<p>As vertebrate embryos grow and develop into adults, their organs must acquire mass and mature tissue architecture to maintain proper homeostasis. While juvenile growth encompasses a significant portion of life, relatively little is known about how individual cells proliferate, with respect to one another, to orchestrate this final maturation. For its simplicity and ease of genetic manipulations, the teleost zebrafish (Danio rerio) was used to understand how the proliferative outputs of individual cells generate an organ from embryogenesis into adulthood. </p><p>To define the proliferative outputs of individual cells, a multicolor clonal labeling approach was taken that visualized a large number of cardiomyocyte clones within the zebrafish heart. This Brainbow technique utilizes Cre-loxP mediated recombination to assign cells upwards of ~90 unique genetic tags. These tags are comprised of the differential expression of 3 fluorescent proteins, which combine to give rise to spectrally distinct colors that represent these genetic tags. Tagging of individual cardiomyocytes was induced early in development, when the wall of the cardiac ventricle is a single myocyte thick. Single cell cardiomyocyte clones within this layer expanded laterally in a developmentally plastic manner into patches of variable shapes and sizes as animals grew into juveniles. As maturation continued into adulthood, a new lineage of cortical muscle appeared at the base of the ventricle and enveloped the ventricle in a wave of proliferation that fortified the wall to make it several myocytes thick. This outer cortical layer was formed from a small number (~8) of dominant cortical myocyte clones that originated from trabecular myocytes. These trabecular myocytes were found to gain access to the ventricular surface through rare breaches within the single cell thick ventricular wall, before proliferating over the surface of the ventricle.</p><p> </p><p>These results demonstrated an unappreciated dynamic juvenile remodeling event that generated the adult ventricular wall. During adult zebrafish heart regeneration, the primary source of regenerating cardiomyocytes stems from this outer wall of muscle. Regenerating cardiomyocytes within this outer layer of muscle are specifically marked by the cardiac transcription factor gene gata4, which they continue to express as they proliferate into the wound area.</p><p>Using heart regeneration to guide investigation of juvenile cortical layer formation, we found that both processes shared similar molecular and tissue specific responses including expression and requirement of gata4. Additional markers suggested that juvenile hearts were under stress and that this stress could play a role to initiate cortical morphogenesis. Indeed, experimental injury or a physiologic increase in stress to juvenile hearts caused the ectopic appearance of cortical muscle, demonstrating that injury could trigger premature morphogenesis.</p><p>These studies detail the cardiomyocyte proliferative events that shape the heart and identify molecular parallels that exist between regeneration and cortical layer formation. They show that adult zebrafish heart regeneration utilizes an injury/stress responsive program that was first used to remodel the heart during juvenile growth.</p> / Dissertation

Next-generation sequencing (NGS) techniques have led to major breakthroughs in the characterization of the chronic lymphocytic leukemia (CLL) genome with discovery of recurrent mutations of potential prognostic and/or predictive relevance. However, before NGS can be introduced into clinical practice, the precision of the techniques needs to be studied in better detail. Furthermore, much remains unknown about the genetic mechanisms leading to aggressive disease and resistance to treatment. Hence, in Paper I, the technical performance of a targeted deep sequencing panel including 9 genes was evaluated in 188 CLL patients. We were able to validate 143/155 (92%) selected mutations through Sanger sequencing and 77/82 mutations were concordant in a second targeted sequencing run, indicating that the technique can be introduced in clinical practice. In Paper II we screened 18 NF-κB pathway genes in 315 CLL patients through targeted deep sequencing which revealed a recurrent 4 base-pair deletion in the NFKBIE gene. Screening of NFKBIE in 377 additional cases identified the mutation in ~6% of all CLL patients. We demonstrate that the lesion lead to aberrant NF-κB signaling through impaired interaction with p65 and is associated with unfavorable clinical outcome. In Paper III we sought to delineate the genetic lesions that leads to relapse after fludarabine, cyclophosphamide, and rituximab treatment. Through whole-exome sequencing of pre-treatment and relapse samples from 41 cases we found evidence of frequent selection of subclones harboring driver mutations and subsequent clonal evolution following treatment. We also detected mutations in the ribosomal protein RPS15 in 8 cases (19.5%) and characterization of the mutations through functional assays point to impaired p53 regulation in cells with mutated RPS15. Paper IV aimed at characterizing 70 patients assigned to three major subsets (#1, #2, and #4) through whole-genome sequencing. Besides recurrent exonic driver mutations, we report non-coding regions significantly enriched for mutations in subset #1 and #2 that may facilitate future molecular studies. Collectively, this thesis supports the potential of targeted sequencing for mutational screening of CLL in clinical practice, provides novel insight into the pathobiology of aggressive CLL, and demonstrates the clinical outcome and cellular effects of NFKBIE and RPS15 mutations.

Neoadjuvant chemotherapy has become standard practice in patients with high-risk early breast cancer as it improves rates of breast conservation surgery and enables prediction of recurrence and survival by using response to treatment as a surrogate. Previous studies have focused on generating molecular datasets to develop prediction models of response, though little is known on how tumours and their microenvironments are modulated by neoadjuvant chemotherapy. The thesis aims at molecularly characterising tumour changes during neoadjuvant chemotherapy in a cohort of 168 patients. Serial tumour samples at diagnosis, and, when available, midway through chemotherapy and on completion of treatment were profiled by shallow whole genome sequencing, deep exome sequencing and transcriptome sequencing, resulting in the generation of an unprecedented genomics dataset with tumours in situ while patients received chemotherapy. Molecular predictors of response to chemotherapy were inferred from the diagnostic biopsy. Several novel observations were made, including previously undescribed associations between copy number alterations, mutational genotypes, neoantigen load, HLA genotypes and intra-tumoural heterogeneity with chemosensitivity. Possible mechanisms of chemoresistance included LOH at the MHC Class I locus, decreased expression of MHC Class I and II genes and drug influx molecules, as well as increased expression of drug efflux pumps. A complex relationship between proliferation, tumour microenvironment composition (TME) and response to treatment was explored by deconvoluting bulk RNAseq data and performing digital pathology orthogonal validation. Clonal and microenvironment dynamic changes induced by/associated with chemotherapy were then modelled. Two types of genomic responses were identified, one in which the clonal composition was stable throughout treatment and another where clonal emergence and/or extinction was evident. Validation by multi-region deep sequencing confirmed the dynamics of the clonal landscape. Clonal emergence was shown to be associated with higher proliferation and decreased immune infiltrate, with an increase in genomic instability and homologous recombination deficiency during treatment. The immune TME composition and activity mirrored response to treatment, with cytolytic activity and innate and adaptive immune infiltrates linearly correlating with the degree of residual disease remaining after chemotherapy. Finally, the circulating tumour DNA (ctDNA) genomic landscape was explored by using shallow whole genome sequencing and targeted sequencing of plasma DNA. Tumour mutations detected on exome sequencing were also detected in ctDNA in plasma, supporting the use of liquid biopsies as a biomarker for monitoring response to therapy and detection of minimal residual disease.

Riparian cottonwoods (populus spp.) are keystone pioneer species that contribute to critical streamside and in-stream habitats, water quality, and aesthetic and recreational value. Land use and river regulation have caused a widespread reduction in the extent and regeneration of this genus. The majority of research on Populus species' reproduction has examined seedling recruitment that dominates in wide alluvial valleys. In contrast, I evaluated reproductive strategies of Populus angustifoliain mountain valleys. Research was conducted in northern Utah on the Little Bear River, a gravel-bedded stream that flows north out of the Bear River Range onto deposits of Ancient Lake Bonneville. I used allozyme electrophoretic data and vegetation mapping to investigate recruitment mechanisms of cottonwoods at two steep, confined mountain valley sites and two wide, alluvial valley sites. Allozyme electrophoretic analysis on samples from sites one through four revealed 60%, 69%, 86%, and 84% clonal recruitment, respectively. The size of cottonwood clones may be on the order of 200-300 m in this system. Vegetation mapping supported allozyme data and suggested that clonal recruitment dominates the system. I also collected data at four sites containing no cottonwoods to compare hydrogeomorphic influences at non-cottonwood (NC) sites and cottonwood recruitment (CR) sites. CR sites possess a distinctive geomorphic template relative to NC sites and this template may facilitate root disturbance resulting in clonal recruitment. A high frequency of woody debris and mid-channel islands characterizes CR sites. Channels at CR sites are less entrenched, more sinuous, and exhibit larger bankfull channel width and width to depth ratios, and finer grain sizes than channels at NC sites. At CR sites, estimates of bankfull average boundary shear stress and unit stream power are less compared to those at NC sites. Cottonwood recruitment models that typically describe seedling reproduction in alluvial valley environments do not apply in narrow, steep-gradient environments. I found clonal recruitment of cottonwoods to correlate spatially and temporally with channel and root disturbance associated with rain on snow events on the order of the 25-30-yr flood event.

The bio-manufacturing of recombinant proteins from mammalian cell cultures requires robust processes that can maximize protein yield while ensuring the efficacy of these proteins as human therapeutics. Recognizing that the challenge of improving protein yield and quality can be met through various approaches, this paper presents three strategies currently being developed in our group. A method for rapidly selecting subpopulations of cells with high production characteristics is proposed. This method combines the efficiency of green fluorescent protein/fluorescence-activated cell sorting (GFP/FACS)–based screening with homologous recombination to generate and select high-producing subclones. Next, the development of chemically defined, protein-free media for enhancing monoclonal antibody production is described. Analysis of culture media effects on the genome-wide transcriptional program of the cell is presented as a means to optimize the culture media and identify potential targets for genetic manipulation. Finally, we propose a method for increasing the extent of intracellular sialylation by improving the transport of CMP-sialic acid into the trans-Golgi. This is hypothesized to increase the sialic acid availability, and may enhance the degree of sialylation in the glycoprotein product. / Singapore-MIT Alliance (SMA)

As shown by recent reports, number of obese people in recent years has been on the increase, there are about 4 million people in Taiwan who are considered to be overweight. World Health Organization (WHO) and United States Center for Disease Control and Prevention (CDC) publicly announced that: Obesity will be the greatest health killer of this century, its damage to personal health is comparable to that of cigarettes. Obesity can cause heart problems, diabetes, artery diseases, high blood pressure, increased chances of cancer occurrence, condition increase and deteriora- tion of Alzheimer¡¦s disease, gall bladder diseases, and shortening of life span. The cause of obesity is due to a fault in adipocytes metabolism functions, and because of this, research into adipocytes molecular regulation is becoming more popular and valued. The process of adipogenesis, the formation of adipose tissue, has become better understood by the studies of several cell types that can be induced to undergo differentiation into adipocytes. The first, and the best characterized, model of adipogenesis in vitro is the 3T3-L1 cell line, a substrain of Swiss 3T3 mouse cell line. 3T3-L1 cells propagated under normal conditions have a fibroblastic phenotype. However, when treated with a combination of dexamethasone, isobutylmethylxanthine (IBMX or MIX) and insulin, 3T3-L1 cells adopt a rounded phenotype and within 5 days begin to accumulate lipids intracellularly in the form of lipid droplets. Treatment of cells with dexamethasone activates the transcription factor CCAAT/enhancer -binding protein £] (C/EBP£]). IBMX inhibits soluble cyclic nucleotide phosphodiesterases and results in increased intracellular cAMP levels. At the nuclear level, treatment with IBMX results in activation of the related transcription factor C/EBP£_. Immediately after exposure to exogenous inducers, the gene expression of C/EBP£] and C/EBP£_ significantly and transiently increases, C/EBP£] and C/EBP£_ may also regulate the expression of C/EBP£\ and PPAR£^. C/EBP£\ and PPAR£^ are considered to play a prominent role in regulating the gene expression of proteins necessary for the development fo the functional mature adipocyte. Within 3 days of exposure to inducers, the cells undergo two rounds of mitosis, termed mitotic clonal expansion, which are required for differentiation. Insulin or insulin-like growth factor-1 promote adipocyte differentiation by activating PI3-kinase and Akt activity. Modulation of the activity of the forkhead transcription factor Foxo1 appears to be necessary for insulin to promote adipocyte differentiation. C/EBP£\ and PPAR£^ direct the final phase of adipogenesis by activating expression of adipocyte-specific genes, such as fatty acid synthetase, fatty acid binding protein, leptin and adiponectin. The identification of regulators of adipogenesis raises the prospect of preventing or reversing obesity through pharmacological means. My research is aimed at investigating the adipocytes differentiation and regeneration adaptive mechanisms of mice 3T3L-1 preadipocytes and human processed lipoaspirate cells (PLA). By using adipocytes culture techniques in conjunction with adipocytes growth induction and gene delivery techniques to further study obesity related genes, POMC and PTEN, and downstream regulators , PPAR£^ and Adiponectin, in regards to their roles in the process of adipocytes differentiation.

Clonal culture is crucial for experimental protocols that require growth or selection of pure populations of cells. Currently, there is no method for deriving neural stem cells (NSCs) clonally from single human embryonic stem cells (hESCs). Bulk derivation of neural progenitors from hESCs for cell therapies can lead to a host of problems including incomplete differentiation leading to proliferation of tumorigenic clusters in vivo. Clonal derivation allows for the screening and selection of only the most suitable cells for culture and expansion. We have developed a clonal, serum free method of generating NSCs and their progenitors directly from hESCs with an efficiency of 1.6%. The NSC colony-forming cell was identified as a TRA-1-60-/SSEA4- cell whose fate becomes specified in maintenance conditions by inhibition of bone morphogenic protein (BMP) signalling. This clonal culture method can be scaled up to produce vast quantities of NSCs for differentiation and use in cell therapies.

Clonal culture is crucial for experimental protocols that require growth or selection of pure populations of cells. Currently, there is no method for deriving neural stem cells (NSCs) clonally from single human embryonic stem cells (hESCs). Bulk derivation of neural progenitors from hESCs for cell therapies can lead to a host of problems including incomplete differentiation leading to proliferation of tumorigenic clusters in vivo. Clonal derivation allows for the screening and selection of only the most suitable cells for culture and expansion. We have developed a clonal, serum free method of generating NSCs and their progenitors directly from hESCs with an efficiency of 1.6%. The NSC colony-forming cell was identified as a TRA-1-60-/SSEA4- cell whose fate becomes specified in maintenance conditions by inhibition of bone morphogenic protein (BMP) signalling. This clonal culture method can be scaled up to produce vast quantities of NSCs for differentiation and use in cell therapies.

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.

Micropropagation methods are often used to supplement existing clonal programmes for Eucalyptus species. However, genotypic differences among clones require the implementation of clone-specific protocols, an expensive and labour-intensive exercise. Hence, this study aimed at determining high-yielding hybrid-specific rather than clone-specific, micropropagation protocols for E. grandis x nitens (GN), E. grandis x nitens (NH), and E. grandis x urophylla (GU). Different conditions for surface sterilisation, bud-break (3 protocols, 2 media), multiplication (4 media), elongation (2 protocols) and rooting (4 media) were tested. A single successful surface sterilisation approach was possible for all clones of the tested hybrids (0.0-11.8% contamination, 0.0-22.9% necrosis). It involved rinsing nodal explants in a fungicide mixture (lg/l Benlate, 1g/1 boric acid, 0.5ml/1 Bravo, Tween 20) for 15 minutes followed by calcium hypochlorite (10g/l with Tween 20) for three minutes. Results at each culture stage were dependent on genotypes, and results indicated here represent ranges in values among the clones of each hybrid. The highest bud-break values for GN clones (87-90%) and NH clones (17-75%) were achieved on a medium containing MS, 0.1mg/1 biotin, 0.1mg/l calcium pantothenate, 0.04mg/1 NAA, 0.11mg/l BAP and 0.05mg/1 kinetin. In GU clones, bud-break values on this medium (84-97%) were not significantly different to those achieved directly on a multiplication medium (80-91%) (MS, 0.1 mg/l biotin, 0.1 mg/l calcium pantothenate, 0.2mg/l BAP, 0.01mg/1 NAA). Shoot multiplication yields for GN clones (4-13 shoots/bud) and GU clones (2-6 shoots/bud) were achieved on a medium consisting of MS, 0.1mg/1 biotin, 0.1 mg/l calcium pantothenate, 0.2mg/1 BAP and 0.01 mg/l NAA. As genotypic effects were highly significant among NH clones, a single multiplication medium for all clones of this hybrid could not be determined. The best method of elongation for clones of all three hybrids involved culturing shoots on MS, 0.1 mg/l calcium pantothenate, 0.1mg/1 biotin, 0.35mg/1 NAA, 0.1mg/l kinetin and 0.1mg/1 IBA, under photoperiod conditions, rather than total darkness, for 6 weeks. This resulted in 82.3-86.6% elongation and shoot lengths increasing by 22.9-35.2 mm for GN clones, 80.2-82.3 % elongation and an increase in length of 24.7-32.2 mm for NH clones and 70.8-78.1 % elongation, and shoot elongation of 21.6-29.3 mm for GU clones from passage 1-2. For all the above stages, media contained 20/25 g/l sucrose and 3.5g/l Gelrite, and cultures were maintained at 25°C ± 2°C day/ 21°C night with a 16 h light/ 8 h dark photoperiod (PPFD 66µmol/m2/s). In terms of rooting, cultures on different media were initially subjected to a 72 hour period of total darkness at room temperature, then a 16 h light/8 h dark photoperiod (PPFD 37µmol/m2 /s) at 24°C day/ 21°C night for 7 days. This was followed by a 16 h light/ 8 h dark photoperiod (PPFD 66µmol/m2/s) at 25°C ± 2°C day/ 21°C night for 21 days. Tested clones of the three hybrids were all rooted successfully (56-93% rooting in GN clones, 36-76% rooting in NH clones and 46-96% rooting in GU clones) on a medium containing ¼ MS, 0.1 mg/l biotin, 0.1 mg/l calcium pantothenate, 0.1mg/l IBA, 0.22g/1 CaCI2 .2H20, 0.185g/l MgS04.7H2O, 15g/l sucrose and 3.5g/1 Gelrite. Predicted yields from the established protocol are also presented (168-667 plants of E. grandis x nitens (GN), 35- 854 plants of E. grandis x nitens (NH) and 54-349 plants of E. grandis x urophylla from 100 initial nodal explants, depending on the clone). Hence, the established protocols can be used successfully for some of the clones, but the implementation of specific media and methods to obtain high yields may still be necessary for certain clones. / Thesis (M.Sc.)-University of Natal, Durban, 2001.