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Genetic instability promotes cancer progression (by increasing the probability of cancerous mutations) as well as hinders it (by imposing a higher cell death rate for cells susceptible to cancerous mutation).With oncogene activation or the loss of tumor suppressor gene functions known to be responsible for a high percentage of breast and colorectal cancer (and a good fraction of lung cancer and other types as well), it is important to understand how genetic instability can be orchestrated toward carcinogenesis. In this context, this research gives a complete characterization of the optimal cell mutation rate for the fastest time to a target cancerous cell population through the loss of both copies of a tumor suppressor gene or through oncogene activation.

Recently studies have shown that in subjects with acute Kawasaki disease (KD), an acute pediatric vasculitis of the coronary artery, there exists a population of myeloid dendritic cells (mDCs) that have the ability to secrete the suppressive cytokine interleukin (IL)-10. Enumeration of the mDC and the regulatory T cell (Treg) populations from 3 cohorts of pediatric subjects: acute KD, acute pediatric febrile controls, and healthy children have been analyzed with an expanded panel of surface markers. From these cohorts of subjects, it was found that a newly described population of tolerogenic mDC is present in circulation and is defined as being CD11c+CD11b+CD14+ILT-4+CD4+ and by the expression of differential amounts of HLA-G. This newly described population of mDCs express the DCs maturation marker CD86, which is a T cell co-receptor. An in vitro assay plating with these newly found tolerogenic mDC population produced IL-10 in a dose dependent fashion when stimulated with scalar doses of purified fragments of the heavy chain constant region of immunoglobulin (Fc). These tolerogenic mDC did not exhibit properties that enable them to polarize T cells into peripherally induced regulatory T cells (iTreg), but the Fc stimulated tolerogenic mDC demonstrated the ability to suppress T helper 1 (Th1) differentiation as demonstrated by reduced levels of interferon (IFN)γ mRNA transcript in naïve T cell co-culture in vitro. In pediatric subjects these mDC express the adenosine 2A receptor (A2AR) which induces intracellular cAMP production, which in turn can cause production of IL-10 in these tolerogenic cells. Expression of CD31 on these tolerogenic mDC can offer a possibility for these cells to directly suppress inflamed tissue by being able to bind to CD38 on the inflamed endothelial cells. This study identifies a new population of tolerogenic mDC in pediatrics who appears to compensate for the lack of a fully developed adaptive immune system in these young children.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. In this study, we investigated the effect of dectin-1 stimulation by yeast-derived particulate β-glucan in MDSC function and differentiation in cancer. In vivo treatment of mice bearing lewis lung carcinoma and mammary cell carcinoma with particulate β-glucan decreased tumor weight and splenomegaly, and reduced the accumulation of polymorphonuclear-MDSC (PMN-MDSC) but not monocytic-MDSC (M-MDSC) in the spleen and tumor. In addition, particulate β-glucan differentially modulated the function of different MDSC subsets; it enhanced PMN-MDSC respiratory burst and apoptosis, and induced the differentiation of M-MDSC into F4/80+CD11c+antigen-presenting cells in a dectin-1 dependent manner. ERK1/2 phosphorylation was also required for the acquisition of APC properties in M-MDSC. Moreover, M-MSDC treated with particulate β-glucan did not promote tumor growth in vivo when inoculated with LLC subcutaneously. To evaluate the effect of particulate β-glucan treatment in humans, patients with non-small cell lung cancer (NSCL) were treated with particulate β-glucan for two weeks prior to any other treatment and surgical excision of the tumor. Strikingly, the frequency of CD14-HLA-DR-CD11b+CD33+ MDSC decreased in the peripheral blood, and arginase-1 expression significantly decreased in a cohort of 15 patients. This study was the first to assess the effect of particulate β-glucan on MDSC in lung cancer patients, towards a future inclusion of particulate β-glucan in combination therapies in lung cancer.

Viruses infect all Kingdoms of life on Earth. Their life cycle represents a constant struggle for survival in hostile cellular environments. To survive the virus must both avoid the host response and replicate in a timely manner. The following dissertation includes investigations into both of these aspects of viral infection focusing on plant-infecting viruses. When infected by viruses, plants respond by initiating defense pathways. One of these pathways is known as RNA silencing. In this pathway host proteins target double-stranded viral RNA intermediates for cleavage. The product of this cleavage, a short-interfering RNA (siRNA), is incorporated into a protein complex that guides sequence specific cleavage of viral RNA. To cause a productive infection, the virus must devise countermeasures to this targeting. They accomplish this by suppressing the RNA silencing pathway by encoding proteins known as viral suppressors of RNA silencing (VSRs). Identifying these VSRs is critical to the understanding of how any plant virus survives in its host. Commonly used assays for identifying VSRs all use reporters expressed in the nucleus. These nuclear-based DNA reporters are being used to assay for the RNA silencing suppression activity of RNA virus proteins. In this thesis I describe the development of a new VSR identification assay that uses a disarmed RNA virus as a reporter, which should be an accurate predictor for VSR activity of other RNA viruses. The reporter is the plant-infecting virus Turnip crinkle virus (TCV) with its previously characterized VSR, the coat protein, replaced with sGFP resulting in a construct termed TCV-sGFP. After validating the use of TCV-sGFP as a reporter for RNA silencing suppression activity TCV-sGFP was used to identify a previously uncharacterized VSR for the plant-infecting virus Red clover necrotic mosaic virus (RCNMV). RCNMV’s replication complex was previously implicated in the suppression of RNA silencing, while the new TCV-sGFP assay allowed us to detect a second suppressor, a protein known to be involved in viral movement, known simply as the movement protein (MP). Domains of MP were analyzed and it was found that the amino acid residues between positions 122 and 277 were required for suppression activity. The RNA silencing pathway has two principal components, siRNAs and host proteins. To disrupt the pathway the MP of RCNMV must interfere with one or both of these components. In this thesis the possibility that the MP suppresses RNA silencing by binding to siRNAs is addressed by employing electrophoretic mobility shift assays to examine MP’s ability to bind to siRNAs. MP was found to have no siRNA binding capability, indicating that it’s mode of action likely relies on binding to or modifiying host proteins. Finally, initial studies into the temporal regulation of RCNMV replication were undertaken. As mentioned, for a productive infection to take place the virus must go beyond evading the host defense response, it must also properly regulate the timing of expression for its many genes. Using real time PCR, the timing of various replication events in the RCNMV life cycle were mapped to more fully understand how RCNMV replicates inside the cell. These time course studies have revealed that virion formation has little effect on RCNMV’s ability to replicate, and that RNA-1 replicates to a slightly higher degree in the absence of RNA-2. Additionally several uncharacterized negative sense replication intermediates that exist during a wildtype infection were identified. This work forms the foundation for future studies into the temporal regulation of RCNMV replication.

Understanding how genes coordinate the organization of cells into organs and how mutations in these genes upset this organization are fundamental questions in both cancer research and developmental biology. PAX factors play important roles to coordinate the development of mammalian organs like the kidney, eye and thyroid. The disruption of PAX activity leads to diseases like renal-coloboma syndrome and cancers in mammals. Dissecting the different functions for these factors has been difficult due to the complexity of organ development. To better understand these processes at a cellular level, I studied the role of a Pax2/5/8 transcription factor, EGL-38 in the developing egg-laying system and in the survival of germline cells of the nematode, Caenorhabditis elegans. egl-38(n578) mutant animals exhibit of high levels of Programmed Cell Death (PCD) in the germline and the soma. Wild-type EGL-38 can transcriptionally regulate the bcl-2 gene, ced-9 in C . elegans. In this dissertation, I performed mosaic analysis for EGL-38 and found that its activity is required in both the germline and soma to promote germline cell survival. EGL-38 is also involved in the development of the C . elegans egg-laying system. The egl-38(n578) mutants are egg-laying defective, so the eggs hatch within the hermaphrodite parent. The latter then bursts to release viable progeny. The C . elegans egg-laying system has two tissues, the somatic gonad and the vulva. The Pax2/5/8 transcription factor, EGL-38 is required for the normal development of the egg-laying system. Previously it was hypothesized to function in one tissue, the vulva to affect the development of both the vulval and somatic gonad tissues of the egg-laying system. While its mammalian counterpart, Pax 2 has independent functions in both the tissues of the metanephric kidney and thereby coordinates development between them. However, our studies involving various mutants and mosaic analysis demonstrate that egl-38 does have distinct functions in both the tissues of the egg-laying system of C . elegans. Furthermore, this influences both the autonomous and non-autonomous functions during the development of this system. Therefore, egl-38 behaves like the mammalian Pax2 by acting in the various tissues of an organ to promote its coordinated development. I also performed a genetic screen to isolate mutations in genes that suppress the egl-38(n578) egg-laying defect. Thereby I isolated four strong suppressor mutations. Two of these were mapped to separate locations on chromosome IV. Both these suppressor mutations suppress both the egg-laying defect and the elevated levels of cell death associated with the egl-38(n578) mutation. These studies have demonstrated that EGL-38 acts in multiple tissues to coordinate development like its mammalian counterpart Pax2 in the developing metanephric kidney. Thus there is conservation of the development logical by which PAX factors coordinate organogenesis. Finally, identifying the genes affected by the suppressor mutations could have implications in understanding development and survival of not only C . elegans but also of higher animals if these genes are evolutionarily conserved.

The endoplasmic reticulum (ER) is a major folding site for secretory proteins. One critical regulatory mechanism of the folding process is the quality control system, which recognizes and retains misfolded conformers in the ER, It has been pro posed that the calnexin/calreticulin (CNX, CRT) cycle is responsible for the folding and quality control of glycoproteins. This cycle consists of glucosidase I (GI), glucosidase II (GII), UDP-glucose:glycoprotein glucosyltransferase (UGGT) and two lectin-like chaperones: CNX and CRT. GI and GII prepare glycoproteins to enter the CNX/CRT cycle by removing two glucose from core glycans (Glc3Man9GlcNAc2). The resulting monoglucosylated glycans (GlcIMan9GlcNAc2) interact with CNX/CRT, thus prolonging the retention of misfolded glycoproteins in the ER, to facilitate their folding. When GII removes the remaining glucose, the glycoprotein dissociates from CNX/CRT and is free to exit the ER, unless it is recognized by UGGT. UGGT only reglucosylates glycans on misfolded conformers to allow their reentry to the CNX/CRT cycle. The alternative actions of deglucosylation and reglucosylation by GII and UGGT continue until the glycoprotein is correctly folded. Despite numerous studies, the significance of this cycle has not yet been demonstrated in multicellular organisms. Through a suppressor screening of a dwarf Arabidopsis mutant plant that has a mutation in the extracellular domain of a plasma membrane localized brassinosteroid (BR) receptor, BRI1 (Brassinosteroid Insensitive 1), I identified two allele-specific suppressors of bri1, ebs1 and ebs2 (EMS mutagenized bri1 suppressor 1 and 2) as the Arabidopsis UGGT and an Arabidopsis CRT, respectively. I demonstrated that UGGT monoglucosylates bril to retain the mutated BR, receptor in the ER, via CRT-G1c1Man9GlcNAc2 interaction. Mutations in either UGGT or CRT significantly compromise the fidelity of the Arabidopsis ER quality control system and allow a significant pool of mutated bril proteins to exit ER and move to the cell surface to transduce BR signal. Thus, this thesis provides the first genetic evidence of the existence of the CNX/CRT cycle in a multicellular organism. Specifically, the results in Chapter II and Chapter III demonstrate the physiological function of UGGT as a protein folding sensor and CRT as a retention factor in vivo.