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Chan Siu Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 149-181). / Abstracts in English and Chinese. / Abstract --- p.ii / Chinese Abstract (摘要) --- p.v / Acknowledgements --- p.viii / Publications --- p.x / Table of contents --- p.xii / List of Figures --- p.xvi / List of Table and Diagram --- p.xx / Abbreviations --- p.xxi / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Major objectives and long-term significance --- p.1 / Chapter 1.2 --- Biological significance of apoptosis --- p.1 / Chapter 1.3 --- Importance of apoptosis in the study of the female reproductive system --- p.3 / Chapter 1.4 --- Specific aims of the present project --- p.3 / Chapter 1.5 --- Experimental approaches --- p.8 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- General experimental methods --- p.11 / Chapter 2.1.1 --- Culture of cells --- p.11 / Chapter 2.1.1.1 --- Culture of rabbit immortalized uterine epithelial cells --- p.11 / Chapter 2.1.1.2 --- Culture of primary mouse uterine epithelial cells --- p.12 / Chapter 2.1.1.3 --- Culture of human ovarian epithelial cancer cells --- p.13 / Chapter 2.1.2 --- Assessment of apoptotic DNA fragmentation --- p.13 / Chapter 2.1.2.1 --- DNA extraction --- p.14 / Chapter 2.1.2.2 --- Assessment of apoptotic DNA --- p.14 / Chapter 2.1.2.3 --- Assessment of apoptotic DNA by CE-LIF --- p.15 / Chapter 2.1.2.4 --- Assessment of apoptosis by Nuclear Hoechst 33248 Staining / Chapter 2.1.3 --- Assessement of protein content --- p.16 / Chapter 2.1.3.1 --- Protein extraction and western blot analysis --- p.16 / Chapter 2.1.4 --- Adenoviral infection of A2780s cells --- p.18 / Chapter 2.2 --- Preparation of solutions --- p.18 / Chapter 2.3 --- Animals and cell lines --- p.25 / Chapter 2.4 --- Statistical analysis --- p.25 / Chapter Chapter 3: --- Literature Review / Chapter 3.1 --- Morphological analysis of physiological cell death --- p.26 / Chapter 3.1.1 --- Characteristics of apoptosis --- p.27 / Chapter 3.2 --- Methods of detecting apoptosis --- p.31 / Chapter 3.3 --- Molecules controlling apoptosis --- p.33 / Chapter 3.3.1 --- Caspases --- p.33 / Chapter 3.3.2 --- The Bcl-2 family proteins --- p.34 / Chapter 3.4 --- Apoptosis signalling --- p.36 / Chapter 3.4.1 --- The death receptor-dependent pathway --- p.36 / Chapter 3.4.2 --- The mitochondria-dependent pathway --- p.38 / Chapter 3.4.3 --- The endoplasmic-reticulum-dependent pathway --- p.39 / Chapter 3.5 --- Importance of apoptosis in the female reproductive system --- p.40 / Chapter 3.5.1 --- Apoptosis in uterus epithelial cells --- p.40 / Chapter 3.5.2 --- Apoptosis in ovarian cancer cells --- p.42 / Chapter 3.6 --- Regulation of apoptosis by nitric oxide/cGMP/protein kinase G --- p.44 / Chapter 3.6.1 --- Regulation of apoptosis by nitric oxide --- p.44 / Chapter 3.6.2 --- Regulation of apoptosis by cGMP --- p.48 / Chapter 3.6.3 --- Regulation of apoptosis by soluble guanyly cyclase activator --- p.50 / Chapter Chapter 4: --- "Apoptotic DNA fragmentation caused by sodium nitroprusside, a nitric oxide donor, in uterine epithelial cells: ultrasensitive quantitation using the new capillary electrophoresis/laser-induced fluorescence (CE-LIF) technology" / Chapter 4.1 --- Abstract --- p.52 / Chapter 4.2 --- Introduction --- p.53 / Chapter 4.3 --- Results --- p.57 / Chapter 4.4 --- Discussion --- p.61 / Chapter 4.5 --- Figures of Chapter 4 --- p.66 / Chapter Chapter 5: --- Guanylyl-cyclase inhibitors NS2028 and ODQ and protein-kinase-G inhibitor KT5823 trigger apoptotic DNA fragmentation in an immortalized uterine epithelial cell line: anti-apoptotic effects of basal cGMP/PKG / Chapter 5.1 --- Abstract --- p.74 / Chapter 5.2 --- Introduction --- p.75 / Chapter 5.3 --- Results --- p.80 / Chapter 5.4 --- Discussion --- p.83 / Chapter 5.5 --- Figures of Chapter 5 --- p.89 / Chapter Chapter 6: --- "Direct, prolonged activation of soluble guanylyl cyclase by YC-1 or protein kinase G by cGMP analogs enhances the level of apoptosis in an immortalized uterine epithelial cell line, HRE-H9 cells" / Chapter 6.1 --- Abstract --- p.100 / Chapter 6.2 --- Introduction --- p.101 / Chapter 6.3 --- Results --- p.105 / Chapter 6.4 --- Discussion --- p.107 / Chapter 6.5 --- Figures of Chapter 6 --- p.114 / Chapter Chapter 7: --- "ODQ，an inhibitor of soluble guanylyl cyclase, down-regulates XIAP expression and induces apoptosis in human ovarian cancer cells" / Chapter 7.1 --- Abstract --- p.124 / Chapter 7.2 --- Introduction --- p.125 / Chapter 7.3 --- Results --- p.129 / Chapter 7.4 --- Discussion --- p.132 / Chapter 7.5 --- Figures of Chapter 7 --- p.138 / Chapter Chapter 8: --- Overall Conclusion --- p.145 / Chapter Chapter 9: --- References --- p.149

by Wong Kin Kwan Kelvin. / Thesis submitted in: September 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 91-101). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.x / List of Figures --- p.xiii / List of Tables --- p.xiv / Chapter Chapter 1 --- Intorduction --- p.1 / Chapter 1.1 --- Alzheimer，s disease --- p.1 / Chapter 1.2 --- Histopathological features --- p.1 / Chapter 1.3 --- Tau protein pathology and AD --- p.4 / Chapter 1.4 --- Tau protein kinase I (TPKI)- GSK-3β --- p.6 / Chapter 1.5 --- Tau protein kinase II (TPKII)- Cyclin dependent kinase 5 (Cdk5) --- p.8 / Chapter 1.6 --- Available treatment --- p.9 / Chapter 1.7 --- Objectives of the present study --- p.12 / Chapter Chapter 2 --- Screening for GSK-3p inhibitors from Traditional Chinese Medicine (TCM) --- p.13 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.1.1 --- Phosphorylation of tau in AD --- p.13 / Chapter 2.1.2 --- Gsk-3p inhibitors --- p.14 / Chapter 2.1.3 --- Screening of GSK-3β inhibitor from TCM --- p.16 / Chapter 2.2 --- Material and Methods --- p.18 / Chapter 2.2.1 --- Preparation of extracts and fractions (AOF1-5) --- p.18 / Chapter 2.2.2 --- General cell culture techniques --- p.21 / Chapter 2.2.3 --- "3-(4,5-dimethyltiazoI-2-yl)-2, 5-diphenyl-tetrazolium (MTT) assay of AOF" --- p.23 / Chapter 2.2.4 --- Recombinant DNA techniques --- p.23 / Chapter 2.2.5 --- Transfection of GSK-3β and tau cDNA into COS7 cells --- p.28 / Chapter 2.2.6 --- Extraction of total proteins from culture cells --- p.28 / Chapter 2.2.7 --- Quantitation of protein by the Bradford method --- p.29 / Chapter 2.2.8 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.9 --- Western blot analysis --- p.31 / Chapter 2.2.10 --- GSK-3β kinase assay --- p.32 / Chapter 2.2.11 --- Determination of lithium content by atomic adsorption spectrophotometry --- p.34 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Establishment of a co-transfected cell model for GSK-3β induced tau hyperphosphorylation --- p.35 / Chapter 2.3.2 --- Preliminary screening results of aqueous and ethanol extracts (AOF1 and AOF2) --- p.37 / Chapter 2.3.3 --- Ethanol extract of AOF inhibits GSK-3p induced tau phosphorylation in COS-7 cells --- p.40 / Chapter 2.3.5 --- Effect of the essential oils of AOF on GSK-3P induced tau phosphorylation --- p.46 / Chapter 2.3.6 --- The effect of AOF essential oil on GSK-3P activity in COS7 --- p.50 / Chapter 2.3.7 --- Lithium content of AOF extracts --- p.52 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 4 --- Evaluation of the in vivo efficacy of cryptotenshinone (CT) in Morris Water Maze Task (WMT) --- p.59 / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.1.1 --- Involvement of Cholinergic system in cognitive dysfunction in AD --- p.59 / Chapter 4.1.2 --- Animal model for Alzheimer's disease --- p.60 / Chapter 4.1.3 --- Morris Watermaze Task (WMT) --- p.61 / Chapter 4.2 --- MATERIAL AND METHODS --- p.64 / Chapter 4.2.1 --- Morris Water maze setup --- p.64 / Chapter 4.2.2 --- Animal model --- p.66 / Chapter 4.2.3 --- Drug preparation --- p.67 / Chapter 4.2.4 --- Toxicity test of CT --- p.67 / Chapter 4.2.5 --- Water maze task (WMT) --- p.68 / Chapter 4.2.6 --- Visual acuity test --- p.73 / Chapter 4.3 --- RESULTS --- p.74 / Chapter 4.3.1 --- Chronic crytotanshinone treatment does not cause hepatic damages to the mice --- p.74 / Chapter 4.3.2 --- Training Session --- p.76 / Chapter 4.4 --- DISCUSSION --- p.85 / Chapter Chapter 5 --- General Discussion and Future Directions --- p.87 / Chapter 5.1 --- "AOF, the potential GSK-3 inhibitor" --- p.87 / Chapter 5.2 --- CT´ؤthe AChEI --- p.88 / References --- p.91 / Appendix --- p.102 / Chapter A1 --- Reagents for SDS-PAGE --- p.103 / Chapter A3 --- Solution components provided by QIAGEN Plasmid Maxipreps kit --- p.108 / Chapter A4 --- Reagents and medium for cell culture --- p.109 / Chapter A5 --- Reagents for kinase assay --- p.110 / Chapter A6 --- Raw data of figures --- p.112 / Chapter A7 --- Plasmid map of PCI-neo --- p.119

Bacillus subtilis est la bactérie modèle des bactéries Gram-positif à bas pourcentage en GC et possède un intérêt marqué en biotechnologie. Par ailleurs, la phosphorylation des protéines est un mécanisme de régulation essentiel chez les bactéries qui reste encore largement à explorer. B. subtilis possède plusieurs ser/thr kinases potentielles (PrkA, YbdM, YabT et PrkC, qui a été déjà largement caractérisée), mais très peu de substrats de ces kinases ont été mis en évidence. Récemment, des études phosphoprotéomiques ont permis d’identifier de nombreux peptides phosphorylés sur des sérines ou des thréonines chez B. subtilis, incluant: a) deux régulateurs globaux de la phase de transition, DegS et AbrB et b) RecA, qui joue un rôle essentiel dans la réparation des cassures double-brin de l’ADN et la recombinaison. Des tests de phosphorylation in vitro nous ont permis d’identifier les ser/thr kinases capables de phosphoryler DegS, RecA et AbrB. La phosphorylation de DegS sur son résidu sérine 76 par la kinase YbdM influence, in vitro et in vivo, son activité kinase vis à vis de son substrat DegU. L’expression chez B. subtilis d’un allèle codant la protéine DegS-S76D (la sérine étant remplacée par un aspartate phosphomimétique) perturbe l’ensemble des processi cellulaires régulés par le système à deux composants DegS/DegU. Ces résultats suggèrent un lien entre la phosphorylation de DegS sur sa sérine 78 et le niveau de phosphorylation de son substrat DegU, cette modification post-traductionnelle représentant un degré supplémentaire de régulation pour ce système à deux composants. Au cours du démarrage de la sporulation, B. subtilis exprime une ser/thr kinase atypique, YabT, qui localise au septum et est activée grâce à la liaison de séquences ADN non spécifiques. YabT activée phosphoryle RecA sur sa sérine 2, ce qui induit la formation de foci RecA. Dans une souche exprimant une protéine RecA non phosphorylable (RecA-S2A) ou inactivée pour yabT, la formation de spores en présence de lésions de l’ADN est diminuée. Ces résultats suggèrent une homologie fonctionnelle au cours du développement entre la phosphorylation de RecA chez B. subtilis et la phosphorylation de son homologue eukaryote Rad51, qui permet leur recrutement sur des lésions de l’ADN. Nous proposons donc que la phosphorylation de RecA serve de signal pour promouvoir la formation de foci au cours de la sporulation. In vitro, le régulateur transcriptionnel AbrB est phosphorylé par les kinases YabT, YbdM et PrkC, L’utilisation de protéines mutées AbrB-S86A (non phosphorylable) et AbrB-S86D (forme phosphomimétique) nous a permis de montrer que la phosphorylation d’AbrB diminue son affinité pour l’ADN cible. L’expression chez B. subtilis des protéines AbrB-S86A et –S86D perturbe des phénomènes mis en place au cours de la phase stationnaire comme la production d’exoprotéases, la compétence et la sporulation via la dérégulation des gènes et opérons AbrB-dépendants correspondants. Nous proposons donc que la phosphorylation d’AbrB par les Hanks-kinases constitue un mécanisme de contrôle supplémentaire nécessaire à l’inactivation de ce régulateur transcriptionnel, qui peut être activateur ou répresseur, pendant la phase de transition. / Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.

Protein phosphorylation is a key cellular regulatory mechanism. Phosphorylation can either activate or inhibit the function of a particular protein. Activation of protein kinases has been implicated in response to light, pathogen attack, growth regulators, stress and nutrient deficiency in plants. Most of the intracellular signaling pathways use protein phosphorylation to create signals and conduct them further. Identification of the physiological substrates for the protein kinase enables the understanding of how the signaling networks function and how they are disturbed under adverse conditions. Identification of the physiological substrates for the kinase is limited by the low stoichiometry of protein phosphorylation inside the cell. Although, recent advances in mass spectrometric techniques have increased the identification of phosphorylated protein in the cell, the precise connection between the kinase and identified phosphorylated protein is not established. Dual-specificity kinases that phosphorylate on serine, threonine and tyrosine residues have been identified and characterized in plants. However, the in vivo substrates for most of these kinases have not been identified. Recently a manganese-dependent dual-specificity STY protein kinase (STYK) has been identified from Arabidopsis thaliana which has been suggested to play a role in plant growth, development and in systemic acquired resistance. The identification of the physiological substrate for AtSTYK may help in understanding the signal transduction pathway the kinase in involved and how it is perturbed in different physiological condition. Therefore, the main objectives of my current study are,  To identify the physiological substrates of the AtSTY dual specificity kinase (STYK). 1) Identification of the substrates by using genetic, proteomic and biochemical approaches. 2) Biochemical characterization of the substrate phosphorylation. 3) Identifying the biochemical function of the substrate protein. 4) Assessing the significance of substrate phosphorylation.

Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis. Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined. In the second part of my thesis, I assess the relationship between Map4k4 and IGF-mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation. The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy.

Yersinia pestis (Y. pestis), as the causative agent of plague, has caused deaths estimated to more than 200 million people in three historical plague pandemics, including the infamous Black Death in medieval Europe. Although infection with Yersinia pestis can mostly be limited by antibiotics and only 2000-5000 cases are observed worldwide each year, this bacterium is still a concern for bioterrorism and recognized as a category A select agent by the Centers for Disease Control and Prevention (CDC). The investigation into the host-pathogen interactions during Y. pestis infection is important to advance and broaden our knowledge about plague pathogenesis for the development of better vaccines and treatments. Y. pestis is an expert at evading innate immune surveillance through multiple strategies, several mediated by its type three secretion system (T3SS). It is known that the bacterium induces rapid and robust cell death in host macrophages and dendritic cells. Although the T3SS effector YopJ has been determined to be the factor inducing cytotoxicity, the specific host cellular pathways which are targeted by YopJ and responsible for cell death remain poorly defined. This thesis research has established the critical roles of caspase-8 and RIP kinases in Y. pestis-induced macrophage cell death. Y. pestis-induced cytotoxicity is completely inhibited in RIP1-/- or RIP3-/-caspase-8-/- macrophages or by specific chemical inhibitors. Strikingly, this work also indicates that macrophages deficient in either RIP1, or caspase-8 and RIP3, have significantly reduced infection-induced production of IL-1β, IL-18, TNFα and IL-6 cytokines; impaired activation of NF-κB signaling pathway and greatly compromised caspase-1 processing; all of which are critical for innate immune responses and contribute to fight against pathogen infection. Y. pestis infection causes severe and often rapid fatal disease before the development of adaptive immunity to the V bacterium, thus the innate immune responses are critical to control Y. pestis infection. Our group has previously established the important roles of key molecules of the innate immune system: TLR4, MyD88, NLRP12, NLRP3, IL-18 and IL-1β, in host responses against Y. pestis and attenuated strains. Yersinia has proven to be a good model for evaluating the innate immune responses during bacterial infection. Using this model, the role of caspase-8 and RIP3 in counteracting bacterial infection has been determined in this thesis work. Mice deficient in caspase-8 and RIP3 are very susceptible to Y. pestis infection and display reduced levels of pro-inflammatory cytokines in spleen and serum, and decreased myeloid cell death. Thus, both in vitro and in vivo results indicate that caspase-8 and RIP kinases are key regulators of macrophage cell death, NF-κB and caspase-1 activation in Yersinia infection. This thesis work defines novel roles for caspase-8 and RIP kinases as the central components in innate immune responses against Y. pestis infection, and provides further insights to the host-pathogen interaction during bacterial challenge.

Human obesity is increasing globally at an impressive rate. The rise in obesity has led to an increase in diseases associated with obesity, such as type 2 diabetes. A major prerequisite for this disease is the development of insulin resistance in the muscle and adipose tissues. Interestingly, experiments in rodent models suggest that adipocytes and macrophages can profoundly influence the development of insulin resistance. Accordingly, the number of adipose tissue macrophages increases substantially during the development of obesity. Numerous research models have demonstrated that macrophages promote insulin resistance by secreting cytokines, like TNFα, which impair whole body insulin sensitivity and adipose tissue function. Additionally, enhancements of murine adipose function, particularly glucose disposal, prevent the development of insulin resistance in mice on a high fat diet. Thus, mechanisms which enhance adipose function or attenuate macrophage inflammation are of interest. Our lab previously identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potent negative regulator of adipocyte function. In these studies, TNFα treatment increased the expression of adipocyte MAP4K4. Furthermore, the use of small interfering RNAs (siRNA) to block the increase in MAP4K4 expression protected adipocytes from some of the adverse effects of TNFα. Because MAP4K4 is a potent negative regulator of adipocyte function, an understanding of the mechanisms by which TNFα regulates MAP4K4 expression is of interest. Thus, for the first part of this thesis, I characterized the signaling pathways utilized by TNFα to regulate MAP4K4 expression in cultured adipocytes. Here I show that TNFα increases MAP4K4 expression through a pathway requiring the transcription factors activating transcription factor 2 (ATF2) and the JUN oncogene (cJUN). Through TNFα receptor 1 (TNFR1), but not TNFR2, TNFα increases MAP4K4 expression. This increase is highly specific to TNFα, as the inflammatory agents IL-1β, IL-6 and LPS did not affect MAP4K4 expression. In agreement, the activation of cJUN and ATF2 by TNFα is sustained over a longer period of time than by IL-1β in adipocytes. Finally, MAP4K4 is unique as the expression of other MAP kinases tested fails to change substantially with TNFα treatment. For the second part of this thesis, I assessed the role of MAP4K4 in macrophage inflammation in vitro and in vivo. To accomplish this task, pure β1,3-D-glucan shells were used to encapsulate siRNA. Glucan shells were utilized because they are effectively taken up by macrophages which express the dectin-1 receptor and they survive oral delivery. I demonstrate that these β1,3-D-glucan encapsulated RNAi particles (GeRPs) are efficiently phagocytosed and capable of mediating the silencing of multiple macrophage genes in vitro and in vivo. Importantly, oral treatment of mice with GeRPs fails to increase plasma IFNγ and TNFα or alter serum AST and ALT levels. Orally administered GeRPs are found in macrophages isolated from the spleen, liver, lung and peritoneal cavity and mediate macrophage gene silencing in these tissues. Utilizing this technology, I reveal that MAP4K4 augments the expression of TNFα in macrophages following LPS treatment. Oral delivery of MAP4K4 siRNA in GeRPs silences MAP4K4 expression by 70% and reduces basal TNFα and IL-1β expression significantly. The depletion of MAP4K4 in macrophages protects 40% of mice from death in the LPS/D- galactosamine (D-GalN) model of septicemia, compared to less than 10% in the control groups. This protection associates with significant decreases in serum TNFα concentrations following LPS/D-GalN challenge. Consistent with reduced macrophage inflammation, hepatocytes from mice treated orally with GeRPs targeting MAP4K4 present less apoptosis following LPS/D-GalN treatment. Thus, MAP4K4 is an important regulator of macrophage TNFα production in response to LPS. The results presented here add to the knowledge of MAP4K4 action in adipocyte and macrophage inflammation substantially. Prior to these studies, the mechanism by which TNFα controlled MAP4K4 expression in adipocytes remained unknown. Considering that MAP4K4 is a negative regulator of adipocyte function, identifying the mechanisms that control MAP4K4 expression was of interest. Furthermore, the role of macrophage MAP4K4 in LPS stimulated TNFα production was also unknown. To address this question in vivo, new technology specifically targeting macrophages was needed. Thus, we developed a technology for non toxic and highly specific macrophage gene silencing in vivo. Considering that macrophages mediate numerous diseases, the application of GeRPs to these disease models is an exciting new possibility.

Breast cancer, as the most prevalent cancer in women, is responsible for more than 15% of new cancer cases and about 6.9% of all cancer-related death in the US. A major cause of therapeutic failure in breast cancer is the development of resistance to chemotherapy, especially for triple-negative breast cancer (TNBC). Therefore, how to overcome chemoresistance is the major challenge to improve the life expectancy of breast cancer patients. Our studies demonstrate that TNBC cells surviving the chronic treatment of chemotherapeutic drugs show significantly higher expression of the dual serine/threonine and tyrosine protein kinase (DSTYK) than non-treated parental cells. In our in vitro cellular models, DSTYK knockout via the CRISPR/Cas9-mediated technique results in apoptotic cell death of chemoresistant cells upon drug treatment. Moreover, DSTYK knockout promotes chemotherapeutic drug-induced tumor cell death in an orthotopic mouse model. These findings suggest that DSTYK exerts an important and previously unknown role in promoting chemoresistance. Our studies provide fundamental insight into the role of DSTYK in chemoresistance in TNBC cells and lay the foundation for the development of new strategies targeting DSTYK for improving TNBC therapy.

No / Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-alpha4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.