In dieser Arbeit werden Techniken beschrieben, die es erlauben (höhere) Ableitungen und Taylorapproximationen solcher Computerprogramme effizient zu berechnen. Auch inbesondere dann, wenn die Programme Algorithmen der numerischen linearen Algebra (NLA) enthalten. Im Gegensatz zur traditionellen algorithmischen Differentiation (AD), bei der die zugrunde liegenden Algorithmen um zusätzliche Befehlere erweitert werden, sind in dieser Arbeit die Zerlegungen durch definierende Gleichungen charakterisiert. Basierend auf den definierenden Gleichungen werden Strukturausnutzende Algorithmen hergeleitet. Genauer, neuartige Algorithmen für die Propagation von Taylorpolynomen durch die QR, Cholesky und reell-symmetrischen Eigenwertzerlegung werden präsentiert. Desweiteren werden Algorithmen für den Rückwärtsmodus der AD hergeleitet, welche im Wesentlichen nur die Faktoren der Zerlegungen benötigen. Im Vergleich zum traditionellen Ansatz, bei dem alle Zwischenergebnisse gespeichert werden, ist dies eine Reduktion von O(N^3) zu O(N^2) für Algorithmen mit O(N^3) Komplexität. N ist hier die Größe der Matrix. Zusätzlich kann bestehende, hoch-optimierte Software verwendet werden. Ein Laufzeitvergleich zeigt, dass dies im Vergleich zum traditionellen Ansatz zu einer Beschleunigung in der Größenordnung 100 führen kann. Da die NLA Funktionen als Black Box betrachtet werden, ist desweiteren auch der Berechnungsgraph um Größenordnungen kleiner. Dies bedeutet, dass Software, welche Operator Overloading benutzt, weniger Overhead hervorruft und auch weniger Speicher benötigt. / This thesis provides a framework for the evaluation of first and higher-order derivatives and Taylor series expansions through large computer programs that contain numerical linear algebra (NLA) functions. It is a generalization of traditional algorithmic differentiation (AD) techniques in that NLA functions are regarded as black boxes where the inputs and outputs are related by defining equations. Based on the defining equations, structure-exploiting algorithms are derived. More precisely, novel algorithms for the propagation of Taylor polynomials through the QR, Cholesky,- and real-symmetric eigenvalue decomposition are shown. Recurrences for the reverse mode of AD, which require essentially only the returned factors of the decomposition, are also derived. Compared to the traditional approach where all intermediates of an algorithm are stored, this is a reduction from O(N^3) to O(N^2) for algorithms with O( N^3) complexity. N denotes the matrix size. The derived algorithms make it possible to use existing high-performance implementations. A runtime comparison shows that the treatment of NLA functions as atomic can be more than one order of magnitude faster than an automatic differentiation of the underlying algorithm. Furthermore, the computational graph is orders of magnitudes smaller. This reduces the additional memory requirements, as well as the overhead, of operator overloading techniques to a fraction.

by Chan Shuk Chong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 135-142). / Statement --- p.i / Acknowledgment --- p.ii / Abbreviations --- p.iii / Abstract --- p.iv / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Haematopoiesis --- p.1 / Chapter 1.1.1 --- Sites of haematopoiesis / Chapter 1.1.1.1 --- Bone marrow stroma / Chapter 1.1.1.2 --- Thymus / Chapter 1.1.1.3 --- Spleen and lymph node / Chapter 1.1.1.3.1 --- Spleen / Chapter 1.1.1.3.2 --- Lymph Nodes / Chapter 1.1.2 --- Blood Cell / Chapter 1.1.2.1 --- Development of T and B cells / Chapter 1.1.2.1.1 --- T cells / Chapter 1.1.2.1.2 --- B cells / Chapter 1.1.2.2 --- Development of Granulocytes and monocytes / Chapter 1.2 --- White Cell Disorder -Leukemia --- p.13 / Chapter 1.2.1 --- Leukemia - general concept / Chapter 1.2.1.1 --- Classification of leukemia / Chapter 1.2.1.2 --- Pathophysiology and Clinical features / Chapter 1.2.1.3 --- Etiology of myeloid leukemia / Chapter 1.2.2 --- Genetic basis of leukemia / Chapter 1.3 --- Acute myeloid leukemia (AML) cell model --- p.19 / Chapter 1.3.1 --- Cell Model for human acute myeloid leukemia / Chapter 1.3.2 --- Murine leukemia cell lines / Chapter 1.4 --- Induction of leukemia cell differentiation --- p.21 / Chapter 1.4.1 --- Overview of different inducers / Chapter 1.4.2 --- Cytokines as Inducers / Chapter 1.5 --- Objectives and Research Strategy --- p.26 / Chapter 1.5.1 --- Objectives / Chapter 1.5.2 --- Research strategy / Chapter Chapter 2 : --- Materials and Methods / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Cell line / Chapter 2.1.2 --- Tissue culture medium / Chapter 2.1.3 --- Tumor necrosis Factor - alpha (TNF-α) / Chapter 2.1.4 --- Interleukin 1- alpha (IL-lα)and Interleukin 1- beta (IL-1β) / Chapter 2.1.5 --- "Monoclonal hamster anti-mouse IL-lα monoclonal hamster anti-mouse IL-1β, and Polyclonal rabbit anti-mouse TNF-α antibodies" / Chapter 2.1.6 --- Lipopolysaccharides (LPS) / Chapter 2.1.7 --- Buffers and solutions / Chapter 2.2 --- Methods : --- p.33 / Chapter 2.2.1 --- Cell culture / Chapter 2.2.2 --- Cytotoxicity assay / Chapter 2.2.3 --- Proliferation assay / Chapter 2.2.4 --- Cell morphology / Chapter 2.2.5 --- Phagocytosis assay / Chapter 2.2.6 --- Preparation of undifferentiated and differentiated murine leukemia WEHI3B (JCS) cells for cell lysate / Chapter 2.2.7 --- Isolation of total cellular RNA / Chapter 2.2.8 --- Extraction of the total RNA / Chapter 2.2.9 --- Spectrophotometry / Chapter 2.2.10 --- Electrophoresis of RNA in agarose gel containing formaldehyde / Chapter 2 2.11 --- First strand cDNA synthesis / Chapter 2.2.12 --- Cytokines phenotyping of the uninduced and induced WEHI 3B (JCS) by The Reverse Trancription Polymerase Chain Reaction method / Chapter 2.2.13 --- Gel electrophoresis of PCR- product / Chapter 2.2.14 --- Southern blot / Chapter 2.2.15 --- Dot blot / Chapter 2.2.16 --- Hybridization with oligonucleotides / Chapter 2.2.17 --- Chemiluminescent detection / Chapter Chapter 3 : --- Growth Inhibitory and Differentiation Effects of Lipopolysaccharides ( LPS ) on WEHI 3B (JCS) cells / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.1.1 --- Chemical structure of LPS / Chapter 3.1.2 --- Biological activity of LPS / Chapter 3.2 --- Results --- p.55 / Chapter 3.2.1 --- Anti-proliferative effects of LPS / Chapter 3.2.2 --- Differentiation inducing effect of LPS on WEHI 3B (JCS) cells / Chapter 3.2.3 --- Phagocytic activity LPS treated WEHI 3B (JCS) cells / Chapter 3.2.4 --- Anti-proliferative effect of TNF-α / Chapter 3.2.5 --- Differentiation inducing effect of TNF-α / Chapter 3.2.6 --- Phagocytic activity of TNF-α treated WEHI3B (JCS) cells / Chapter 3.3 --- Discussion --- p.67 / Chapter 3.4 --- Summary --- p.69 / Chapter Chapter 4 : --- The Cytokine Genes Expression of the TNF-α and LPS Treated WEHI 3B (JCS) cells / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.1.1 --- Differentiation of leukemia cell line / Chapter 4.1.2 --- Study of the cytokine genes expression of WEHI 3B (JCS) cells / Chapter 4.2 --- Results --- p.72 / Chapter 4.2.1 --- Isolation of total RNA from uniduced and induced WEHI 3B (JCS) cells / Chapter 4.2.2 --- The cytokine genes expression during differentiation / Chapter 4.2.2.1 --- "Up-regulation of IL-lα, IL-1β,TNF-α and IFN-γ in both TNF-α induced and LPS induced WEHI 3B (JCS) cells" / Chapter 4.2.2.1.1 --- Southern blot / Chapter 4.2.2.1.2 --- Semi-quantitation of PCR-products by gel electrophoresis and dot-blot hybridization / Chapter 4.2.2.2 --- up-regulation of GM-CSF and G-CSF in LPS induced WEHI 3B (JCS) cells / Chapter 4.3 --- Discussion --- p.92 / Chapter 4.4 --- Summary --- p.95 / Chapter Chapter 5 : --- Growth inhibitory and Differentiation Inducing Effect of IL-l( IL-1α and IL-1β) on WEHI 3B (JCS) cells / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.1.1 --- The interleukin 1 (IL-1) family / Chapter 5.1.1.1 --- Structure of IL-1 / Chapter 5.1.1.2 --- The biological function of IL-1 / Chapter 5.1.2 --- Tumor necrosis factor - alpha ( TNF-α) / Chapter 5.1.2.1 --- Structure of TNF-α / Chapter 5.1.2.2 --- Biological functions of TNF-α / Chapter 5.1.3 --- The similarity between TNF and IL-1 / Chapter 5.2 --- Results --- p.102 / Chapter 5.2.1 --- Anti-proliferative effect of IL-1 / Chapter 5.2.2 --- Differentiation inducing effect of IL-1 / Chapter 5.2.3 --- Phagocytic activity of IL-1 treated JCS cells / Chapter 5.2.4 --- "Role of endogenously produced IL-lα, IL-1β and TNF-α in LPS cytokines differentiation of WEHI 3B (JCS) cells" / Chapter 5.2.4.1. --- "Effect of neutralizing anti- ILl-α,anti - IL-l-β, and anti-TNF-α antibodies on the growth inihbition of the treated WEHI 3B (JCS) cells" / Chapter 5.2.4.2 --- "Effects of neutralizing anti-IL-lα, anti- IL-1β, and anti-TNF-α antibodies on differentiation of the treated WEHI 3B (JCS) cells" / Chapter 5.3 --- Discussion --- p.124 / Chapter 5.4 --- Summary --- p.127 / Chapter Chapter 6 --- : Concluding Discussion --- p.128 / References --- p.135

Choi, Wing-Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 55-61). / Abstract and questionnaire also in Chinese.

Dietary phosphate is essential for normal fracture healing and bone growth. Previous studies have established that mice given a phosphate deficient diet after a fracture demonstrate delayed cartilage maturation and callus mineralization, as well as changes in gene expression consistent with oxidative phosphorylation dysfunction. This study was undertaken in order to examine the role of inorganic and organic phosphate availability on chondrocyte differentiation and mineralization, and to define the relationship between these processes and changes in chondrocyte metabolic function. ATDC5 murine chondroprogenitor cell line, which has been shown to undergo in vitro differentiation and extracellular matrix mineralization, was cultured under both differentiating and non-differentiating media conditions under conditions in 1mM -0.25mM sodium phosphate monobasic (inorganic phosphate) in the presence or absence of 4mM β-glycerol phosphate (organic phosphate). In the first series of studies, overall cell growth (total DNA and protein contents), mineralization (calcium accumulation), and cell-normalized oxidative metabolism (basal respiration, maximal respiration, ATP turnover, spare capacity, proton leak, and non-mitochondrial respiration rates) were measured over a 28 day time course in cultures grown in differentiating (ascorbic acid, insulin-transferrin-selenium, and β-glycerol phosphate) conditions in 1mM phosphate. These studies found that when the cells were induced to differentiate, there was a measurable increase in protein content while DNA content decreased by 30%, indicating a fraction of the cells underwent cell death. Differentiation was further associated with an overall two-fold increase in oxidative respiration. Next we assessed how differentiation, the promotion of matrix mineralization, and inorganic phosphate availability affected oxidative respiration. When differentiation was not induced with ascorbic acid and β-glycerol phosphate, there was no over growth in the cultures nor any change in total extracellular matrix mineralization or oxidative respiration. In the absence only of β-glycerol phosphate, differentiation proceeded but matrix mineralization did not occur. However, overall protein content and oxidative respiration were statistically two- and 1.5-fold higher, respectively, independent of the inorganic phosphate contents of the growth media. These results suggest that both differentiation and overall protein accumulation are strongly associated with increased oxidative metabolism while mineralization of the matrix decreased oxidative function. Only at the lowest phosphate levels were changes in basal oxidative function observed. These results are consistent with previous in vivo findings suggesting that diminished expression of mitochondrial associated genes in callus tissues from hypophosphatemic mice were associated with an overall decrease in chondrocyte differentiation.

INTRODUCTION: Stem cells have become promising treatments for osteoarthritis due to the cells’ ability to regenerate cartilage and availability from bone marrow. Various studies have established the chondrogenic potential of human marrow stromal cells (hMSCs) upon treatment with transforming growth factor β1 (TGF-β1), yet the difference in potential between cells derived from young subjects and those derived from elder subjects has not been confirmed. OBJECTIVES: This study seeks to establish whether the chondrogenic potential of hMSCs changes with age and sex. This study used a high-density 2D model to measure the acute response of hMSCs to chondrogenic induction over a short time course and various treatment levels. The experiments investigated the expression of chondrogenic genes and expression of TGF-β1 receptors (ALK5) in hMSCs after TGF-β1 treatment to determine whether pediatric hMSCs have more potential for chondrocyte differentiation than adult hMSCs. METHODS: With IRB approval, nine bone marrow samples were obtained from discarded tissue of adults undergoing total hip replacement and juveniles requiring bone graft for alveolar cleft repair. Subject ages ranged from age 8 to 66. Low-density mononucleated cells were cultured in plastic tissue culture dishes. Adherent hMSCs were expanded in monolayer culture with phenol red-free α-MEM medium with 10% fetal bovine serum. After 48 hours of treatment with TGF-β1, cells were collected for RNA extraction and RT-PCR analysis of chondrogenic genes and TGF-β1 receptor levels. Alcian blue staining in 24-well plates of hMSCs was performed after 10 days to compare the effects of different concentrations of TGF-β1, and the effects of another inducer of chondrogenesis, kartogenin (KGN) on matrix accumulation. RESULTS: Gel electrophoresis of PCR products revealed no consistent trend in chondrogenic mRNA expression in pediatric cells compared to adult cells, or female cells compared to male cells. The data indicate that the change in chondrogenic potential of hMSCs with age and sex is inconsistent. KGN showed no consistent effect on hMSCs. Cells with high baseline levels of TGF-β1 receptor (ALK5) showed no upregulation of ALK5 after TGF-β1 treatment, while samples with low basal expression of TGF-β1 receptors showed upregulation after TGF-β1 treatment. CONCLUSIONS: There is still much debate in the literature regarding the potential of adult hMSC chondrogenesis compared to juveniles. This study confirms the irreproducibility of displaying differences between young and adult hMSCs. A larger sample size is needed to establish a correlation between age and chondrogenic potential. Further in vitro studies will consider the optimum time course and concentration of TGF-β1 to observe differences in gene expression of cells, and will identify other clinical determinants of differentiation potential.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumour in adults, as well as the most common. The current standard therapy is maximal safe resection, followed by radiotherapy in combination with the alkylating agent, temozolomide. Despite this multi-model treatment approach, median survival is just 14.6 months and new therapies are urgently needed. Glioblastoma stem cells (GSCs) are a highly tumourigenic subpopulation of GBM cells believed to promote therapeutic resistance along with angiogenesis and metastasis. Cancer stem cells share crucial characteristics with normal stem cells such as their ability to self- renew, maintain proliferation and differentiate multi-potently. One strategy to target GSCs is to force them to differentiate into post-mitotic cells, as this would cause them to lose their long-term repopulation potential and would therefore limit tumour growth. In this study, I investigated if the transcription factor ASCL1 could drive neuronal differentiation in GSCs. ASCL1 is a key regulator of neurogenesis in the developing CNS and is sufficient to reprogramme fibroblasts, astrocytes and induced pluripotent cells into neurons. However, ASCL1 also promotes proliferation, and in GSCs, it has been shown to drive tumourigenesis by upregulating Wnt signaling. Given its role in two opposing functions, ASCL1 is tightly regulated by multi-site phosphorylation on serine-proline resides. It is phosphorylated when driving proliferation in cycling cells and is un(der)phosphorylated when activating the transcription of its downstream targets involved in differentiation. I found that endogenous ASCL1 was phosphorylated in GSC lines and hypothesized that dephosphorylating ASCL1 may drive differentiation. I tested this by overexpressing a phosphomutant form of ASCL1, and found that it drove cell cycle exit through the downregulation of Cyclin D2, CDK4 and CDK6. However, ASCL1 did not drive overt differentiation which suggests GSCs may not respond to differentiation cues.

by Tang Shuk Chun. / "September 1998." / Thesis (Ph.D.)--Chinese University of Hong kong, 1998. / Includes bibliographical references (p. 157-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Pathways and mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a novel, multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naïve, central and effector memory, and terminal effector subsets is contingent on both differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is dependent on CD4+ or CD8+ T cell lineage, subset differentiation and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting new pathways for human T cell fate determination and homeostasis. Memory T cells can remain in tissues as non-circulating, resident memory populations, which provide optimal protection against infection at barrier surfaces. Lung-resident memory T cells (TRM) mediate in situ protection to respiratory pathogens, though mechanisms for their maintenance and retention are unknown. Through whole transcriptome profiling, we identify a cohesive network of genes enriched in lung CD4+ TRM, including Itgad (CD11d), Cd69, and IFN-associated responders. We find that upregulation of CD11d enhances CD69 expression through type I IFN signaling downstream of homotypic cell adhesion, and is required for optimal T cell differentiation and lung retention. Moreover, blockade of IFNαR1 reduces CD11d expression and retention of influenza-generated lung TRM, suggesting that CD11d-dependent type I IFN signaling promotes TRM establishment. Our results implicate CD11d and type I IFN in retaining lung CD4+ TRM cells, and identify potential targets for modulating tissue immunity.

Embryonic stem (ES) cell maintenance and differentiation are dynamic processes controlled by various intrinsic and extrinsic factors. Identifying these factors will enhance the understanding about developmental process and improve the application of stem cells in clinic. Previous studies highlight a shift between non-oxidative and oxidative energy metabolism to play roles during differentiation. Oxidative metabolism is a major source of reactive oxygen species (ROS) which is regulated by a cytoprotective transcription factor, Nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, this study investigate relationship between metabolism, ROS, and Nrf2 during mouse ES cell differentiation. In vitro models representing early lineage differentiation were used. By measuring metabolic profiles, ROS, and Nrf2 levels from the models, Nrf2 was found related to pluripotency and ROS. However, relationship among metabolism and Nrf2 or ROS could not be detected. Gain- and loss-of-function experiments by pharmacological activator, short hairpin RNA knockdown, and CRISPR-Cas9 genome editing showed that Nrf2 could promote pluripotency and inhibit differentiation, especially during early differentiation toward neural lineage. This study suggested a new player in transcription control that governs pluripotency and differentiation.

In mammalian development, endoderm formation occurs in two phases and the fate of these populations is different. In the blastocyst, inner cell mass (ICM) cells generate the primitive endoderm (PrE), which will give rise to the extra-embryonic parietal (PE) and visceral endoderm (VE). Hematopoietically expressed homeobox (Hhex) protein is initially expressed throughout the PrE and subsequently becomes restricted to the anterior visceral endoderm (AVE), one of two important early embryonic signalling centres in the mouse. During gastrulation a second wave of endoderm differentiation occurs, the definitive endoderm (DE), generating the foregut. Immediately following the induction of DE, regional identity is initially established in the anterior region with the expression of Hhex. One of the earliest specification events in this lineage is the specification of anterior fate by Hhex, this time in a second signalling centre, the anterior definitive endoderm (ADE). The ADE is both important for embryonic patterning, and as the precursor population for differentiating to the foregut and its derivatives the thyroid, liver and pancreas. The literature surrounding these early embryonic patterning events is covered in depth in chapter 1. Embryonic stem cells (ESCs) are normal cell lines derived from the mammalian blastocyst at the time that it is making PrE. A number of laboratories have generated protocols to make endoderm from ESCs and in my thesis I define approaches to distinguish between PrE and DE. I generated a new ESC reporter line utilising a gene normally expressed in both the PrE and later in hepatic endoderm; this reporter contains a GFP in the first exon of the Hnf4α locus. This was combined with a second fluorescent reporter containing DSRed in the Hhex locus. This cell line is described and characterised in chapter 3. As Hnf4α is initially expressed in PrE prior to Hhex, but in the DE following Hhex, I was able to use the temporal expression of this reporter to distinguish the induction of PrE from DE. As Activin and Wnt are known to induce endoderm from ESCs, I was then able to ask what sort of endoderm the combination of these two signals induced. In chapter 4 I found that normal ESCs would readily differentiate to iPrE in the presence of Activin and Wnt3a. While this has not been described previously, my analysis suggests that ESC protocols applying these cytokines directly to ESCs have produced PrE. Given that ESCs are derived from the blastocyst, the generation of iPrE from Wnt3a/Activin treatment fits with developmental paradigms. However, Act/Wnt3a is used routinely on Human ESCs (hESCs) and so I attempted to reconcile these observations. HESCs, while derived from the blastocyst, appear to progress developmentally in vitro, to a stage closer to the epiblast, immediately prior to gastrulation. I therefore assessed the effect of Activin and Wnt3a on mouse stem cell lines derived from the epiblast (Epiblast Stem Cells, EpiSCs), that are grown under similar conditions to hESCs. When Wnt3a/Act is applied to these cells I found that they made DE rather than PrE, which I describe in chapter 4. Taken together my observations suggest that Act/Wnt3a are general endoderm inducers that induce context specific differentiation in vitro. The cell type derived in response to this treatment depends on the developmental stage of the starting stem cell culture. During the course of this work, I also observed that PrE was growing under Activin/Wnt3a treatment. As a number of cell culture systems have been established that reflect PE, but not truly bipotent PrE, I investigated the conditions under which PrE can be expanded. In chapter 5 I characterize a new PrE culture system, in which bipotent extra-embryonic endoderm can be expanded indefinitely in culture. I also explore a bit more precisely the nature of the starting cells that initially become exposed to Activin/Wnt3a treatment. Previous work has extensively characterized the existence of a primed population of PrE in ESC culture and in chapter 6 I explore the existence of a primed DE population in EpiSC culture. Taken together, my thesis is the first demonstration that Activin/Wnt3a can induce different endoderm populations in different embryonic stem cell populations. It underlies the notion that the evolutionary origin of both cell types is the same and that the pathways evolved for extra-embryonic development in mammals just exploit the ancient modes of germ layer specification that evolved with gastrulation.