Cardiovascular diseases contribute a large amount of morbidity and mortality in developing and developed countries all around the world. In conditions such as the myocardial infarction, a significant amount of cardiomyocytes die leading to an impaired function of the heart. One promising method for replacing these cardiomyocytes would be with the use of cardiomyocytes derived from embryonic stem cells. However, a large number of cardiomyocytes and a highly efficient method for obtaining cardiomyocytes are needed. Using the principles of small molecule treatment to induce differentiation in a serum-free based differentiation protocol, I have demonstrated that the induction of canonical Wnt signalling via CHIR 99021, and subsequent addition of bone morphogenetic protein 4 was best able to induce cardiomyogenesis in mouse embryonic stem cells. While improvements in efficiency are still required, the manipulation of the Wnt and BMP4 signalling pathways hold great promise in improving cardiomyogenesis in mESCs.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第14264号 / 工博第3015号 / 新制||工||1448(附属図書館) / 26591 / UT51-2008-T24 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 伊藤 紳三郎, 教授 大嶋 正裕 / 学位規則第4条第1項該当

M. Tech. / Stem cells are regarded as undifferentiated cells that are capable of selfrenewal, proliferation, production of a great number of differentiated progeny, and regeneration of tissues (Blau et al., 2001). The therapeutic potential of multilineage stem cells for tissue engineering (TE) applications is vast. Two general types of stem cells are potentially useful for this application: embryonic stem cells (ESCs) and adult (autologous) stem cells (Zuk et al., 2001). Traditionally, ESCs are isolated from the inner cell mass (ICM) of blastocysts, however harvesting of these cells results in the death of the embryo, which has led to ethical, religious and political issues (Moore, 2007). In contrast, adult stem cells, by virtue of their nature, are immunocompatible and have no ethical issues associated with their use (Zuk et al., 2001). Subcutaneous adipose tissue is an active and highly complex tissue composed of several different cell types, and is derived from the mesodermal germ layer and contains a supportive stromal vascular fraction (SVF) that can be easily isolated. This SVF contains a heterogeneous mixture of cells including preadipocytes (Raposio et al., 2007; Schäffler and Büchler, 2007; Jurgens et al., 2008). The preadipoctyes are considered as the multipotent stem cells termed adipose derived stem cells (ADSCs) that have similar properties to bone marrow mesenchymal stem cells (BM-MSCs) (Fraser et al., 2006). ADSCs are idyllic for cellular therapy applications due to various factors: they can be harvested, multiplied and handled easily, efficiently and noninvasively, they have a pluripotential and proliferative capacity comparable to BM-MSCs, and morbidity to donors is considerably less, requiring only local anaesthesia and a short wound healing time. Human ADSCs (hADSCs) can be expanded in an undifferentiated state and have multipotential differentiation capacity along the classical mesenchymal lineages of adipogenesis, osteogenesis, chondrogenesis and myogenesis (de Villiers et al., 2009).

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Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of 129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 or Day-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues) showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Polycomb proteins are epigenetic regulators that are critical in mediating gene repression at critical stages during development. Core and accessory proteins make up the Polycomb Repressive Complex 2 (PRC2), which is responsible for trimethylation of lysine 27 on histone 3 (H3K27me3), leading to maintenance of chromatin compaction and sustained gene repression. Classically, Polycomb accessory proteins are often thought of as having minor roles in fine-tuning the repressive action of PRC2. Their actions have often been attributed to chromatin recognition, targeting to specific loci and enhancing methyltransferase activity. In our previous work in mouse embryonic stem cells (ESCs), we showed that Polycomb-like 2 (Mtf2/Pcl2) is critical for PRC2-mediated regulation of stem cell self-renewal through feed-forward control of the pluripotency network. In moving beyond the ESC model system, we sought to interrogate the role of Mtf2 in vivo by creating a gene-targeted knockout mouse model. Surprisingly, we discovered a tissue-specific role for Mtf2 in controlling erythroid maturation and hematopoietic stem cell self-renewal. Via its regulation of other PRC2 members, Mtf2 is critical for global H3K27me3 methylation at promoter-proximal sites in developing erythroblasts. Thus, Mtf2 is required for proper maturation of erythroblasts. Loss of Mtf2 also reduces HSC self-renewal leading to stem cell pool exhaustion. Additionally, misregulation of Mtf2 in leukemia models contributes to massive leukemic blast expansion at the expense of leukemic stem cell self-renewal. In the developing hematopoietic system, Mtf2 functions as a core complex member, controlling epigenetic regulation of self-renewal and maturation of both stem and committed cells.

Repeated, intense, cutaneous stimulation results in the gradual development of inhibition of spinal interneurones. This change in neuronal activity could not be demonstrated in rats whose spinal cords had been transected, and was considered to be the consequence of supraspinal mechanisms. . Experiments sere carried out to determine which areas of the brain were involved. Unitary recordings from neurones situated in nucleus reticularis pontis-caudalis, nucleus reticularis giganto-cellularis, nucleus reticularis parvocellularis and nucleus medulla oblongata pars ventralis demonstrated a progressively increasing excitatory response to repeated intense cutaneous stimulation. These areas were shown to have direct projections to the spinal cord, by retrograde transport of horseradish peroxidase. Cells in nucleus reticularis gigantocellularis, which demonstrated a progressively increasing excitatory response, could also be antidromically activated from the spinal cord. Repeated stimulation of some of these areas produced a progressive inhibition of spinal interneurones which was similar to that resulting from cutaneous stimulation. It would appear that nucleus reticularis gigantocellularis and nucleus reticularis pontis-caudalis are involved in the development of a progressive inhibition of spinal interneurones. A similar role for other reticular and raphe nuclei can not be excluded on the basis of evidence presently available. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Despite all the national efforts to increase STEM enrollment in the United States, the gap between the U.S. and other developed countries in terms of STEM graduates has widened over the last 20 years. Researchers have studied factors such as gender, race, high school GPA, and the student’s socioeconomic status for their impact on STEM enrollment. This study offers another possible explanation of why students might choose, or not choose, to enroll in STEM majors by examining the relationship between personality and STEM enrollment. the sample included 2,745 respondents to the 2008 Cooperative Institutional Research Program freshman survey at a large research university in the southwestern United States. Factor analysis was used to create four personality scales, based on John Holland’s theory of personality types, with items selected from the survey. Logistic regression was utilized to answer three research questions: Are students classified as a strong investigative personality type more likely to enroll in STEM majors than students classified as a weak investigative personality type? Are there differences in their likelihood to enroll in STEM majors among students of investigative-social, investigative-artistic, and investigative-enterprising personality types? What effect does personality have on students’ self-selection into a biological versus a physical STEM major? Results suggested that students with a combined investigative and social personality were more likely to enroll in STEM majors whereas students with a combined investigative and artistic personality were less likely to do so. Additionally, STEM students with an enterprising personality were more likely to choose a biological STEM major than a physical STEM major. These results should benefit educators and policy makers who seek to strengthen the pipeline into STEM fields.

Plantations of Eucalyptus spp. are expanding world-wide to serve growing global requirements for timber and pulp products. Together with this expansion, there has been a concomitant increase in diseases affecting these trees. Most of these are caused by fungi but there are a growing number of diseases caused by bacterial pathogens. Very little is known about them and the focus of this study was to consider species in the genus Pantoea and their association with diseases on Eucalyptus. Pantoea spp. are known pathogens of agricultural crops in South Africa and elsewhere in the world. They are also ubiquitous occurring in diverse ecological niches. Despite their prevalence, little is known about their association with plants, particularly where they occur as pathogens. The first chapter of this thesis presents an overview of the important aspects concerning the identification and classification of bacterial pathogens. Different techniques used for bacterial identification and classification were considered. These techniques are classified into different levels, based on their complexity and level of data resolution. As techniques have developed and been refined, our understanding of how organisms are related to each other has increased. An overview of the taxonomic history of the genus Pantoea was used to illustrate this point. An interaction between two Pantoea spp. and Colletogloeopsis zuluense, a serious fungal pathogen of Eucalyptus has been reported in the past. In the second chapter of this thesis, I considered the view that pathogenicity of C. zuluense is enhanced when infection occurs in conjunction with the two Pantoea spp. The identities of the two Pantoea spp. were confirmed as Pantoea ananatis and Pantoea stewartii subsp. indologenes. Both greenhouse and field inoculation trials with the two Pantoea spp. and C. zuluense failed to confirm that there is an increase in pathogenicity of C. zuluense when these bacteria are present. Studies in chapter three of this thesis, considered the identity of bacteria associated with diseased Eucalyptus leaf material from Uganda, Thailand and Uruguay. Symptoms observed in these countries were very similar and they were also similar to those of bacterial blight observed in South Africa. The majority of isolates obtained from Thailand were identified as Pantoea dispersa, based on both phenotypic and DNA-based data. This is the first report of Pantoea dispersa associated with disease on Eucalyptus. Uganda isolates were identified as Pantoea vagens prov.nom., a new species in the genus Pantoea. The majority of Uruguay isolates were identified as either Pantoea ananatis or Pantoea vagens prov.nom. The remaining isolates from Uruguay were found to belong to Pantoea eucalyptii prov.nom., a proposed new Pantoea sp., as well as Pantoea agglomerans. Pathogenicity results showed that the majority of isolates from all three countries were moderately pathogenic, eliciting moderate reactions in both tobacco and susceptible Eucalyptus grandis x nitens hybrid clones. Overall, results of studies presented in this thesis showed that Pantoea spp. can exist in complex interactions with both plants and fungi. These interactions are, however, not necessary for bacterial survival. We believe that the majority of Pantoea spp. are opportunistic pathogens based on their ability to selectively enter into interactions as well as occur epiphytically on plants. Variability in pathogenicity, both observed in this study and previously reported, further supports this view. Additional studies are needed to determine the conditions conducive to disease development in order to fully understand the threat these pathogens pose to global forestry. / Dissertation (MSc (Microbiology))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Basal cells (BCs) are multipotent tissue-specific stem cells of a variety of organs including the skin, digestive and respiratory tract. BCs are broadly identified by expression of Krt5, Krt14 and the transcription factor p63. In the adult airways, BCs are not only important for normal maintenance but also crucial for epithelial repair after injury. However, the embryonic origin of these adult stem cells remains elusive. Previous reports showed that p63+ cells appear early during airway development, but these do not express markers of adult BCs, raising the question whether these cells represent BC precursors. Moreover, little was known whether embryonic BCs have an impact in the adult pool of progenitors that mediate responses of the lung to injury or pulmonary diseases. The goal of this thesis is to address these gaps of knowledge using a variety of technologies, including functional and lineage tracing analysis in vivo in mouse genetic models, injury modeling, high-throughput profiling and gene regulation approaches. This thesis is to comprehensively characterize airway BCs in development, injury-repair, and homeostasis. These studies revealed a previously unrecognized broader role of embryonic p63+ cells in the establishment of the stem cell pools of the lung pre and postnatally. Surprisingly, lineage analysis showed that early in development these cells were able to generate all epithelial cell types of the airways and alveolar compartment. However, as development proceeds, they underwent two sequential lineage segregation events to finally generate two regionally distinct adult stem cell pools. One of these became the well-known BCs that populate extrapulmonary airways through an undescribed maturation process from the perinatal stage to adulthood, and the other was identified as a rare stem cell pool in the pseudostratified epithelium of intrapulmonary airways which maintained immature and quiescent throughout lifetime. Moreover, the latter responded uniquely to lung injury induced by H1N1 viral infection. Recent studies have demonstrated that BC-like p63+ Krt5+ cell clusters (“Krt5+ pods”) are ectopically present in the areas of severe alveolar injury by H1N1 viral infection. The presence of these pods has been associated with pathological scars in several human pulmonary diseases including idiopathic pulmonary fibrosis (IPF) and acute respiratory distress syndrome (ARDS). However, their cellular origin has been intensely debated. This thesis showed that this rare progenitor pool is established during embryonic development when airways are still branching. Further characterization demonstrated a p63 gene dosage dependency in the specification/maintenance of this rare progenitor pool. By utilizing multiple lineage-tracing lines, an underappreciated diversity of this pool was revealed by showing a novel subpopulation carrying secretory lineage marker spatially restricted to intrapulmonary airways. Further molecular characterization and genetic manipulation of this rare progenitor pool may provide valuable cues to understand the pathogenesis about pulmonary disorders and to develop effective therapies. Moreover, the molecular signatures of tracheal embryonic E18.5 preBCs and adult TrBCs were generated through high-throughput profiling, which provided hints about the genetic regulation of airway BC maturation process and generated potential molecular landmarks for the in vitro ES/iPS cell differentiation towards airway BCs. In addition, single cell RNA-sequencing analyses revealed heterogeneity of adult BCs in the tracheal and esophageal epithelia. Lastly, candidate master regulators of their differentiation programs in homeostatic and metaplastic states were identified through unbiased systems biology algorithms, which will be further validated in functional assays in the near future. Taken together, the studies in this thesis comprehensively characterized airway BCs in development, injury-repair and homeostasis. This thesis work showed the newly identified p63+ airway progenitors before E10.5 are multipotent for all lung epithelial lineages and this multipotency gets restricted to proximal fate at E10.5. In the adult injury-repair, this thesis work for the first time revealed that the H1N1-induced Krt5+ pods are generated by bronchial p63+ Krt5- progenitors, which originate from a subpopulation of E13.5 intrapulmonary p63+ progenitors. At homeostasis, this thesis work uncovered a previously underappreciated heterogeneity of BCs in both airways and esophagus, and provided molecular foundations for further explorations into the mechanistic perspectives of BC cellular identity maintenance.