Theses and Dissertations from UMD
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Item Role of Rv3167c in necrosis and autophagy induction by and in virulence of Mycobacterium tuberculosis(2015) Srinivasan, Lalitha; Briken, Volker; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mycobacterium tuberculosis (Mtb), an intracellular pathogen targeting lung phagocytes has been reported to manipulate apoptotic and necrotic host cell death. Mtb induces necrosis allowing for bacteria to escape from the host cell and infect naive cell populations. Mtb genes that influence host cell death responses as well as host cell pathways that are implicated in survival outcomes during mycobacterial infection represent interesting areas of investigation. In this study we identified the Mtb gene Rv3167c to be required for the inhibition of Mtb induced, caspase-independent necrotic cell death. The Rv3167c mutant strain induced higher levels of necrosis in macrophages compared to Mtb or the complement strain. Using a combination of chemical inhibitors and bone marrow derived macrophages from different knock-out mice, we screened for host factors required for the Rv3167c mutant mediated cell death and were able to eliminate involvement of known regulated necrosis pathways. Necrosis induction by the mutant strain was however found to be dependent on reactive oxygen species (ROS) generated by mitochondria and on inhibition of Akt activation. Apart from necrosis, Rv3167c was also found to be required for inhibition of host cell autophagy. Mitochondrial ROS, inhibition of Akt activation and upregulation of JNK activation were required for Rv3167c mutant -induced autophagy. Recent studies on Mtb induced autophagy have shown that Mtb co-localizes with autophagosomes, leading to lysosomal mediated bacterial destruction. Surprisingly, we did not observe co-localization of Mtb or the mutant strain with autophagosomes indicating Mtb has the ability to inhibit selective autophagy mediated killing. Deletion of Rv3167c results in an increase in Mtb virulence as demonstrated by the higher bacterial burden in the lungs and extra-pulmonary organs and the lower median survival time observed in the Rv3167c mutant infected mice compared to Mtb and complement strain infected control animals. In conclusion, this work indicates that Mtb utilizes Rv3167c to regulate necrosis and autophagy induction and to temper its own virulence.Item THE ANTIVIRAL ROLES OF ATG1 IN DROSOPHILA MELANOGASTER: IMMUNE RESPONSES AGAINST DROSOPHILA X VIRUS(2014) Wang, Qian; Wu, Louisa; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In mammals, autophagy is important for the immune response against select viruses and is responsible for delivering virus to the lysosome for degradation. In Drosophila melanogaster, the roles of autophagy genes in an antiviral immune response are not fully understood. Here we identify a novel antiviral role for Atg1 in Drosophila melanogaster upon infection with Drosophila X virus (DXV). Flies with a decreased level of Atg1 expression in the fat body developed an increased susceptibility to DXV and have a higher viral load compared to wildtype. However, silencing of other autophagy components (Atg7, Atg8) does not have the same effect. Moreover, we could find no evidence that classical autophagy is directly associated with DXV upon viral infection. This suggests that the antiviral function of Atg1 may be independent of classical autophagy. To address this, we examined the effect of Atg1 knockdown on the fly transcriptome in both DXV infected and uninfected flies. Interestingly, lipid droplet lipolysis and β-oxidation, two major processes responsible for energy production, are induced upon DXV infection. Facilitating lipolysis by knocking down lsd2, a positive regulator of lipase bmm, results in an increased host susceptibility to DXV, together with an increased viral load. In contrast, blocking lipolysis in the negative regulator lsd1 null mutant renders the host more resistant to the virus. This indicates that the increased energy production favors the virus for active replication and does not favor the elimination of virus. Surprisingly, silencing of Atg1, even in the absence of infection, also increases the rates of lipolysis and &beta-oxidation, shown by an increased expression of genes that are involved in lipid metabolism and an decreased lipid droplet size in the Atg1-silenced flies. The differences in gene expression and lipid droplet size between Atg1 RNAi flies and WT flies become more apparent as the infection progresses. In summary, we identify a novel role for Atg1 in restricting energy production and limiting DXV replication. This finding may shed light on antiviral studies against other dsRNA viruses that manipulate host energy homeostasis. Finally, our data reveal an important and unexpected role for Atg1 in innate immune antiviral responses independent of autophagy.Item REGULATION OF MACROAUTOPHAGY BY VITAMIN A/ RETINOIDS(2013) Rajawat, Yogendra Singh; Bossis, Ioannis; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Retinoic acids (RAs) have diverse biologic effects and regulate several cellular functions. Here, we investigated the role of RA on autophagy by studying its effects on autophagosome (AUT) maturation, as well as on upstream regulators of autophagosome biogenesis. Our studies, based on the use of pH-sensitive fluorescent reporter markers, suggest that RA promotes AUT acidification and maturation. By using competitive inhibitors and specific agonists, we demonstrated that this effect is not mediated by the classic Retinoic Acid Receptor (RAR) and Retinoid X Receptors (RXR). RA did not affect the protein expression levels of upstream regulators of autophagy, such as Beclin-1, phospho-mTOR, and phospho-Akt1, but induced redistribution of both endogenous cation-independent mannose-6-phosphate receptor CI-MPR and transiently transfected GFP and RFP full-length CI-MPR fusion proteins from the trans-Golgi region to acidified AUT structures. Those structures were found to be amphisomes (acidified AUTs) and not autophagolysosomes. The critical role of CI-MPR in AUT maturation was further demonstrated by siRNA-mediated silencing of endogenous CI-MPR. Transient CI-MPR knockdown resulted in remarkable accumulation of nonacidified AUTs, a process that could not be reversed with RA.These results suggest that RA induces AUT acidification and maturation by regulating CI-MPR subcellular location, a process critical in the cellular autophagic mechanism.Item FUNCTIONAL CHARACTERIZATION OF THE VIRAL FLICE INHIBITORY PROTEIN OF RHESUS MONKEY RHADINOVIRUS(2011) Ritthipichai, Krit; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus closely related to Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Here we found that RRV viral FLICE inhibitory protein (vFLIP) inhibits apoptosis. In HeLa cells with vFLIP expression, cleavage of poly [ADP-ribose] polymerase 1 (PARP-1) and activities of caspase 3, 7, and 9 were much lower than controls. RRV vFLIP was able to enhance cell survival under starved condition or apoptosis induction. After apoptosis induction, autophagosome formation was enhanced in cells with vFLIP expression and when autophagy was inhibited, these cells underwent apoptosis. Moreover, RRV latent infection of BJAB B-lymphoblastoid cells protects the cells against apoptosis. Knockdown of vFLIP expression in the RRV-infected BJAB cells with siRNA abolished the protection against apoptosis. These findings indicate that RRV vFLIP protects cells against apoptosis by enhancing autophagosome formation.Item Role and Regulation of Autophagy During Developmental Cell Death in Drosophila Melanogaster(2010) McPhee, Christina Kary; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Two prominent morphological forms of programmed cell death occur during development, apoptosis and autophagic cell death. Improper regulation of cell death can lead to a variety of diseases, including cancer. Autophagy is required for survival in response to starvation, but has also been associated with cell death. It is unclear how autophagy is regulated under specific cell contexts in multi-cellular organisms, and what may distinguish autophagy function during cell survival versus cell death. Autophagic cell death is characterized by cells that die in synchrony, with autophagic vacuoles in the cytoplasm, and phagocytosis of the dying cells is not observed. However, little is known about this form of cell death. Autophagic cell death is observed during mammalian development, during regression of the corpus luteum and involution of the mammary and prostate glands. Autophagic cell death is also observed during development of the fruitfly Drosophila melanogaster, during larval salivary gland cell death. Drosophila is an excellent genetic model system to study developmental cell death in vivo. Cells use two main catabolic processes to degrade and recycle cellular contents, the ubiquitin/proteasome system (UPS) and autophagy. Here I investigate the role of the UPS and autophagy in developmental cell death using Drosophila larval salivary glands as an in vivo model. Proteasome inhibitors are being used in anti-cancer therapies; however the cellular effects of proteasome inhibition have not been studied in vivo. Here I demonstrate that the UPS is impaired during developmental cell death in vivo. Taking a proteomics approach to identify proteins enriched in salivary glands during developmental cell death and in response to proteasome impairment, I identify several novel genes required for salivary gland cell death, including Cop9 signalsome subunit 6 and the engulfment receptor Draper. Here I show that the engulfment receptor Draper is required for salivary gland degradation. This is the first example of an engulfment factor that is autonomously required for self-clearance. Surprisingly, I find that Draper is cell-autonomously required for autophagy during cell death, but not for starvation-induced autophagy. Draper is the first factor to be identified that genetically distinguishes autophagy that is associated with cell death from cell survival.Item THE RELATIONSHIP BETWEEN AUTOPHAGY, CELL SURVIVAL AND CELL DEATH IN A MODEL OF NEURODEGENERATION AND DEVELOPMENT.(2009) Batlevi, Yakup; Pick, Leslie; Baehrecke, Eric H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The catabolic degradation of proteins is vital for the proper function and homeostasis of all cells. Autophagy is one of the major catabolic systems, and it is involved in processes that are as diverse as cell survival, cell death, immune reaction, cancer and neurodegeneration. Neurodegenerative diseases often have the pathology of protein accumulation in inclusions, but it is unclear whether these inclusions cause cell toxicity. Here I show that autophagy has protective functions in a model of a polyglutamine neurodegenerative disease in Drosophila. Inhibition of autophagy in this model enhances polyglutmine-induced degeneration, while activation of autophagy suppresses degeneration. Moreover, I observed similar protein aggregates in the larval salivary glands of a Drosophila dynein light chain mutant. This dynein light chain mutant is defective in autophagy, and their salivary glands fail to execute developmentally regulated programmed cell death. Ectopic activation of autophagy is sufficient to suppress the protein accumulation in dynein light chain mutant salivary glands. Both neurons and salivary glands are long-lived post-mitotic cells, and these cells are likely to have unique catabolic needs. Our data indicate that defects in catabolism are responsible for the neurodegenerative and salivary gland cell death defects that I observed, and could explain the association of autophagy with neurodegenerative diseases.Item Genetic regulation of autophagic cell death in Drosophila Melanogester(2008-11-20) Dutta, Sudeshna; Baehrecke, Eric H; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Apoptosis and autophagic cell death are the two most prominent morphological forms of programmed cell death that occur during animal development. While much is known about the mechanisms that regulate apoptosis, relatively little is known about autophagic cell death. The steroid hormone ecdysone coordinates multiple cellular processes during metamorphosis in Drosophila, including cell differentiation, morphogenesis and death. E93 is necessary and sufficient for larval tissue cell death during metamorphosis, including autophagic cell death of salivary glands. Here we characterize new mutant alleles of a dominant wing vein mutation Vein-off (Vno), and provide evidence that E93 and Vno are related. Our data also indicate that E93 functions in steroid regulation of both cell development and death during metamorphosis. E93 encodes a helix-turn-helix DNA binding motif and binds to specific regions of salivary gland polytene chromosomes. We have used genetic and genomic approaches to identify downstream targets of E93. We have identified numerous candidate E93 target genes using DNA microarrays, and have generated transgenic animals to identify downstream target genes of E93 by chromatin immune precipitation. We show that one putative E93 target gene, hippo (hpo), is required for salivary gland cell death. The Wts/Hpo tumor-suppressor pathway is a critical regulator of tissue growth in animals, but it is not clear how this signaling pathway controls cell growth. Our data indicate that salivary gland degradation requires genes in the Wts/Hpo pathway. Wts is required for cell growth arrest and autophagy in dying salivary glands, and regulates the degradation of this tissue in a PI3K-dependent manner.Item Identification of autophagic cell death and implications for therapy(2004-05-04) Alva, Ajjai Shivaram; Baehrecke, Eric H; Cell Biology & Molecular GeneticsAutophagy is an evolutionarily conserved mechanism of bulk protein and organelle degradation that requires the ATG class of genes. Although autophagy has been frequently observed in dying cells in several species, a causative role for autophagy in cell death has not been demonstrated. We show that inhibition of caspase-8 in mouse L929 fibroblast cells causes cell death with the morphology of autophagy. Autophagic cell death in L929 cells is dependent on ATG genes and involves the receptor interacting protein (RIP) and the activation of the MAP kinase kinase 7(MKK7) - Jun N-terminal kinase (JNK) - cJUN pathway. We also show that autophagy occurs in many primary human tumors including cancer of the breast, lung and pancreas. Our findings validate autophagic cell death and might explain the role of autophagy in development, viral infections, neurodegenerative diseases and cancer.