Duchenne muscular dystrophy (DMD) is a devastating primary muscle disease with pathological changes in skeletal muscle that are ongoing at the time of birth. Progressive deterioration in striated muscle function in affected individuals ultimately results in early death due to cardio-pulmonary failure. Since affected individuals can be identified prior to birth by prenatal genetic testing for DMD, gene replacement treatment can be started in utero. This approach offers the possibility of preventing pathological changes in muscle that begin early in life. Previous studies with systemic in utero adenoassociated viral (AAV) vector serotype 1 gene delivery to embryonic day 16 (E-16) pups resulted in high levels of transduction in diaphragm and intercostal muscles, but no detectable levels in limb muscle. Recently newer AAV serotypes such as AAV8 have demonstrated widespread and high transgene expression in skeletal muscles and diaphragm by systemic delivery in adults and neonatal mice. In this study I tested AAV8 vector gene delivery by intraperitoneal administration in E-16 mice in utero. Using an AAV8 vector carrying a lacZ transgene, I observed high level transduction of diaphragm and more moderate transduction of multiple limb muscles and heart. Encouraged with these results I tested in utero gene transfer in the mdx mouse model of DMD, a minidystrophin gene driven by the human cytomegalovirus promoter was delivered systemically by an intraperitoneal injection to the fetus at embryonic day 16. Treated mdx mice studied at 9 weeks after birth demonstrated widespread expression of recombinant dystrophin in skeletal muscle, restoration of the dystrophin associated glycoprotein complex in dystrophin-expressing muscle fibers, improved muscle pathology, and functional benefit to the transduced diaphragm compared to untreated littermate controls. In order to further extend these studies, AAV9 carrying a minidytsrophin gene was also tested. Robust expression in heart and muscles were seen at 4 weeks post treatment by in utero gene delivery. Furthermore robust heart expression persisted as long as 3 months post treatment. These results support the potential of AAV8 and AAV9 vectors to efficiently cross the blood vessel barrier to achieve systemic gene transfer to skeletal muscle in utero in a mouse model of muscular dystrophy, to significantly improve the dystrophic phenotype and to ameliorate the processes that lead to exhaustion of the skeletal muscle regenerative capacity.

Human T-cell leukemia virus type I (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL). Its encoded oncoprotein Tax plays the key roles in HTLV-I-mediated cell transformation and pathogenesis. Although the mechanisms by which the HTLV-I Tax deregulates cellular signaling for oncogenesis have been extensively studied, how Tax itself is regulated remains largely unknown. Here we showed that PDZ-LIM domain-containing protein 2 (PDLIM2, SLIM or Mystique) negatively regulated Tax by promoting poly-ubiquitination and proteasomal degradation of Tax, so that to suppress Tax-mediated signaling activation, cell transformation and oncogenesis both in vitro and in animal. To further define the molecular determinant responsible for PDLIM2 mediated Tax suppression, we characterized that a putative á-helix motif of PDLIM2 at amino acids 236-254 was crucial for the interaction between PDLIM2 and Tax. PDLIM2 with selective disruption of this short helix lost the tumor suppression function and failed in altering Tax subcellular distribution as well as promoting Tax proteasomal degradation. Additionally, the expression of PDLIM2 was down-regulated in HTLV-I-transformed T cells and primary ATL samples, and the re-introduction of PDLIM2 reversed the tumorigenicity of the malignant cells. The evidence indicated that the counterbalance between HTLV-I Tax and PDLIM2 might determine the outcome of HTLV-I infection. Meanwhile, in those HTLV-I-transformed T cells, we found that DNA methyltransferases (DNMT) 1 and 3b but not 3a were over-expressed, suggesting the involvement of DNA methylation in PDLIM2 repression. Consistently, the hypomethylating agent 5-aza-2-deoxycytidine (5-aza-dC) restored PDLIM2 expression and induced death of these malignant cells. Our studies provided important insights into the function of PDLIM2 in HTLV-I leukemogenicity, long latency and cancer heath disparities. Given the efficient antitumor activity with no obvious toxicity of 5-aza-dC, our studies also suggest potential therapeutic approaches for ATL, a disease with poor treatments.

Tumor cells usually express specific antigens that are potentially immunogenic; however, established tumors primarily induce immune tolerance. In the last decade, a population of small membrane vesicles, termed "exosomes", has gained increasing attention for their potential role in tumor immune regulation. Exosomes are formed in the late endocytic compartments and are released upon their fusion with the plasma membrane. They are secreted by various cell types, especially tumor cells and cells in the hematopoietic system. Although tumor-derived exosomes usually contain tumor antigens, they have been shown to exert diverse immunosuppressive effects. However, the ability of tumor-derived exosomes to induce antigen-specific immunosuppression has not been well examined. Also, the immunoregulatory effect of exosome-like vesicles in the blood circulation of tumor-bearing hosts remains unclear. <br><br>In this thesis, we first investigate the role of tumor-derived exosomes in mediating antigen-specific immune suppression using ovalbumin (OVA) as a model tumor antigen. We demonstrate that exosomes derived from OVA-expressing tumor cell lines potently suppress OVA-specific delayed-type hypersensitivity (DTH) response. We also show that exosomes are mostly taken up by dendritic cells (DCs) after local administration, and the mRNA levels of TGF-beta1 and IL-4 in the draining lymph node were significantly elevated in correlation with suppression of the DTH response. Furthermore, tumor-derived exosomes affect the function of DCs in vitro by inhibiting their maturation and inducing TGF-beta1 production. These results suggest that tumor-derived exosomes are able to confer antigen-specific immune suppression possibly by DC-mediated mechanism. <br><br>We further investigate the immunoregulatory effect of plasma-derived exosomes and demonstrate that plasma-derived exosomes isolated from mice bearing OVA-expressing tumors are able to suppress the OVA-specific DTH response. However, enrichment of tumor-derived exosomes in blood plasma was not identified and the suppressive effect is partially mediated by the MHC class II+ vesicle portion. The third part of the thesis discusses the B cell stimulatory and the consequent T cell inhibitory effect of exosomes derived from tumor cells with mycoplasma infection. <br><br>The work presented in this thesis increases our understanding of the immunoregulatory role of tumor-derived exosomes, circulating exosomes in tumor-bearing hosts as well as exosomes derived from pathogen-infected tumor cells.

Hematopoiesis proceeds from a bone marrow resident population of stem cells responsible for generation of all lineages within the blood. Distinct molecular programs within hematopoietic stem cells regulate maintenance of this population under homeostatic conditions, however the coordination of these programs remains largely undefined. DNA methylation is an epigenetic means of gene regulation in a mammals carried out by a family of DNA methyltransferases. Of these, the de novo methyltransferase is highly expressed in hematopoietic stem cells as compared to other members of this family and somatic mutations in DNMT3b lead to a syndrone characterized by immunodeficiency. Therefore we hypothesized that DNMT3b regulates hematopoietic stem cells via its ability to methylate DNA. By knock-down of DNMT3b with a retrovirally delivered shRNA or Cre mediated recombination of floxed DNMT3b alleles work presented in this thesis demonstrates a critical role for DNMT3b in hematopoiesis in mice. Loss of DNMT3b leads to limited reconstitution of hematopoiesis in irradiated recipients associated with a proliferative defect in vitro and a failure of hematopoietic stem cell self-renewal in vivo. Targeted deletion of DNMT3b in hematopoietic stem cells leads to decreased engraftment following transplantation and decreased proliferation in vitro. DNMT3bs function in hematopoietic cells requires the methyltransferase activity of the enzyme and the defects in hematopoiesis are associated with loss of DNA methylation and decreased expression of MLL. Therefore DNMT3b is necessary for maintenance of the proliferative ability and engraftment capacity of hematopoietic cells and hematopoiesis is dependent upon appropriate DNA methylation.

Mitotic motors have gained considerable interest as anticancer targets given their often essential functions during mitosis. Furthermore, mitotic motors are thought to represent ideal targets because their functions are believed to be confined to mitosis; thus, only rapidly dividing cells would be susceptible to inhibitors of mitotic motors. The work presented herein challenges the concept of mitotic motors as specific targets of dividing cells by exploring the interphase function of three mitotic motors Kid, Eg5, and MKLP1. Our results demonstrate that all three motors associate with the nucleolus and with the ribosomal subunits. Furthermore, it is demonstrated Eg5 functions to increase the processivity of the ribosome, the first cellular factor to be characterized with that property. Additionally, as loss of Kid results in an increase in focal adhesion proteins throughout the cell and increased protein synthesis in its absence, our data are consistent with a role for Kid in mRNA silencing and transport of mRNAs for site-specific translation. Also, evidence is presented that suggests a role for Kid in ribosome biogenesis and/or ribosomal function, similar to nucleophosmin. Finally, both Kid and Eg5 participate in stress granule dynamics, with Kid and Eg5 functioning in stress granule formation, and Eg5 participating in stress granule coalescence, transport and dissolution. Collectively these findings demonstrate diverse interphase functions for these mitotic motors in nearly all phases of the ribosomes life cycle. These studies not only call into question the potential safety of mitotic motor inhibitors for the treatment of cancer, but also open a new avenue of exploring polypeptide synthesis.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the gene coding for dystrophin protein, which give rise to a dysfunctional protein in skeletal muscle. Dystrophic muscle progressively degenerates. In addition, necrotic muscle fibers undergo high levels of inflammation that in turn promote the pathology that is associated with this devastating disease. Therefore, treatments that 1) restore expression a functional dystrophin protein in dystrophic muscles, and 2) lower the ongoing inflammation in the necrotic muscle tissue, are both important in ameliorating DMD phenotype. Transfer of a functional dystrophin gene using a viral vector can help restore the missing dystrophin protein in dystrophic muscles. The host immune system, however, is a major barrier to successful vector-mediated dystrophin protein expression in a dystrophic host, as anti-dystrophin immune response leads to rejection of the protein. Here I show that temporal elimination of the host immune system by irradiation in the mdx mouse, a murine model of DMD, prior to vector-mediated dystrophin gene delivery, leads to a delayed and diminished host anti-dystrophin immune response. These findings are important for a better evaluation of anti-dystrophin immunity in a dystrophic host. In the case of lowering inflammation in dystrophic muscles, I investigated the effects of rapamycin, a potent immunosuppressant, on both dystrophic phenotype and dystrophin gene transfer in mdx mice. Treatment of adult mdx muscles with rapamycin lead to significantly lower levels of muscle fiber necrosis and reduced effector T cell infiltration in dystrophic muscles. These events correlated with a difference in activation of the mammalian target of rapamycin (mTOR) in the diaphragm muscle, but not the TA muscle, suggesting a differential regulation of mTOR activation in the two tissues. Rapamycin treatment, however, did not allow for a higher level of vector-mediated dystrophin protein expression in treated muscles. In general, these findings shed more light on the effects of manipulating the immune system in a dystrophic host in terms of both reducing the inflammation that is associated with DMD and reducing anti-dystrophin responses following gene therapy, suggesting that regulation of the immune system is essential in ameliorating DMD.

Alpha 1-antitrypsin deficiency (ATD) is an autosomal recessive disorder characterized by mutations in SERPINA1. Since α1-antitrypsin (α1-AT) is the predominant serine peptidase inhibitor in extracellular fluids, decreased α1-AT secretion results in a loss-of-function phenotype manifested by peptidase-inhibitor imbalance, connective tissue matrix destruction, and susceptibility to chronic obstructive lung disease. In contrast, the accumulation of aggregation-prone alpha-1 antitrypsin-Z (ATZ) variant in liver cells leads to a toxic gain-of-function phenotype characterized by liver failure and carcinogenesis. Curiously, only ~10% of all ATZ homozygous individuals develop severe liver disease. This observation suggests that additional genetic disease modifiers and/or environmental conditions make an important contribution to disease severity and outcome. Our current understanding of these factors is incomplete and effective therapeutic options are very limited. I report here the development of a C. elegans model of ATZ deficiency that recapitulates the ER transport defect associated with ATD. Furthermore, we identified key components of the disposal mechanism of ATZ in C. elegans, which parallels those in mammalian systems. Lastly, I developed a semi-automated high content genome-wide RNAi screen technology and used this method to identify <100 genes that modify the accumulation of ATZ. Many of these genes implicated in playing a role in the disposal of ATZ are novel and provide a source of potential ATZ genetic modifiers for future study.