple mutations that are physically linked in the genome, so their fates are mutually dependent and selection only acts on these loci in their entirety. This aspect has been largely overlooked in the study of asexual or somatic evolution and plays a major role in the evolution of bacterial and viral infections and cancer. In this thesis, we put forward a theoretical description for a minimal model of evolutionary dynamics to identify driver mutations, which carry a large positive fitness effect, among passenger mutations that hitchhike on successful genomes. We examine the effect this mode of selection has on genomic patterns of variation to infer the location of driver mutations and estimate their selection coefficient from time series of mutation frequencies. We then present a probabilistic model to reconstruct genotypically distinct lineages in mixed cell populations from DNA sequencing. This method uses Hidden Markov Models for the deconvolution of genetically diverse populations and can be applied to clonal admixtures of genomes in any asexual population, from evolving pathogens to the somatic evolution of cancer. To understand the effects of selection on rapidly adapting populations, we constructed sequence ensembles in a recombinant library of budding yeast (S. cerevisiae). Using DNA sequencing, we characterised the directed evolution of these populations under selective inhibition of rate-limiting steps of the cell cycle. We observed recurrent patterns of adaptive mutations and characterised common mutational processes, but the spectrum of mutations at the molecular level remained stochastic. Finally, we investigated the effect of genetic variation on the fate of new mutations, which gives rise to complex evolutionary dynamics. We demonstrate that the fitness variance of the population can set a selective threshold on new mutations, setting a limit to the efficiency of selection. In summary, we combined statistical analyses of genomic sequences, mathematical models of evolutionary dynamics and experiments in molecular evolution to advance our understanding of rapid adaptation. Our results open new avenues in our understanding of population dynamics that can be translated to a range of biological systems.

In the field of biologics production, productivity and stability of the transfected gene of interest are two very important attributes that dictate if a production process is viable. To further understand and improve these two traits, we would need to further our understanding of the factors affecting them. These would include integration site of the gene, gene copy number, cell phenotypic variation and cell environment. As these factors play different parts in the development process, they lead to variable productivity and stability of the transfected gene between clones, the well-known phenomenon of “clonal variation”. A study of this phenomenon and how the various factors contribute to it will thus shed light on strategies to improve productivity and stability in the production cell line. Of the four factors, the site of gene integration appears to be one of the most important. Hence, it is proposed that work is done on studying how different integration sites affect the productivity and stability of transfected genes in the development process. For the study to be more industrially relevant, it is proposed that the Chinese Hamster Ovary dhfr-deficient cell line, CHO-DG44, is used as the model system. / Singapore-MIT Alliance (SMA)

Questions: 1. Are there differences among species in their preference for coniferous vs. deciduous forest? 2. Are tree and shrub species better colonizers of recent forest stands than herbaceous species? 3. Do colonization patterns of plant species groups depend on tree species composition? Location: Three deciduous and one coniferous recent forest areas in Brandenburg, NE Germany. Methods: In 34 and 21 transects in coniferous and deciduous stands, respectively, we studied the occurrence and percentage cover of vascular plants in a total of 150 plots in ancient stands, 315 in recent stands and 55 at the ecotone. Habitat preference, diaspore weight, generative dispersal potential and clonal extension were used to explain mechanisms of local migration. Regression analysis was conducted to test whether migration distance was related to species’ life-history traits. Results: 25 species were significantly associated with ancient stands and ten species were significantly more frequent in recent stands. Tree and shrub species were good colonizers of recent coniferous and deciduous stands. In the coniferous stands, all herbaceous species showed a strong dispersal limitation during colonization, whereas in the deciduous stands generalist species may have survived in the grasslands which were present prior to afforestation. Conclusions: The fast colonization of recent stands by trees and shrubs can be explained by their effective dispersal via wind and animals. This, and the comparably efficient migration of herbaceous forest specialists into recent coniferous stands, implies that the conversion of coniferous into deciduous stands adjacent to ancient deciduous forests is promising even without planting of trees.

Most clonal plants exhibit a modular structure at multiple levels. At the level of the organs, they are characterized by functional modules, such as, internodes, leaves, branches. At the level of the genetic individual (clone or genet), they possess independent evolutionary and physiological units (ramets). These evolutionary units arise through the widespread phenomenon of clonal reproduction, achieved in a variety of ways including rhizomes, stolons, bulbils, or lateral roots. The focus of this study was Populus tremuloides, trembling aspen, a dioecious tree that reproduces sexually by seed and asexually through lateral roots. Local forest patches in western populations of Populus tremuloides consisted largely of multiple genotypes. Multi-clonal patches were dominated by a single genotype, and in one population (Riske Creek) we found several patches (five out of 17) consisting of a single genotype. A second consequence of modularity is that during the repeated cycle of ramet birth, development and death, somatic mutations have the opportunity to occur. Eventually, the clone becomes a mosaic of mutant and non-mutant cell lineages. We found that neutral somatic mutations accumulated across 14 microsatellite loci at a rate of between 10^-6 and 10^-5 per locus per year. We suggest that neutral genetic divergence, under a star phylogeny model of clonal growth, is an alternative way to estimate clone age. Previous estimates of clone age couple the mean growth rate per year of shoots with the area covered by the clone. This assumes a positive linear relationship between clone age and clone size. We found, however, no repeatable pattern across our populations in terms of the relationship of either shape or size to the number of somatic changes. A final consequence of modularity is that during clonal growth, natural selection is relaxed for traits involving sexual function. This means that mutations deleterious to sexual function can accumulate, reducing the overall sexual fitness of a clone. We coupled neutral genetic divergence within clones with pollen fitness data to infer the rate and effect of mildly deleterious mutations. Mutations reduced relative sexual fitness in clonal aspen populations by about 0.12x10^-3 to 1.01x10^-3 per year. Furthermore, the decline in sexual function with clone age is evidence that clonal organisms are vulnerable to the effects of senescence.

Although semi-natural grasslands in Europe are declining there is often a time delay in the local extinction of grassland species due to development of remnant populations, i.e., populations with an extended persistence despite a negative growth rate. The objectives of this study were to examine the occurrence of remnant populations after abandonment of semi-natural grasslands and to examine functional traits of plants associated with the development of remnant populations. We surveyed six managed semi-natural grasslands and 20 former semi-natural grasslands where management ceased 60-100 years ago, and assessed species response to abandonment, assuming a space-for-time substitution. The response of species was related to nine traits representing life cycle, clonality, leaf traits, seed dispersal and seed mass. Of the 67 species for which data allowed analysis, 44 species declined after grassland abandonment but still occurred at the sites, probably as remnant populations. Five traits were associated with the response to abandonment. The declining but still occurring species were characterized by high plant height, a perennial life form, possession of a perennial bud bank, high clonal ability, and lack of dispersal attributes promoting long-distance dispersal. Traits allowing plants to maintain populations by utilizing only a part of their life cycle, such as clonal propagation, are most important for the capacity to develop remnant populations and delay local extinction. A considerable fraction of the species inhabiting semi-natural grasslands maintain what is most likely remnant populations after more than 60 years of spontaneous succession from managed semi-natural grasslands to forest. / <p>authorCount :3</p>

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

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.

Enterococci are fecal indicator bacteria (FIB) that are used worldwide for water quality assessment. However, evidence of high densities and extended survival of enterococci in sediments and submerged aquatic vegetation (SAV) has caused uncertainty about their reliability in predicting human health risks from recreational activities in environmental waters. To address the concern that sediments and SAV may harbor large reservoirs of enterococci that can affect water column concentrations, aquatic mesocosms and environmental sampling were employed to investigate patterns of enterococci densities and population structure across the Tampa Bay watershed. In mesocosm experiments and environmental samples, SAV harbored higher densities of enterococci, per mass of substrate, than sediments, and sediments harbored higher densities than water. Population structure assessed by BOX-PCR genotyping was relatively unique in each sample, although slight similarities among samples suggested grouping primarily by location rather than substrate or season. Strain diversity was highly variable, and many samples had low diversity, including nearly monoclonal structure throughout the mesocosm experiments and in several of the environmental samples. Several strains were highly abundant and cosmopolitan (found across sites, seasons, and substrates), and may represent highly naturalized and reproducing indicator bacteria populations that are not directly related to pollution events. When the enterococci densities were viewed from the perspective of the entire aquatic system, SAV-associated enterococci did not comprise a major proportion of the total population, due to the typically large differences in volume of each substrate (SAV vs. sediments vs. water). Instead, the largest proportions of enterococci were typically found in the water or the sediments, depending on the relative volume of substrate or the enterococci density associated with each substrate. Modeling results illustrate that the relative importance of each substrate in terms of FIB populations can shift dramatically over time and space due to changes such as vegetation cover, tidal cycles, and bacteria densities. Furthermore, at several sites within the watershed, estimates of sediment and bacteria resuspension from sediments were very low, suggesting that this process rarely, if ever, significantly affects water column concentrations of enterococci in the watershed.

Acute myeloid leukemia (AML) is a clonal neoplastic disorder organized as a cellular hierarchy, with the self-renewing leukemia stem cell (LSC) at the apex. Recurrent mutations in transcription factors (TF) and epigenetic regulators suggest that AML is driven by aberrant transcriptional circuits, but these circuits have not been fully defined in an LSC model. To study transcriptional mechanisms relevant to leukemogenesis in vivo, we generated a murine serial transplantation model of MLL-AF9-driven, myelomonocytic leukemia with genetically- and phenotypically-defined LSCs. Using this model, we pursued two related lines of investigation. First, we performed an in vivo RNA interference (RNAi) screen to identify transcription factors required for LSC function. This screen highlighted the circadian rhythm TFs, Clock and Bmal1, as genes essential for the survival of murine leukemia cells, and we validated this finding with CRISPR/Cas-based genome editing and knockdown studies in AML cell lines. Utilizing luciferase reporter mice to track expression of the circadian target gene Per2, we demonstrated that both leukemic and normal hematopoietic cells have the capacity for oscillating, circadian-dependent gene expression. Importantly, using murine knockout models, we found that normal hematopoietic stem and progenitor cells (HSPC), in contrast to leukemia cells, do not depend on Bmal1. We further demonstrated that selective depletion of LSCs following circadian perturbation is mediated through enhanced myeloid differentiation. ChIP-Seq studies revealed that the circadian rhythm network is integrally connected to the LSC self-renewal circuitry and highlighted putative Clock/Bmal1 targets in leukemia, providing a mechanistic basis for our findings. Second, we performed a functional and genomic characterization of our MLL-AF9 serial transplantation model to explore mechanisms of disease evolution and clonal selection in AML. Limiting dilution studies demonstrated that serial transplantation results in a reduction in disease latency, dramatic enrichment of leukemia-initiating cells (LIC), and reconfiguration of the LSC hierarchy. While mutations in known AML-associated genes were not linked to disease progression, RNA-sequencing (RNA-Seq) demonstrated that the increase in LIC frequency in serially transplanted leukemias is driven by changes in cell cycle and differentiation. In aggregate, these studies offer insights into the biological mechanisms regulating LSC self-renewal and disease evolution in AML.