Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Dr. John Cairney. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Many problems in the biological sciences reduce to questions of genetic evolution. Entire classes of medical pathology, such as malignant neoplasia or infectious disease, can be viewed in the light of Darwinian competition of genomes. With the benefit of today's maturing sequencing technologies we can observe and quantify genetic evolution with nucleotide resolution. This provides a molecular view of genetic material that has adapted, or is in the process of adapting, to its local selection pressures. A series of problems will be discussed in this thesis, all involving the mathematical modeling of genomic data derived from clonally evolving populations. We use a variety of computational approaches to characterize over-represented features in the data, with the underlying hypothesis that we may be detecting fitness-conferring features of the biology. In Part I we consider the cross-sectional sampling of human tumors via RNA-sequencing, and devise computational pipelines for detecting oncogenic gene fusions and oncovirus infections. Genomic translocation and oncovirus infection can each be a highly penetrant alteration in a tumor's evolutionary history, with famous examples of both populating the cancer biology literature. In order to exert a transforming influence over the host cell, gene fusions and viral genetic programs need to be expressed and thus can be detected via whole transcriptome sequencing of a malignant cell population. We describe our approaches to predicting oncogenic gene fusions (Chapter 2) and quantifying host-viral interactions (Chapter 3) in large panels of human tumor tissue. The alterations that we characterize prompt the larger question of how the genetics of tumors and viruses might vary in time, leading us to the study of serially sampled populations. In Part II we consider longitudinal sampling of a clonally evolving population. Phylogenetic trees are the standard representation of a clonal process, an evolutionary picture as old as Darwin's voyages on the Beagle. Chapter 4 first reviews phylogenetic inference and then introduces a certain phylogenetic tree space that forms the starting point of our work on the topic. Specifically, Chapter 4 describes the construction of our projective tree space along with an explicit implementation for visualizing point clouds of rescaled trees. The Chapter finishes by defining a method for stable dimensionality reduction of large phylogenies, which is useful for analyzing long genomic time series. In Chapter 5 we consider medically relevant instances of clonal evolution and the longitudinal genetic data sets to which they give rise. We analyze data from (i) the sequencing of cancers along their therapeutic course, (ii) the passaging of a xenografted tumor through a mouse model, and (iii) the seasonal surveillance of H3N2 influenza's hemagglutinin segment. A novel approach to predicting influenza vaccine effectiveness is demonstrated using statistics of point clouds in tree spaces. Our investigations into clonal processes may be extended beyond naturally occurring genomes. In Part III we focus on the directed clonal evolution of populations of synthetic RNAs in vitro. Analogous to the selection pressures exerted upon malignant cells or viral particles, these synthetic RNA genomes can be evolved against a desired fitness objective. We investigate fitness objectives related to reprogramming ribosomal translation. Chapter 6 identifies high fitness RNA pseudoknot geometries capable of inducing ribosomal frameshift, while Chapter 7 takes an unbiased approach to evolving sequence and structural elements that promote stop codon readthrough.

The response of three Eucalyptus spp. clones (GC550, GU210 and TAG14) to water availability was assessed in terms of growth, plant water status, leaf gas exchange, whole plant hydraulic characteristics (both root and shoot), stem xylem vulnerability. Furthermore, to experimentally assess the influence of hydraulic conductance on leaf physiology and plant growth, specimens of two of the clones were subjected to long-term root chilling. Prior to harvesting data were collected on the diurnal variation in leaf water potential (ΨL), transpiration rate (E), stomatal conductance (gs) and net CO2 assimilation rate (A). Main stem xylem vulnerability was assessed using ultrasonic acoustic emissions (UAE). Vulnerability of the main stem was assessed as the leaf water potential corresponding to the maximum rate of acoustic emissions (ΨL, EPHmax), and as the critical water potential triggering cavitation events, calculated as the mean of the water potentials of data points lying between 5 and 10% of the total accumulated emissions (ΨCAV,cUAE,%). Hydraulic conductance was measured on roots and shoots using the high-pressure flow meter (HPFM). Root data were expressed per unit root dry mass (Kr/trdw) and per unit leaf areas (Kr/LA), shoot data expressed per unit shoot dry mass (Ks/tsdw) and per unit leaf area (Ks/LA), and whole plant conductance was expressed per unit leaf area (KP/LA). Soil-to-leaf hydraulic conductance was also assessed as the inverse of the slope of the relationship between leaf water potential and transpiration rate (the evaporative flux, EF, method). A field study was undertaken on three month old TAGl4 and GU210 plants. Diurnal values of leaf water potential ΨL, E and gs were consistently higher in TAG14 than GU210, but A did not differ among the clones. Main stem xylem vulnerability (ΨCAV, cUAE,%) was higher in TAG14 than GU210. In both clones midday ΨL fell below ΨcAv,cUAE,%, suggesting lack of stomatal control of xylem cavitation. Kr/LA was higher in TAGl4 than GU210, whereas, Ks/LA and Ks/tsdw was higher in GU210 than TAGI4. A greater proportion of hydraulic resistances resided in the roots, particularly in GU210. Kp/LA was higher in TAGl4 than GU210 clone, although the significance was marginal (P=0.089). However, all the physiological measurements, were consistent with the concept of higher hydraulic conductances in TAGl4 leading to lower leaf level water stress. Above ground biomass was higher in TAG14 than GU210, in agreement with this concept, although this clone was more vulnerable than GU210. Material grown for 14 months in 25 l pots clones showed no differences in ΨSoil between the high and low watering supply, indicating that even the 'high' supply was inadequate to prevent water stress. In accordance with this, diurnal values of ΨL, gs, E and A did not differ significantly between treatments and clones. Early stomatal closure was apparent, maintaining ΨL constant during the middle of the day. Stem xylem vulnerability, assessed as both ΨL,EPHrnax and ΨCAV,cUAE,% showed that the main stem of GC550 was more vulnerable than other two clones, and that low watered plants were more resistant to xylem cavitation than those receiving high water. Midday ΨL fell below the vulnerability values assessed by both measures across treatments and clones, suggesting lack of stomatal control preventing stem xylem cavitation. There was no relationship between stem xylem cavitation and the shoot hydraulic conductances. Root pressures did not differ between either treatment or clones. Kr/LA was marginally higher in high watered plants, and Ks/LA and Ks/tsdw were higher in low watered plants, possibly by adjustment of leaf hydraulic architecture, and there were no clonal differences. Kr/LA was much lower than Ks/LA. Kp/LA did not differ between the watering treatment, but there was a clonal effect. Growth in dry mass was higher in high watered than low watered plants, but there were no differences among clones. As KP/LA was not affected by watering treatment there was no relationship between KP/LA and growth in total biomass. In plants grown for 21 months in 85 l pots low water treatment decreased midday ΨL, gs, E and A relative to high watered plants. Interclonal differences occurred at midday. Stem xylem vulnerability assessed as ΨCAV,cUAE,% and as ΨL,EPHrnax show similar trends as in the 14 months saplings, clonal differences being significant in ΨL, EPHmax. There was a 1:1 relationship between minimum leaf water potential and ΨL, EPHmax, suggesting that the water potential developed was limited by stem vulnerability. This implies stomatal control to reduce transpiration rates to prevent extensive cavitation occurring. These plants did not develop positive root pressures, indicating that recovery from xylem cavitations occurred through some other process. Kr/LA was higher in high watered plants than those receiving low water, and clonal differences were observed in Kr/trdw. There was no treatment effect in KS/LA and KS/tsdw, but a clonal effect was apparent. KP/LA was significantly different between treatment, and was reduced by low water in two clones, and increased by this in TAGI4. Reduced water availability reduced biomass production, with a greater effect on roots than shoots, such that low watering reduced root:shoot ratios. There was a weak but significant relationship between whole plant hydraulic conductance and maximum stomatal conductance, and between plant conductance and total biomass produced; these data are consistent with the concept of some hydraulic limitation to growth. Root chilling (achieved through chilling the soil) of two of the clones was used to experimentally manipulate hydraulic conductance to test the hydraulic limitation hypothesis. Short-term root chilling decreased both Kr/LA and KP/LA in both clones, but had marginal effects on leaf gas exchange. With long-term chilling the decrease in Kr/LA was observed only in GU210, with TAGl4 showing some adjustment to the treatment. As the roots constitute the major hydraulic resistance, KP/LA largely reflected those of the roots. Long-term root chilling significantly affected leaf physiological characteristics, despite the lack of effect on hydraulic conductance in TAGI4. Long term chilling decreased the whole plant dry mass, but the effect was smaller in TAGI4, and this clone also showed morphological adjustment, in that root growth was less adversely affected than shoot growth. The data from GU210 support the hydraulic limitation hypothesis; because of the morphological and physiological adjustment to long-term root chilling in TAGI4, the data are unsuitable to directly assess the hypothesis. / Thesis (Ph.D.)-University of Natal, Durban, 2002.

Thesis (Ph.D.)--Ohio University, March, 2005. / Includes bibliographical references (p. 139-146)

Thesis (Ph.D.)--Ohio University, March, 2005. / Title from PDF t.p. Includes bibliographical references (p. 139-146)

This research studied recombinant DNA-derived protein expression utilising expression vectors containing IRES sequences to link the gene of interest with the gene encoding selectable marker in mammalian cell cultures. Polycistronic expression constructs utilising internal ribosome entry site (IRES) can link unrelated genes under control of a single promoter. Transient study on the IRESlinked gene expression was performed. It was possible to standardise the level of protein expression to plasmid number by determining the number of free plasmids in the cytoplasm. The expression of a selectable marker when downstream of IRES was reduced in comparison to the monocistronic construct. Importantly when IRES was used, there were no negative effects on recombinant gene expression upstream of IRES. Down-regulating the selectable marker gene expression has been shown to enhance the probability of obtaining highly expressing clones. To investigate the effects of down-regulating fusion metallothionein green fluorescent protein (MTGFP), new constructs were created to combine metal inducible M2.6 promoter to drive the expression of human growth hormone linked to MTGFP by an attenuated IRES. This resulted in less MTGFP expression, reduced survivability and mean fluorescence in the presence of heavy metal. The increased metal sensitivity lengthened the initial selection period using reduced metal concentration in comparison to cells transfected with wildtype MTGFP. FACS can be used to select for resistance conferred by MTGFP despite reduced expression. FACS enrichment and sorting increased the hGH expression, which was correlated with mean fluorescence of the population; therefore fluorescence can be used as an indication of the final recombinant protein expression. Different approaches to isolate suitable clones were also investigated. It is preferable to select the transfected pool in low metal concentration for two weeks, sort for cells of high-fluorescence, and allow for recovery and proliferation. It is then possible to amplify gene expression by culturing the clones in increasing metal, resulting in further improvement of recombinant protein expression.

Homeobox genes encode homeodomain (HD) proteins which function as transcription factors and play an important role in plant and animal development by controlling cell specification and pattern formation. (Knotted1 in Arabidopsis thaliana) KNAT1-like mRNAs referred to as PtKN1(HD+) and mRNA sequences which lack HD region referred as PtKN1(hd-) were cloned from embryos of loblolly pine (Pinus taeda L.). Production of PtKN1(hd-) mRNAs is developmentally regulated and their encoded protein is abundant in mature pine embryos. Both forms of PtKN1 are produced by the same gene which has 5 exons; the regulatory dynamic is between cleavage-polyadenylation or termination within intron 3 to produce PtKN1 mRNA lacking HD sequences and splicing of exon 3 to exon 4 which excludes the 3'UTR/exon3 sequence to create an mRNA which encodes a HD. KNAT1 mRNA in Arabidopsis which lacks HD sequences was identified and characterized. While KNAT1 has been studied for many years, this is the first report of a KNAT1 mRNA lacking HD. KNAT1 mRNA lacking HD sequences was identified for the RS1 gene of maize, a monocotyledon. This is the first report of splicing of KNAT1 genes to produce mRNAs lacking HD sequences. The phenomenon appears to be ubiquitous as it is observed in gymnosperms, and both dicotyledonous and monocotyledonous angiosperms.

Thesis (M.Sc.)--Acadia University, 1999. / Includes bibliographical references (leaves 44-59). Also available on the Internet via the World Wide Web.

Thesis (M.Sc.)--Acadia University, 1999. / Includes bibliographical references (leaves 44-59). Also available on the Internet via the World Wide Web.