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Mathematical modeling of biological systems can be useful to reveal new insights into biological observations. Here we apply mathematical modeling to study the underlying molecular networks driving observed behaviors of two systems. First, we apply systems biology and dynamic systems theory techniques to reveal new insights into the process of hematopoiesis. More specifically, we search the literature to deduce the underlying molecular mechanism that drives cell fate determination in granulocyte-monocyte progenitor (GMP) cells that are exposed to various cytokines. By converting this molecular mechanism into a set of ordinary differential equations (ODEs), we acquired new insights into the behavior of differentiating GMP cells. Next, we explore the cell cycle of the model prokaryotic organism, Caulobacter crescentus. Caulobacter is a uniquely successful oligotrophic bacterium, found abundantly in freshwater systems. While it is not a pathogenic species, Caulobacter is extremely well studied due to its distinguishable asymmetrical morphology and the ability to synchronize populations by cell cycle stage. We built a detailed mathematical model of the molecular mechanism driving the cell cycle. This research suggests a previously unknown role for the unknown form of the master regulator, CtrA, in regulating the G1-S transition. Furthermore, we incorporate a nutrient signaling model into the cell cycle model to investigate how Caulobacter responds to nutrient deprivation. We find that regulation of DivK phosphorylation is an essential component of the nutrient signaling pathway and demonstrate how starvation signals work together in synergy to manifest in observed cell cycle response. / Doctor of Philosophy / Every cell in the human body has the same DNA, yet there are cells of all kinds with different jobs, appearances and behaviors. This simple concept is a consequence of complex regulatory systems within cells that dictate what genes are expressed and when. This dissertation breaks down the molecular mechanisms that regulate gene expression in cells and how these mechanisms result in the interesting behaviors and morphologies that have been observed experimentally. By deriving mathematical equations to describe the molecular mechanisms, we simulate how cell behavior might change under different conditions to make novel discoveries. More specifically, we utilize these techniques to study the freshwater bacterium, Caulobacter crescentus, and human cells of the white blood cell lineage. We utilize our models to identify previously unknown aspects of the molecular mechanisms, develop explanations for mysterious cell behaviors and provide interesting predictions that have not been explored experimentally.

The focus of this research is to describe the production and characterization of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in insect cells, using Autographa californica buclear polyhedrosis virus (AcNPV) as an expression vector. All three forms of biological activity of hGM-CSF. Following N-glycanase treatment, the two glycosylated hGM-CSF proteins (15.5 and 16.5 KDa) which bound to Concanavalin A affinity column ran as a 14.5-15.5 KDa band on SDS-PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 KDa species. The N-terminal amino acid sequence of the recombinant hGM-CSF was identical to that of natural hGM-CSF deduced from cDNA. These results demonstrate that baculovirus-produced hGM-CSF could be N-glycosylated in Sf9 cells, the signal peptide of recombinant hGM-CSF could be recognized and cleaved by infected insect cells and the resultant molecule secreted into the medium.

Indiana University-Purdue University Indianapolis (IUPUI) / The innate and adaptive immune responses are critical for host survival. The TNFα/NF-κB signaling pathway is a major regulator of the immune response. The TNFα/NF-κB signaling pathway has also been proposed to play a role in the regulation of hematopoiesis. In the TNFα signaling pathway, full induction of NF-κB (specifically the p65 subunit) dependent transcription is regulated by a co-activator SIMPL. The biological significance of SIMPL in TNFα dependent responses is poorly understood. To study SIMPL in vitro and in vivo in mammalian cells, a knockdown system utilizing shRNA (short hairpin RNA) was used. Analysis of hematopoietic progenitor cells infected with a retrovirus encoding the SIMPL shRNA was used to study the role of SIMPL in hematopoiesis. The ability of progenitor cells lacking SIMPL to grow and differentiate was not compromised. In contrast in the progenitors cells lacking SIMPL, TNFα mediated inhibition of colony formation was significantly enhanced. These growth inhibitory effects of SIMPL were not due to an increase in apoptosis. The enhanced inhibitory affects were specific for TNFα and not found in other common hematopoietic inhibitors (TGF-β1 and IFNγ). Results of this work reveal that SIMPL is a component of the hematopoiesis that is required for TNFα dependent effects upon myeloid progenitors.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurofibromatosis type 1 is a genetic disease that results from either heritable or spontaneous autosomal dominant mutations in the NF1 gene, which encodes a protein serving, at least in part, to accelerate the intrinsic hydrolysis of active Ras-GTP to inactive Ras-GDP. A second-hit NF1 mutation precedes predominant NF1 neoplasms, including juvenile myelomoncytic leukemia (JMML) and plexiform neurofibroma formation, potentially fatal conditions with no medical therapy. While NF1 loss of heterozygosity (LOH) in myeloid progenitor cells sufficiently engenders leukemogenesis, plexiform neurofibroma formation depends on LOH in Schwann cells and Nf1 heterozygosity in the hematopoietic system. Specifically, recruited Nf1+/- mast cells accelerate tumorigenesis through secreted cytokines and growth factors. Nf1+/- mast cells depend upon deregulated signaling in c-kit pathways, a receptor system conserved in hematopoietic stem cells (HSCs). Accordingly, Nf1-/- myeloid progenitor cells, which can induce a JMML-like disease in mice, also demonstrate deregulated c-kit receptor signaling. C-kit-activated Nf1+/- mast cells and Nf1-/- myeloid progenitors both show increased latency and potency of active Erk1 and Erk2, the principal cytosolic-to-nuclear effectors of canonical Ras-Raf-Mek signaling. Thus, Erk represents a potential regulator of leukemogenesis and tumor-associated inflammation. However, single and combined Erk1 and Erk2 roles in HSC function, myelopoiesis, and mature mast cell physiology remain unknown, and recent hematopoietic studies relying on chemical Mek-Erk inhibitors have produced conflicting results. Here, we show that hematopoietic stability, myelopoiesis, and mast cell generation require Erk1 or Erk2, but individual isoforms are largely dispensable. Principally, Erk-disrupted hematopoietic stem cells incorporate BrdU but are incapable of dividing, a novel and cell type-specific Erk function. Similarly, mast cell proliferation requires Erk but cytokine production proceeds through other pathways, elucidating molecule-specific functions within the c-kit cascade. Based on these findings, we have reduced tumor mast cell infiltration by treating genetically-engineered tumor model mice with PD0325901, a preclinical Mek-Erk inhibitor. Moreover, we have devised a quadruple transgenic HSC transplantation model to examine dual Erk disruption in the context of Nf1 nullizygosity, testing whether diseased hematopoiesis requires Erk. These insights illuminate cell-specific Erk functions in normal and Nf1-deficient hematopoiesis, informing the feasibility of targeting Mek-Erk in NF1-associated disease.

Hematopoiesis in the adult organism is maintained by a complex, hierarchically organized cascade of differentiating cells that ultimately originate from adult hematopoietic stem cells (HSCs). HSCs have been extensively studied in perturbative settings (e.g transplantation assays), which are well suited for exploring cell potential but tell very little about cell behaviour in a physiological state. Novel \textit{in situ} techniques have made it possible to observe hematopoiesis in its natural environment, yet many questions about native HSC behaviour remain controversial. It is yet unclear whether and to what extent HSCs contribute to steady-state blood production, and whether they represent a reserve population that can be activated upon emergency. As, to date, common definitions of HSCs comprise cells of heterogeneous function, incoherent results are likely linked by another fundamental question: what is the identity of the HSC population residing on the apex of the hematopoietic hierarchy? To answer these questions is essential to gain a deeper understanding of fatal human diseases like leukemia or aplastic anemia. We describe an integrative approach combining non-invasive experimental methods with mathematical inference to uncover underlying pathways and differentiation patterns of steady state hematopoiesis. By this, we identify a population of Sca-1\textsuperscript{hi} CD201(EPCR)\textsuperscript{hi} HSCs as the hithertho elusive apex population, and show that they contribute marginally, but continuously, to native steady-state hematopoiesis. Further, we clarify the architecture of a shortcut route of thrombopoiesis, which emanates directly from HSCs and links them trough a previously undefined CD48\textsuperscript{-/low} megakaryocyte progenitor population directly to platelets. Finally, we provide an extensive analysis of the dynamic label propagation and proliferation behaviour of hematopoietic stem and progenitor cells in prototypical stress situations mimicking blood loss, infection and inflammation. We find that apex stem cells do not directly contribute to emergency hematopoiesis and are only activated upon severe myeloablation and chronic inflammation. Further, innate immune training did not influence HSC contribution in response to reoccurring stimulation. In sum, we demonstrate that primitive HSCs do neither represent a major contributor to steady-state blood production, nor a reservoir for emergency supply. We thus question current dogma of hematopoiesis, and argue that the primary function of HSCs in the adult organism remains yet to be discovered.:Contents Abstract iv Zusammenfassung vii List of Figures xii List of Tables xiii List of Abbreviations xv 1 Introduction 1 1.1 Hematopoiesis and the hematopoietic system 1 1.1.1 Developmental origins 1 1.1.2 The hematopoietic hierarchy 3 1.2 Hematopoietic stem cells 5 1.2.1 Characteristics of hematopoietic stem cells 7 1.2.2 Identification of hematopoietic stem and progenitor cells 7 1.2.3 HSC heterogeneity 11 1.3 Novel genetic tools to analyse native hematopoiesis 12 1.3.1 Conditional gene targeting 12 1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15 1.4.1 Models of native Hematopoiesis 16 1.4.2 Models of HSC quiescence 23 1.4.3 Aged hematopoiesis 25 1.5 Hematopoietic stem cell behaviour in stress response 26 1.5.1 Inflammation and Infection 27 1.5.2 Myeloablation 30 1.5.3 Innate immune training 32 2 Aim of Thesis 35 3 Material and Methods 37 3.1 Materials 37 3.1.1 Antibodies 37 3.1.2 Buffer and Solutions 38 3.1.3 Chemicals and Reagents 39 3.1.4 Cultivation Media 40 3.1.5 Kit Systems 41 3.1.6 Software 41 3.2 Mice 42 3.2.1 Animal housing and husbandry 42 3.2.2 Mouse strains 42 3.3 Genotyping of mice 43 3.4 Treatments of mice 46 3.4.1 Reporter induction 46 3.4.2 Hematopoietic stress induction 46 3.5 Hemograms 48 3.6 Transplantation and in vivo repopulation assays 49 3.6.1 Preconditioning and transplantation of recipients 49 3.6.2 Competitive repopulation assay 50 3.6.3 Limiting dilution assay 50 3.6.4 Transplantation into sublethally irradiated hosts 51 3.7 Cell preparation for transplantation and flow cytometry 51 3.7.1 Bone marrow 51 3.7.2 Splenocytes 51 3.7.3 Peripheral blood 51 3.7.4 Peritoneal cells 52 3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52 3.8 Flow Cytometry 53 3.8.1 Cell staining 54 3.8.2 Cell counting 55 3.8.3 Cell identification and gating 55 3.8.4 Chimerism analysis 57 3.8.5 Cell sorting 58 3.9 Cytokine detection assay 58 3.10 in vitro single cell expansion assay 59 3.11 Sequencing 61 3.11.1 Single cell RNA sequencing 61 3.11.2 Bulk RNA sequencing 62 3.11.3 Single cell and bulk transcriptome analysis 62 3.12 Mathematical modelling 62 3.13 Data Normalization 63 3.14 Statistical Analysis 64 4 Results 67 4.1 Identification of an apex HSC population 67 4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68 4.1.2 ES HSCs reside at the top of the hierarchy 71 4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76 4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77 4.2.2 The myeloid lineage diverges within phenotypic HSCs 80 4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82 4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82 4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88 4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92 4.4 Ageing modulates the dynamics of fate mapping 95 4.5 HSCs in times of crisis: Contribution to stress recovery 99 4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100 4.5.2 Severe myeloablation provokes HSC activation 105 4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107 4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113 4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118 4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120 5 Discussion 123 5.1 Labels matter: What is a true HSC? 123 5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124 5.1.2 ES HSCs reside at the top of the hierarchy 126 5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127 5.1.4 The myeloid lineage diverges within phenotypic HSCs 130 5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132 5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132 5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135 5.3 Fundamental dynamics of label propagation are preserved in aged mice 136 5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138 5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139 5.4.2 Severe myeloablation provokes HSC activation 143 5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145 5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149 5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150 6 Conclusion 153 References 155 Acknowledgements 185 Appendices 187 Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189 Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190 / Die Blutbildung in erwachsenen Organismen wird durch eine komplexe, hierarchisch organisierte Kaskade von sich fortwährend differenzierenden Zellen aufrechterhalten, und hat ihren Ursprung in adulten h\'amatopoietischen Stammzellen (HSZs). HSZs wurden ausgiebig unter unphysiologischen Extrembedingungen (z.B. Transplantation) untersucht, in welchen sich viel über das maximale potential einer Zelle, aber wenig über ihr Verhalten in ungestörtem Zustand sagen lässt. Obwohl durch moderne \textit-Techniken mittlerweile h\'amatopoietische Differenzierung in ihrer ursprünglichen Umgebung beobachtet werden kann, bleiben etliche Fragen zum nativen Verhalten von HSZs offen. So ist es noch unklar, ob und in welchem Ausmaß HSZs überhaupt zur stationären Blutbildung beitragen, oder ob sie eine Reservepopulation darstellen, die im Notfall aktiviert werden kann. Da bis dato gebr\'auchliche Definitionen von HSZs verschiedene Sub-Populationen mit heterogener Funktionalität umfassen, sind die inkohärenten Ergebnisse wahrscheinlich mit einer anderen grundlegenden Frage verknüpft: Was ist die wahre Identit\'at der HSZ-Population, die an der Spitze der h\'amatopoetischen Hierarchie steht? Die Beantwortung dieser Fragen ist essenziell für ein besseres Verst\'andnis von fatalen humanen Krankheiten, wie beispielsweise Leuk\'amie oder aplastische An\'amie. Wir beschreiben einen integrativen Ansatz, der nicht-invasive experimentelle Methoden mit mathematischer Modellierung vereint, um die zugrundeliegenden Pfade und Differenzierungsmuster der station\'arer H\'amatopoese zu ergründen. Auf diese Weise können wir eine Population von Sca-1\textsuperscript{hi} und CD201(EPCR)\textsuperscript{hi} HSZs als die bisher unbekannte Apex-Population identifizieren, und zeigen, dass diese Zellen geringf\'ugig, aber kontinuierlich, zur nativen H\'amatopoese beitragen. Darüber hinaus klären wir die Architektur eines direkten thrombozytischen Differenzierungspfades, welcher unmittelbar von HSZs abzweigt und diese über eine neu definierte CD48\textsuperscript{-/lo} Megakaryozyten-Vorläuferpopulation direkt mit Thrombozyten ver- bindet. Schließlich liefern wir eine umfassende Analyse des dynamischen Differenzierungs- und Proliferationsverhaltens von hämatopoetischen Stamm- und Vorläuferzellen in prototypischen Stresssituationen, die Blutverlust, Infektion und Entzündung nachahmen. Wir zeigen, dass primitive Stammzellen nicht direkt zur Notfall-Hämatopoese beitragen und nur bei schwerer Myeloablation und chronischer Entzündung aktiviert werden. Ebenfalls hat das Training des angeborenen Immunsystems keinen Einfluss auf die Aktivität von HSZs bei wiederholter Stimulation. Unsere Ergebnisse belegen, dass primitive HSZs weder einen wesentlichen Beitrag zur stationären Blutbildung leisten, noch ein Reservoir für die Notfallversorgung darstellen. Wir stellen somit das derzeitige Dogma der Hämatopoese in Frage und argumentieren, dass die primäre Funktion der HSZ im erwachsenen Organismus noch zu entdecken ist.:Contents Abstract iv Zusammenfassung vii List of Figures xii List of Tables xiii List of Abbreviations xv 1 Introduction 1 1.1 Hematopoiesis and the hematopoietic system 1 1.1.1 Developmental origins 1 1.1.2 The hematopoietic hierarchy 3 1.2 Hematopoietic stem cells 5 1.2.1 Characteristics of hematopoietic stem cells 7 1.2.2 Identification of hematopoietic stem and progenitor cells 7 1.2.3 HSC heterogeneity 11 1.3 Novel genetic tools to analyse native hematopoiesis 12 1.3.1 Conditional gene targeting 12 1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15 1.4.1 Models of native Hematopoiesis 16 1.4.2 Models of HSC quiescence 23 1.4.3 Aged hematopoiesis 25 1.5 Hematopoietic stem cell behaviour in stress response 26 1.5.1 Inflammation and Infection 27 1.5.2 Myeloablation 30 1.5.3 Innate immune training 32 2 Aim of Thesis 35 3 Material and Methods 37 3.1 Materials 37 3.1.1 Antibodies 37 3.1.2 Buffer and Solutions 38 3.1.3 Chemicals and Reagents 39 3.1.4 Cultivation Media 40 3.1.5 Kit Systems 41 3.1.6 Software 41 3.2 Mice 42 3.2.1 Animal housing and husbandry 42 3.2.2 Mouse strains 42 3.3 Genotyping of mice 43 3.4 Treatments of mice 46 3.4.1 Reporter induction 46 3.4.2 Hematopoietic stress induction 46 3.5 Hemograms 48 3.6 Transplantation and in vivo repopulation assays 49 3.6.1 Preconditioning and transplantation of recipients 49 3.6.2 Competitive repopulation assay 50 3.6.3 Limiting dilution assay 50 3.6.4 Transplantation into sublethally irradiated hosts 51 3.7 Cell preparation for transplantation and flow cytometry 51 3.7.1 Bone marrow 51 3.7.2 Splenocytes 51 3.7.3 Peripheral blood 51 3.7.4 Peritoneal cells 52 3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52 3.8 Flow Cytometry 53 3.8.1 Cell staining 54 3.8.2 Cell counting 55 3.8.3 Cell identification and gating 55 3.8.4 Chimerism analysis 57 3.8.5 Cell sorting 58 3.9 Cytokine detection assay 58 3.10 in vitro single cell expansion assay 59 3.11 Sequencing 61 3.11.1 Single cell RNA sequencing 61 3.11.2 Bulk RNA sequencing 62 3.11.3 Single cell and bulk transcriptome analysis 62 3.12 Mathematical modelling 62 3.13 Data Normalization 63 3.14 Statistical Analysis 64 4 Results 67 4.1 Identification of an apex HSC population 67 4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68 4.1.2 ES HSCs reside at the top of the hierarchy 71 4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76 4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77 4.2.2 The myeloid lineage diverges within phenotypic HSCs 80 4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82 4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82 4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88 4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92 4.4 Ageing modulates the dynamics of fate mapping 95 4.5 HSCs in times of crisis: Contribution to stress recovery 99 4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100 4.5.2 Severe myeloablation provokes HSC activation 105 4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107 4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113 4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118 4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120 5 Discussion 123 5.1 Labels matter: What is a true HSC? 123 5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124 5.1.2 ES HSCs reside at the top of the hierarchy 126 5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127 5.1.4 The myeloid lineage diverges within phenotypic HSCs 130 5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132 5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132 5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135 5.3 Fundamental dynamics of label propagation are preserved in aged mice 136 5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138 5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139 5.4.2 Severe myeloablation provokes HSC activation 143 5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145 5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149 5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150 6 Conclusion 153 References 155 Acknowledgements 185 Appendices 187 Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189 Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190

Blood cells or hemocytes play important roles in immunity. They are a major source of many immune-related molecules such as antibodies in adaptive immunity of vertebrates and prophenoloxidase (proPO) in invertebrates. In the crayfish Pacifastacus leniusculus, the proPO-system has been reported to be an important component of immune responses against microorganisms. In this study, several mutant strains of Aeromonas hydrophila were used to reveal that LPS (lipopolysaccharide) is an important factor for the pathogenicity of A. hydrophila, strongly inducing the proPO system and melanization. This proPO activating system is a multistep process, which has to be tightly controlled to avoid the harmful side effects of toxic intermediates. Many regulating factors have been reported to fine-tune the proPO-system. In this study, the cleavage of caspase-1-like activity was shown to be a novel negative regulator of PO activity in crayfish. Moreover, the fragments obtained by cleavage of proPO by the proPO-activating enzyme and caspase-1-like protein increased bacterial clearance. Thus, the peptides generated also have important biological functions. In addition to being a source of immune proteins, hemocytes also participate in phagocytosis, encapsulation, and nodulation. An infection normally causes a reduction of hemocyte numbers. Consequently, hemocyte homeostasis is important for maintaining appropriate hemocyte numbers in the circulation of the animal. This study shows that the reactive oxygen species level in the anterior proliferation center of crayfish hematopoietic tissue (HPT), together with cell proliferation, was increased during infection. Pl-β-thymosins were proposed to be involved in hemocyte homeostasis by increasing stem cell migration and thus increasing the circulating hemocyte number. Crayfish hemocyte numbers, as well astakine (Ast1 and Ast2) expression in hemocytes and HPT, were previously shown to be under circadian regulation. Here, we show that Ast1, Ast2, and proPO exhibit rhythmic expression in the crayfish brain similarly to their orthologs, prokineticin 1, prokineticin 2 and tyrosinase, respectively, in the zebrafish brain. Tyrosinase expression was detected in zebrafish brain cells while PO-positive cells were identified as hemocytes that had infiltrated into the crayfish brain. Therefore, this information suggests a close relationship between crayfish hemocytes and the crayfish brain as well as vertebrate neurons.

Previous studies in our lab identified a novel gene, latexin (Lxn), that regulates murine hematopoietic stem cells through balancing apoptosis, self-renewal and proliferation. In these dissertation studies, I performed a series of experiments to examine the function of Lxn using a Lxn conventional knockout mouse, and characterize Lxn’s role in the presence of hematopoietic stresses such as ionizing radiation, cytokines induced-mobilization, and hematopoietic malignancy. The first series of experiments was designed to determine the role of Lxn in hematopoiesis under homeostatic conditions. I found that Lxn-/- mice exhibited hyperproliferative hematopoiesis, a repopulation advantage and elevated self-renewal capacity which was intrinsic to the Lxn-/- hematopoietic cells. Furthermore, I identified a reduction in apoptotic frequency in Lxn-/- hematopoietic progenitors, which may account for the expansion seen in the progenitor population. In a second series of experiments, I discovered a role of Lxn in the radio-sensitivity of hematopoietic cells. I found that loss of Lxn in mice confers resistance to ionizing radiation. Lxn-/- mice showed rapid hematological recoveries after radiation exposure at the stem and progenitor cell (HSPC) level. The ablation of Lxn hindered irradiation-induced apoptosis which may underlie the radiation resistance through regulating hematopoietic recovery. In a third series of experiments, I studied the interaction of Lxn-/- stem and progenitor cells with their microenvironment. Using a granulocyte colony-stimulating factor-induced mobilization model, I determined that the ability of HSPCs to mobilize into the bloodstream was significantly increased in Lxn-/- mice. The adhesive properties of hematopoietic cells were compromised in Lxn-/- animals. Gene expression studies on progenitor cells identified cell-to-ECM interactions were down-regulated upon Lxn deletion, implying the enhanced mobilization efficiency of hematopoietic cells from Lxn-/- mice correlated with reduced adhesion of hematopoietic progenitor cells to stroma. Last, but not least, I performed a series of experiments to study the putative tumor suppressor role of Lxn in hematological malignancy. I found that Lxn expression was down-regulated in primary tumor and tumor cell lines by promoter methylation. Overexpression of Lxn inhibited lymphoma cell growth both in vitro and in vivo. Overexpressed Lxn increased apoptosis frequency by suppressing the expression of several anti-apoptotic genes, and therefore reduced the tumor growth.