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Expression analysis of DEK protein in normal and autophagy-deficient liverJanuary 2021 (has links)
archives@tulane.edu / 1 / Niani Bailey
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Autophagy During and Following Dynamic Exercise in Young Adults: Influence of Exercise IntensityCôté, Melissa D. 24 January 2022 (has links)
Autophagy plays a critical role in the cell’s response to exercise-induced cell stress. Exercise intensity and elevations in tissue temperature are known to modulate autophagic activation, however, the relationship is still unclear. Thus, we investigated the effect of increasing intensities of exercise, as well as evaluated high intensity exercise performed in a hot environment on autophagy and associated cellular stress pathways (the heat shock response [HSR], apoptosis, and the acute inflammatory response). To evaluate the hypothesis that greater autophagic activation occurs with increasing exercise intensity, which is be exacerbated by exercise performed in the heat, 9 young men (mean [SD]; 23 [2] years) cycled for 30 minutes at 40, 55 and 70% of VO2max at an ambient temperature of 25°C and at 70% of VO2max at an ambient temperature of 40°C. Peripheral blood mononuclear cells were harvested before exercise, as well as at 0, 3 and 6 hours post-exercise. Changes in protein content were analyzed via Western blotting. With each increase in exercise intensity, there was a concomitant increase in mean body temperature and heart rate, both of which were further increased in the heated condition. A significant increase (p≤0.05) in LC3-II and LC3-II/I protein expression was observed immediately post high-, but not low- or moderate-intensity exercise. Additionally, elevations in LC3-II immediately following high intensity exercise in a hot environment were significantly greater than those in the non-heat stress environment. Taken together, this may indicate a threshold volume of exercise- and heat-induced cellular stress required to stimulate elevations in autophagic activity.
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Biochemical and pharmacological characterization of the Atg8 conjugation system in toxoplasma gondiiVarberg, Joseph M. 28 June 2017 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii is an important human pathogen that infects millions of people worldwide and causing severe and potentially lethal disease in immunocompromised individuals. Recently, a homologue for the autophagy protein Atg8 (TgAtg8) was identified in Toxoplasma that is required for both canonical and noncanonical processes essential for parasite viability. Importantly, TgAtg8 functionality requires its conjugation to phosphatidylethanolamine through the activity of the Atg8 conjugation system. In this thesis, we characterized the proteins that interact with TgAtg8 and TgAtg3, a component of the Atg8 conjugation system, to further define their functions in Toxoplasma and identify opportunities for targeted inhibition of Atg8-related processes. We previously identified that TgAtg8 is acetylated at lysine 23 (K23) and assessed the role of this modification in this thesis. Using mutagenesis, we showed that K23 acetylation did not modulate the interaction with TgAtg3, but appeared to promote TgAtg8 protein stability. Additionally, endogenous mutation of K23 to the nonacetylatable amino acid arginine resulted in severe impairment of parasite replication and spontaneous differentiation into bradyzoites. To gain insight into the role of TgAtg8 in Toxoplasma biology, we next characterized TgAtg8 and TgAtg3 interacting proteins using affinity purification and mass spectrometry. We identified a novel group of interacting proteins that are unique to Toxoplasma, including the dynamin-related protein DrpC. Functional characterization of DrpC identified a potential role of TgAtg8 in trafficking of membrane from the Golgi to the nascent daughter parasites during replication. Lastly, we examined a group of small molecules recently identified as Atg3-Atg8 inhibitors in Plasmodium falciparum and assessed their activity against Toxoplasma. Although the compounds effectively inhibited Toxoplasma replication, they did so through novel mechanisms of action unrelated to the disruption of the TgAtg3-Atg8 interaction. Together, this work provides insight into the function of the Atg8 conjugation system in Toxoplasma that will help guide the future development of novel therapeutics targeting Atg8-related processes.
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RNA editing and autophagy in Drosophila melanogasterParo, Simona January 2012 (has links)
Post-transcriptional regulation of gene expression involves a diverse set of mechanisms such as RNA splicing, RNA localization, and RNA turn-over. Adenosine to Inosine (A-to-I) RNA editing is an additional post-transcriptional regulatory mechanism. Temporally, it occurs after transcription and before RNA splicing and has been shown in some instances to possibly modulate alternative splicing events. This is the case for example, with the pre-mRNA encoding the GluR- 2 subunit of AMPA receptor, a glutamate-activated ion channel. ADAR (Adenosine deaminase acting on RNA) proteins bind to double-stranded regions in pre-messenger RNAs. They deaminate specific adenosines, generating inosines; if the editing event occurs within the coding region, inosine is then interpreted as guanosine by the ribosomal translational machinery, changing codon meaning. These editing events can increase the repertoire of translated proteins, generating molecular diversity and modifying protein function. In mammals there are four ADAR genes: ADAR1, ADAR2, ADAR3 and TENR. ADAR3 and TENR are enzymatically inactive. All the proteins have two types of functional domains: (i) the catalytic deaminase domain at the carboxyl-terminus and (ii) the double stranded RNA binding domains, dsRBDs, at the amino terminus. ADAR1 and ADAR2 differ significantly at the amino terminus, by the number of the dsRNA binding domains (three and two dsRBDs for ADAR1 and ADAR2 protein, respectively). The differences observed between ADAR1 and ADAR2 are likely to reflect the different repertoires of substrates edited by these two enzymes. Data concerning the conservation of Adar genes throughout evolution suggest that Drosophila melanogaster has a unique Adar gene which is a true ortholog of human ADAR2 rather than an invertebrate gene ancestral for both vertebrate genes. Flies that are null mutants for Adar (Adar5G1 mutants) display profound behavioral and locomotive deficits. Impairment in motor activity of the mutants is succeeded by age-dependent neurodegeneration, characterized by swelling within the Adar-null mutant fly brain. The initial focus of my thesis was to elucidate what causes Adar mutant phenotypes or, whether it is possible, to suppress them. I took advantage of Drosophila genetics to establish a forward genetic screen for suppressors of reduced Adar5G1 viability which is approximately 20-30% in comparison to control flies at eclosion. The results from an interaction screen on Chromosome 2L were further confirmed using Exelixis P-element insertion lines. The screen revealed that decreasing Tor (Target of rapamycin) expression suppresses Adar mutant phenotypes. TOR plays a role in maintaining cellular homeostasis by balancing the metabolic processes. It controls anabolic events by phosphorylating eukaryotic translation initiation factor 4E-binding protein (4E-BP) and p70 S6 kinase (S6K) and inducing cap-mediated translation. However, different types of stress, signals or increased demand in catabolic processes, converge to reduce TOR enzymatic activity. This results in long-lived proteins and organelles being engulfed in double-membrane vesicles and degraded; this bulk degradation process is called (macro)autophagy. The second aim of my thesis was to clarify which pathway, downstream to TOR, was responsible for the suppression of Adar-null phenotypes. I mimicked the effect of reduced Tor expression by manipulating genetically the cap-dependent translation and the autophagy pathways. Interestingly, boosting the expression of Atg (autophagy specific genes) genes, such as, Atg1 and Atg5, thereby increasing the activation rate of the autophagy pathway, suppresses Adar5G1 phenotypes. Finally, I found that Adar5G1 mutant flies have an increased level of autophagy that is observable from the larval stage. I investigated possible stresses affecting our mutants; Adar-mutant larval fat cells show ER stress triggering an unfolded protein response as indicated by expression of XbpI-eGFP reporter. Thus, ER stress might induce increased autophagy and it can lead to locomotive impairments and neurodegeneration in Adar-null mutants. These results suggest a function for the Adar gene in regulating cellular stress.
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Study of the roles of autophagy in human follicular and diffuse large B-cell lymphomaMcCarthy, Áine Claire January 2014 (has links)
Autophagy, a cellular self-degradation process, plays important roles in cancer development and progression. Autophagy can be inhibited by the anti-apoptotic protein BCL-2 which binds and sequesters the autophagy essential protein Beclin-1, therefore preventing autophagy induction. It is currently unclear whether BCL-2 inhibits autophagy as well as apoptosis in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) which frequently express BCL-2 at high levels. This study aimed to determine (1) the role of BCL-2 in the basal level autophagy status and autophagy flux in primary FL and DLBCL samples, and lymphoma cell lines at both the gene and protein levels; (2) whether aberrant autophagy activity in these lymphoma patients is associated with clinical outcome. We initially found that the BCL-2 inhibitor ABT-737 concurrently induced autophagy and apoptosis in BCL-2HIGH DLBCL cell lines. Blocking autophagy degradation with chloroquine sensitised BCL-2HIGH cells to ABT-737-induced cell death, indicating that acquired autophagy acts as a cytoprotective mechanism in these cells. Expression levels of autophagy-related genes were analyzed by qRT-PCR. The BCL-2HIGH cell line Su-DHL4 showed up-regulation of more autophagy machinery genes at both the basal level and following starvation-induced autophagy compared with the BCL-2LOW cell line. These results suggest that inhibition of BCL-2 can induce cytoprotective autophagy, but overexpression of BCL-2 alone may increase basal level autophagy by inhibiting apoptosis. The expression levels of autophagy-related genes were examined in purified primary FL and DLBCL B cells or un-purified whole FL and DLBCL tumour tissue biopsies and compared with non-malignant reactive lymph nodes. A number of autophagy machinery genes were significantly up-regulated in malignant samples. In particular, more autophagy machinery genes showed significantly increased expression in both purified and un-purified FL samples, indicating that despite frequently overexpressing BCL-2, FL appears to have increased basal level autophagy activity. 4 Expression of the key autophagy proteins p62, Beclin-1, LC3 and BCL-2 were determined in FL and DLBCL using tissue microarrays and immunohistochemistry. Both p62 and LC3, substrates of autophagic degradation, served as markers for autophagy activity. Significantly decreased expression of p62 and LC3, indicating active autophagy, was observed in FL samples (n=117). DLBCL samples (n=109) showed a heterogeneous expression pattern of these four proteins. We identified p62 as an independent prognostic biomarker in DLBCL with decreased expression predicting shorter overall, disease specific and progression-free survival. DLBCL patients with lower p62 or LC3 expression and higher levels of BCL-2, i.e. active autophagy and inhibited apoptosis, had the worst prognosis. Beclin-1 expression was significantly reduced in both FL and DLBCL, where lower levels were significantly associated with shorter overall and disease-specific survival. In summary, this study demonstrates that FL, characterised by overexpression of BCL-2, shows increased autophagy activity, indicating that BCL-2 may not inhibit basal level autophagy in this indolent lymphoma. High levels of BCL-2 and active autophagy did not affect the clinical outcome of FL, but significantly shortened the survival rates of DLBCL patients. Our data propose that active autophagy could be used as a biomarker for DLBCL prognosis, but not FL.
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Deregulation Of Selective Autophagy And Sirtuin 3 Expression In Lung Aging And Pulmonary FibrosisJanuary 2016 (has links)
Meredith L Sosulski
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The Role of P62 in Autophagy of Salmonella Enterica Serovar TyphimuriumZheng, Yiyu Terrence 03 January 2011 (has links)
Autophagy, a cellular degradative pathway, plays a key role in protecting the cytosol from bacterial colonization, but the mechanisms of bacterial recognition by this pathway are unclear. Autophagy is also known to degrade cargo tagged by ubiquitinated proteins, including aggregates of misfolded proteins, and peroxisomes. Autophagy of ubiquitinated cargo requires p62, an adaptor protein with multiple protein-protein interaction domains. Previous studies demonstrated that the intracellular bacterial pathogen S. typhimurium is targeted by autophagy during infection of host cells. Here I show that p62 is recruited to S. typhimurium targeted by autophagy, and that the recruitment of p62 is associated with ubiquitinated proteins localized to the bacteria. Expression of p62 is required for efficient autophagy of bacteria, and restriction of their intracellular replication. My study demonstrates that the surveillance of misfolded proteins and bacteria occurs via a conserved pathway and reveals a novel function of p62 in innate immunity.
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The Role of P62 in Autophagy of Salmonella Enterica Serovar TyphimuriumZheng, Yiyu Terrence 03 January 2011 (has links)
Autophagy, a cellular degradative pathway, plays a key role in protecting the cytosol from bacterial colonization, but the mechanisms of bacterial recognition by this pathway are unclear. Autophagy is also known to degrade cargo tagged by ubiquitinated proteins, including aggregates of misfolded proteins, and peroxisomes. Autophagy of ubiquitinated cargo requires p62, an adaptor protein with multiple protein-protein interaction domains. Previous studies demonstrated that the intracellular bacterial pathogen S. typhimurium is targeted by autophagy during infection of host cells. Here I show that p62 is recruited to S. typhimurium targeted by autophagy, and that the recruitment of p62 is associated with ubiquitinated proteins localized to the bacteria. Expression of p62 is required for efficient autophagy of bacteria, and restriction of their intracellular replication. My study demonstrates that the surveillance of misfolded proteins and bacteria occurs via a conserved pathway and reveals a novel function of p62 in innate immunity.
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KSHV vGPCR: A VIRAL ONCOPROTEIN THAT TRIGGERS AUTOPHAGY AND CELLULAR SENESCENCECyr, David P 16 June 2011 (has links)
Autophagy (literally to ‘self-eat’) is an intracellular, catabolic mechanism to
degrade and recycle cytoplasmic contents in response to metabolic, oxidative, and
genotoxic stresses. Autophagy plays an important role in cellular homeostasis, and
dysfunctional autophagic activity has been implicated in an array of human diseases.
Importantly, autophagy has recently been identified to function in host defence against
intracellular pathogens, including viruses. For this reason, many viruses have evolved
strategies to subvert or exploit autophagy and block its antiviral effects. Kaposi’s
sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma
(KS), an AIDS-related cancer of the endothelium. KSHV gene products have evolved to
support viral replication and evade immune surveillance. Some of these same gene
products impact KS tumourigenesis, but the precise mechanisms have yet to be
elucidated. Furthermore, the impact of autophagy on KSHV replication and KS
tumourigenesis remains unexplored.
The KSHV viral G-protein-coupled receptor (vGPCR) is a constitutively active
signalling molecule that stimulates a number of host regulatory pathways that would be
expected to impact autophagy, including PI3K/Akt/mTOR and JNK. Moreover, vGPCR
is expressed during lytic replication when the antiviral autophagic response may threaten
virion production. Here, vGPCR activity has been definitively shown to trigger
autophagy in endothelial cells using immunoblot analysis, fluorescent reporter proteins,
and transmission electron microscopy. Furthermore, preliminary data suggest that this
stimulatory effect is evoked through JNK activation. Taken together, these findings
indicate that vGPCR likely elicits autophagic responses during KSHV lytic replication.
Recently, autophagy has been recognized as a molecular barrier to
tumourigenesis, influencing cell survival, cell death, or a form of cell cycle arrest called
oncogene-induced senescence (OIS). Remarkably, like many host oncogenes, ectopic
expression of vGPCR triggers OIS in endothelial cells. This response is dependent on
vGPCR signalling activity, as an inactive form of vGPCR (R143A) fails to trigger OIS.
Furthermore, vGPCR OIS is atypical in that it does not involve DNA damage responses
(DDRs). Together, these autophagic and senescence responses to ectopic vGPCR
expression illustrate the potency of its oncogenic potential.
The significance of vGPCR-induced autophagy and senescence during KSHV
replication and KS development is presently unclear. I speculate that autophagy
represents a hurdle that the virus must overcome in vivo. In my working model, potent
vGPCR oncogenic signalling activity sets off the alarm, eliciting autophagic responses. It
seems likely that additional lytic viral gene products may serve to undermine these
autophagic responses and permit viral replication and dissemination in vivo.
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THE ROLE OF A DEFECT IN THE CDP-ETHANOLAMINE PATHWAY IN AUTOPHAGYPereira, Leanne 11 December 2012 (has links)
Autophagy is the process that degrades cytosolic constituents into products that can be recycled for use in energy generation and other processes. The endoplasmic reticulum is responsible for the bulk synthesis of the phospholipid phosphatidylethanolamine (PE) via the CDP-ethanolamine pathway. The aim of the present study was to determine the role of PE synthesis and the CDP-ethanolamine pathway in autophagy. This objective was examined through the use of two novel models deficient in Pcyt2, a gene that encodes the rate-limiting enzyme CTP-ethanolamine cytidyltransferase (ET) in the CDP-ethanolamine pathway. PCYT2 knockdown in human fibroblast cells did not respond normally to starvation conditions that activate autophagy. Similarly, Pcyt2 knockout in mice showed differences in autophagy induction in/between muscle, liver, and adipose tissue based on metabolic state (fasting/feeding). Pcyt2 knockout mice display evidence of metabolic syndrome at an older age and experiments with these mice determined that there was an effect of age (healthy young mice versus obese older mice) on autophagy induction. It was concluded based on in vitro and in vivo studies that autophagy induction is affected by impairment to the CDP-ethanolamine pathway and subsequent PE synthesis.
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