A number of cyclic peptides containing a positively charged ring composed of arginine residues attached to hydrophobic tail made of tryptophan residues through a lysine linker namely [R5K]W5, [R6K]W5, [R5K]W6, [R7K]W5, [R5K]W7, [R6K]W6, and [R7K]W7 were synthesized and evaluated as molecular transporters. The peptides were evaluated for their ability to deliver, fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI), and fluorescent labeled anti-HIV drugs (F′-FTC and F′-d4T). The results indicated that the presence of positively charged arginine residues on the ring and hydrophobic tryptophan residues in a sequential linear outside the ring was an optimal approach to improve the intracellular uptake of cargo molecules through non-covalent interactions. Some of these peptides were also evaluated for their efficiency for intracellular delivery of siRNA to triple-negative breast cancer cell lines in the presence and absence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). [R6K]W6 and [R5K]W5 were found to be very efficient in the delivery of siRNA. Furthermore, co-formulation of peptides with lipid DOPE significantly enhanced the efficiency of siRNA delivery compared to peptide alone. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. A chemotherapeutic drug, doxorubicin (Dox) was covalently conjugated to the cyclic peptide [R5K]W7A and linear peptide R5KW7A, and the biological activity was evaluated in cell-based assays. Comparative antiproliferative assays between covalently conjugated peptide-Dox and the corresponding noncovalent physical mixtures of the peptides and Dox were performed. The conjugation of Dox with cyclic [R5K]W7A-Dox exhibited similar antiproliferative activity compared to Dox alone after 72 h incubation time in all cancer cell lines, such as leukemia, ovarian and gastric cancer cells. However, [R5K]W7A-Dox significantly reduced the cell cytotoxicity in normal cell lines such as normal heart muscle and normal kidney cells after 72 h when compared with Dox alone. These results revealed that this cyclic peptide prodrug can be used as a potential candidate for the treatment of cancer cells with reduced side effects against normal cells in the body.

Long-range chromosomal interactions have been discovered in a number of organisms, suggesting that gene regulation through direct physical association with regulatory elements and/or other genes is a common and conserved phenomenon. This thesis investigates the relationship between direct physical contact of genomic loci and how these interactions may play a role in gene regulation. Analysis of such levels of chromosomal organization has been made possible in part by the emergence of Chromosome Conformation Capture (3C). This technique makes use of formaldehyde crosslinking to trap interacting chromosomal fragments, which can be detected after a number of manipulations. By adapting the 3C protocol for use in intact Saccharomyces cerevisiaecells, one can determine the structure of a chromosome or chromosomal region without introducing artifacts due to the harsh isolation of nuclei. A number of 3C-based technologies, such as 4C (Circular 3C or 3C-on-Chip) and 5C (3C Carbon Copy) have added to the knowledge of physical association of genes with regulatory elements and/or other genes. Here, we present a new non-biased technology that allows for determination of chromosomal interactions between all fragments throughout a genome. We present two-dimensional heatmaps of chromosomal interactions for all 16 chromosomes in yeast. These techniques promise to shed light onto the biochemical process by which clustering of genes and elements can result in up- or down-gene expression, which is still poorly understood. To understand how chromosomal interactions play a role in gene regulation, we study clustering of heterochromatic loci. Clustering of heterochromatic loci in silenced nuclear compartments is a phenomenon that has been observed throughout evolution. These clusters are thought to represent nuclear sub-compartments that are enriched in silencing proteins, while the rest of the nucleus is depleted in such factors. Chromosome III in Saccharomyces cerevisiae contains four heterochromatic regions: the two telomeres and the silent mating type loci, HML and HMR, located on either end of the chromosome. Our work describes a long-range interaction between the heterochromatic regions on chromosome III. We analyze the mechanism that drive these interactions and reveal roles for silencing proteins and proper nucleosome assembly in mediating heterochromatic clustering. In addition we identify a novel step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions.

Chronic myeloid leukemia (CML) is a disease characterized by the expansion of granulocytic cells. The BCR-ABL tyrosine kinase inhibitor imatinib, the frontline treatment for Ph+ leukemias, can induce complete hematologic and cytogenetic response in most chronic phase CML patients. Despite the remarkable initial clinic effects, it is now recognized that imatinib will unlikely cure patients because a small cell population containing leukemic stem cells (LSCs) with self-renewal capacity is insensitive to tyrosine kinase inhibitors. In Chapter I, I briefly review the BCR-ABL kinase and its related signaling pathways. BCR-ABL kinase activates several signaling pathways including MAPK, STAT, and JNK/SAPK. BCR-ABL also mediates kinase-independent pathways through SRC family kinases. I will also discuss pathways involving β-catenin, hedgehog, FoxO and Alox5 are critical to the regulation of self-renewal and differentiation in LSC of CML. As detailed in Chapter II, I describe our work evaluating the effects of omacetaxine, a novel CML drug inducing cell apoptosis by inhibition of protein synthesis, on self-renewal and differentiation of LSCs and BCR-ABL-induced CML and acute lymphoblastic leukemia (B-ALL) in mice. We found that treatment with omacetaxine decreased the number of LSCs and prolonged the survival of mice with CML or B-ALL. In chapter III, I describe that Alox5 is an essential gene in the function of LSCs and CML development. We show evidence that Alox5 affects differentiation, cell division, and survival of long-term LSCs. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly and prolonged survival. In chapter IV, I present evidence of our work showing a further dissection the Alox5 pathway by comparing the gene expression profiles of wild type and Alox5-/- LSCs. We show that Msr1 deletion causes acceleration of CML development. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-catenin. Taken together, these results demonstrate that some pathways including Alox5 and Msr1 play an important role in regulating the self-renewal and differentiation of LSC. More efforts should be put into developing the novel strategies that may effectively target LSCs and thus cure CML.

The Hedgehog (Hh) family of proteins are secreted growth factors that play an essential role in the embryonic development of all organisms and the main components in the pathway are conserved from insects to humans. These proteins affect patterning and morphogenesis of multiple tissues. Therefore, mutations in the Hh pathway can result in a wide range of developmental defects and oncogenic diseases. Because the main components in the pathway are conserved from insects to humans, Drosophilahas been shown to provide a genetically tractable system to gain insight into the processes that Hh is involved in. In this study, the roles of Hh cholesterol modification and endocytosis during gradient fonnation are explored in the Drosophila larval wing imaginal disc. To exclude the possibility of looking at a redistribution of preexisting Hh instead of Hh movement, a spatially and temporally regulated system has been developed to induce Hh expression. Functional Hh-GFP with and without the cholesterol-modification was expressed in a wild-type or shi-tslendocytosis mutant background. The Gal80 system was used to temporally express (pulse) the Hh-GFP transgenes to look at the rate of Hh gradient formation over time and determine whether this process was affected by cholesterol modification and/or endocytosis. Hh with and without cholesterol were both largely detected in punctate structures and the spreading of the different forms of Hh was quantified by measuring distances of these particles from the expressing cells. Hh without cholesterol showed a greater range of distribution, but a lower percentage of particles near the source. Loss of endocytosis blocked formation of intracellular Hh particles, but did not dramatically alter its movement to target cells. Staining for Hh, its receptor Ptc and cortical actin revealed that these punctate structures could be classified into four types of Hh containing particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Cholesterol is specifically required for the formation of cytoplasmic particles lacking Ptc. While previous studies have shown discrepancies in the localization of Hh following a block in endocytosis, Hh with and without cholesterol is detected at both apical and basolateral surfaces, but not at basal surfaces. In the absence of cholesterol and endocytosis, Hh particles can be observed in the extracellular space. Through three-dimensional reconstruction and quantitative analysis, this study concludes that the cholesterol modification is required to restrict Hh movement. In addition, the cholesterol modification promotes Ptc-independent internalization. This study also observes that Dynamin-dependent endocytosis is necessary for internalization but does not play an essential role in Hh distribution. The data in this thesis supports the model in which Hh movement occurs via planar diffusion.

Cancer is the second leading cause of death among men and women after heart disease. Though our knowledge associated with the complexities of the cancer network has significantly improved over the past several decades, we have only recently started to get a more complete molecular understanding of the disease. To better comprehend signaling pathways that prevent disease development, we focused our efforts on investigating endogenous tumor suppression networks in controlling effectors of cancer cell survival and proliferation. Survivin is one such effector molecule that controls both cell proliferation and survival. In order to identify how this protein is overexpressed in cancer cells as opposed to normal cells, we looked at signaling molecules that negatively regulate this inhibitor of apoptosis protein. PTEN and caspase 2 are two of the identified proteins that utilize their enzymatic activity to suppress tumor growth by inhibiting downstream cell survival effectors, namely survivin. PTEN uses its phosphatase activity to suppress the PI3K/AKT pathway and maintain cellular homeostasis. In the absence of AKT activity, FOXO transcription factors are able to target downstream gene expression and regulate cell proliferation and survival. Here we have identified survivin as a novel gene target of FOXO, which binds to a specific promoter region of survivin and suppresses its transcription. Alternatively, caspase 2 uses its catalytic activity to suppress survivin gene expression by targeting the NFκB pathway. Caspase 2 acts by cleaving a novel substrate known as RIP1 that prevents NFκB from entering the nucleus, thus inhibiting target gene transcription. Interestingly, survivin is known to be a direct gene target of NFκB that controls cancer cell survival. In our investigation, we found that survivin is downregulated upon caspase 2 activation via the NFκB pathway, resulting in decreased cell cycle kinetics, increased apoptotic threshold and suppressed tumor growth in mice. These studies conclude that survivin is a common effector molecule that is regulated by tumor suppressors to maintain cellular homeostasis. However, upon deactivation of the tumor suppressor pathway, survivin is deregulated and contributes significantly to disease progression. These observations may lead to potential therapeutic implications and novel targeting strategies that will help eradicate harmful cancer cells and spare surrounding healthy cells; often the most persistent problem of most conventional chemotherapy.

Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNAs have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate. Historically, siRNAs were believed to arise only from exogenous double-stranded RNA triggers in organisms lacking RNA-dependent RNA polymerases. However, the discovery of endogenous siRNAs in flies expanded the biological significance of siRNAs beyond viral defense. By high throughput sequencing we identified Drosophila endosiRNAs as 21 nt small RNAs, bearing a 2´-O-methyl group at their 3´ ends, and depleted in dicer-2 mutants. Methylation of small RNAs at the 3´ end in the soma, is a consequence of assembly into a mature Argonaute2-RNA induced silencing complex. In addition to endo-siRNAs, we observed certain miRNAs or their miRNA* partners loading into Argonaute2. We discovered, that irrespective of its biogenesis, a miRNA duplex can load into either Argonaute (Ago1 or Ago2), contingent on its structural and sequence features, followed by assignment of one of the strands in the duplex as the functional or guide strand. Usually the miRNA strand is selected as the guide in complex with Ago1 and miRNA* strand with Ago2. In our efforts towards finding 3´ modified small RNAs in the fly soma, we also discovered 24-28nt small RNAs in certain fly genotypes, particularly ago2 and dcr-2mutants. 24-28nt small RNAs share many features with piRNAs present in the germline, and a significant fraction of the 24-28nt small RNAs originate from similar transposon clusters as somatic endo-siRNAs. Therefore the same RNA can potentially act as a precursor for both endo-siRNA and piRNA-like small RNA biogenesis. We are analyzing the genomic regions that spawn somatic small RNAs in order to understand the triggers for their production. Ultimately, we want to attain insight into the underlying complexity that interconnects these small RNA pathways. Dysregulation of small RNAs leads to defects in germline development, organogenesis, cell growth and differentiation. This thesis research provides vital insight into the network of interactions that fine-tune the small RNA pathways. Understanding the flow of information between the small RNA pathways, a great deal of which has been revealed only in the recent years, will help us comprehend how the pathways compete and collaborate with each other, enabling each other’s optimum function.

Werner syndrome (WS) is an autosomal recessive disorder associated with premature aging and cancer predisposition. WS cells show increased genomic instability and are hypersensitive to DNA-damaging agents. WS is caused by mutations of the WRN gene. WRN protein is a member of RecQ DNA helicase family. In addition to a conserved 3’–5’ helicase activity, the WRN protein contains unique 3’–5’ exonuclease activity. WRN recognizes specific DNA structures as substrates that are intermediates of DNA metabolism. WRN physically and functionally interacts with many other proteins that function in telomere maintenance, DNA replication, and DNA repair. The function of WRN is regulated by post–translational modifications that include phosphorylation, acetylation, and sumoylation. SIRT1 is a NAD-dependent histone deacetylase (HDAC) that deacetylates histones and a numbers of cellular proteins. SIRT1 regulates the functions of many proteins, which are important for apoptosis, cell proliferation, cellular metabolism, and DNA repair. SIRT1 is also regulated by other proteins or molecules from different levels to activate or inhibit its deacetylase activity. In this study, we found that SIRT1 interacts with and deacetylates WRN. We further identified the major acetylation sites at six lysine residues of the WRN protein and made a WRN acetylation mutant for functional analysis. We found that WRN acetylation increases its protein stability. Deacetylation of WRN by SIRT1 reverses this effect. CREB-binding protein (CBP) dramatically increased the half-life of wild-type WRN, while this increase was abrogated with the WRN acetylation mutant. We further found that WRN stability is regulated by the ubiquitination pathway, and that WRN acetylation by CBP dramatically reduces its ubiquitination level. We also found that acetylation of WRN decreases its helicase and exonuclease activities, and that SIRT1 reverses this effect. Acetylation of WRN alters its nuclear distribution. Down-regulation of SIRT1 increases WRN acetylation level and prevents WRN protein translocating back to nucleolus after DNA damage. Importantly, we found that WRN protein is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells that were stably expressing WRN acetylation mutant display significantly higher sensitivity to MMC than the cells expressing wild-type WRN. Taken together, these data demonstrated that acetylation pathway plays an important role in regulating WRN function in response to DNA damage. A model has been proposed based on our discoveries.

Pim (Provirus Integration site for Moloney murine leukemia virus) kinases are a family of three serine/threonine kinases involved in cell cycle, survival and metabolism. These kinases were first identified in malignant cells and are most often associated with their role in cancer. Their role in immunity and lymphocytes is less well known. To date, it has been shown that Pim 1 and/or Pim 2 are important for T lymphocyte survival and activation when the Akt signaling pathway is inhibited by rapamycin. In addition, our laboratory has shown that Pim 2 is critical for BLyS-mediated naive B lymphocyte survival in the presence of rapamycin. This thesis extends the role(s) for Pim 1 and/or 2 to include functions during B cell activation and the generation of immune responses. We found that during in vitro activation of purified resting splenic B cells from wild type mice with a variety of activators that use multiple signaling pathways, including the BCR, TLR and CD40 receptors, both Pim 1 and 2 kinases were induced by 48 hours post-activation, suggesting that they could play a role in B cell activation and differentiation to antibody secreting or memory B cells. Immunization of Pim 1-/-2-/- knockout mice with T cell dependent antigens showed impairment in antibody and antibody secreting cell generation as well as lack of germinal center formation clearly demonstrating an involvement of Pim 1 and/or 2 in the immune response. FACS examination of B cell populations from naive Pim 1-/-2-/- knockout mice revealed normal levels of splenic marginal zone and follicular B cells and T cells, however, decreased numbers of all peritoneal B cell populations and decreased B cells in Peyer's Patches was seen. An examination of serum antibody found in naive Pim 1-/-2-/- knockout mice showed decreased levels of natural antibody, which is likely due to loss of the peritoneal B1 cells but does not explain the significantly decreased TD immune response. To determine whether the defect was B cell intrinsic or a more complex interaction between B and T cells, we determined whether Pim 1-/-2-/- mice would respond to T cell independent, TI-1 and TI-2, antigens. Antibody production and antibody secreting cell formation were also significantly decreased in these mice supporting our notion of a B cell intrinsic defect. To further examine the B cell response problem, we attempted to establish chimeric mice using either bone marrow derived cells or fetal liver cells from WT or Pim 1-/-2-/- donors so that the B cells were derived from Pim 1-/-2-/- mice and the T cells would be WT. Unfortunately, we were not able to consistently engraft and develop mature Pim 1-/-2-/- B cells, which indicate that there is a stem cell defect in these knockout mice that requires further investigation. Because one of the major failures in activated Pim 1-/-2-/- B cells is the generation of antibody secreting cells, an analysis of the expression of transcription factors IRF-4 and BLIMP-1, known to play a role in this process was carried out. Although IRF-4 induction was not affected by the loss of Pim 1 and 2, the number of cells able to increase BLIMP-1 expression was significantly decreased, revealing a partial block in the generation of ASCs. Taken together the data presented in this thesis reveals a new and critical role for Pim 1 and 2 kinases in the humoral immune response.

Faithful duplication and segregation of undamaged DNA is critical to the survival of all organisms and prevention of oncogenesis in multicellular organisms. To ensure inheritance of intact DNA, cells rely on checkpoints. Checkpoints alter cellular processes in the presence of DNA damage preventing cell cycle transitions until replication is completed or DNA damage is repaired. Several checkpoints are specific to S-phase. The S-M replication checkpoint prevents mitosis in the presence of unreplicated DNA. Rather than outright halting replication, the S-phase DNA damage checkpoint slows replication in response to DNA damage. This checkpoint utilizes two general mechanisms to slow replication. First, this checkpoint prevents origin firing thus limiting the number of replication forks traversing the genome in the presence of damaged DNA. Second, this checkpoint slows the progression of the replication forks. Inhibition of origin firing in response to DNA damage is well established, however when this thesis work began, slowing of replication fork progression was controversial. Fission yeast slow replication in response to DNA damage utilizing an evolutionarily conserved kinase cascade. Slowing requires the checkpoint kinases Rad3 (hATR) and Cds1 (hChk2) as well as additional checkpoint components, the Rad9-Rad1-Hus1 complex and the Mre11-Rad50-Nbs1 (MRN) recombinational repair complex. The exact role MRN serves to slow replication is obscure due to its many roles in DNA metabolism and checkpoint response to damage. However, fission yeast MRN mutants display defects in recombination in yeast and, upon beginning this project, were described in vertebrates to display S-phase DNA damage checkpoint defects independent of origin firing. Due to these observations, I initially hypothesized that recombination was required for replication slowing. However, two observations forced a paradigm shift in how I thought replication slowing to occur and how replication fork metabolism was altered in response to DNA damage. We found rhp51Δ mutants (mutant for the central mitotic recombinase similar to Rad51 and RecA) to slow well. We observed that the RecQ helicase Rqh1, implicated in negatively regulating recombination, was required for slowing. Therefore, deregulated recombination appeared to actually be responsible for slowing failures exhibited by the rqh1Δ recombination regulator mutant. Thereafter, I began a search for additional regulators required for slowing and developed the epistasis grouping described in Chapters II and V. We found a wide variety of mutants which either completely or partially failed to slow replication in response to DNA damage. The three members of the MRN complex, nbs1Δ, rad32Δ and rad50Δ displayed a partial defect in slowing, as did the helicase rqh1Δ and Rhp51-mediator sfr1Δ mutants. We found the mus81Δ and eme1Δ endonuclease complex and the smc6-xhypomorph to completely fail to slow. We were able to identify at least three epistasis groups due to genetic interaction between these mutants and recombinase mutants. Interestingly, not all mutants’ phenotypes were suppressed by abrogation of recombination. As introduced in Chapters II, III and IV checkpoint kinase cds1Δ, mus81Δ endonuclease, and smc6-x mutant slowing defects were not suppressed by abrogation of recombination, while the sfr1Δ, rqh1Δ, rad2Δ and nbs1Δ mutant slowing defects were. Additionally, data shows replication slowing in fission yeast is primarily due to proteins acting locally at sites of DNA damage. We show that replication slowing is lesion density-dependent, prevention of origin firing representing a global response to insult contributes little to slowing, and constitutive checkpoint activation is not sufficient to induce DNA damage-independent slowing. Collectively, our data strongly suggest that slowing of replication in response to DNA damage in fission yeast is due to the slowing of replication forks traversing damaged template. We show slowing must be primarily a local response to checkpoint activation and all mutants found to fail to slow are implicated in replication fork metabolism, and recombination is responsible for some mutant slowing defects.

Myosin II and actin are two major components of the ingressing cortex during cytokinesis. However, their structural dynamics and functions during cytokinesis are still poorly understood. To study the role of myosin II in cortical actin turnover, dividing normal rat kidney (NRK) cells were treated with blebbistatin, a potent inhibitor of the non-muscle myosin II ATPase. Blebbistatin caused a strong inhibition of actin filament turnover and cytokinesis. Local release of blebbistatin at the equator caused inhibition of cytokinesis, while treatment in the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a global role. These observations indicate that myosin II ATPase is essential for actin turnover and remodeling during cytokinesis. To further study the mechanism of myosin II and actin recruitment to the cytokinetic furrow, equatorial cortex were observed with total internal reflection fluorescence microscope (TIRF-M) coupled with spatial temporal image correlation spectroscopy (STICS) and a new approach termed temporal differential microscopy (TDM). The results indicated at least partially independent mechanisms for the early equatorial recruitment of myosin II and actin filaments. Cortical myosin II showed no detectable directional flow toward the equator. In addition to de novo equatorial assembly, localized inhibition of disassembly appeared to contribute to the formation of the equatorial myosin II band. In contrast, actin filaments underwent a striking, myosin II dependent flux toward the equator. However, myosin II was not required for equatorial actin concentration, suggesting that there was a flux-independent, de novo mechanism. The study was then extended to retraction fibers found typically on mitotic adherent cells, to address the hypothesis that they may facilitate post-mitotic spreading. Cells with retraction fibers showed increased spreading speed in post-mitotic spreading compared to cells without retraction fibers. In addition, micromanipulation study suggested that retraction fibers may guide the direction of post-mitotic spreading. Focal adhesion proteins were present at the tips of retraction fibers, and may act as small nucleators for focal adhesions reassembly that help cell quickly respread and regrow focal adhesions. These findings may suggest a general mechanism utilized by adherent cells to facilitate post-mitotic spreading and reoccupy their previous territory.