Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. ATG16L1 polymorphisms have been linked to the development of Crohn’s disease (CD) and phosphorylation of CD-associated ATG16L1 (caATG16L1) has been hypothesized to contribute to cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation. Moreover, we show that ULK1-mediated phosphorylation drives the destabilization of caATG16L1 in response to stress. Additionally, we found that phosphorylated ATG16L1 was specifically localized to the site of internalized bacteria indicating a role for ATG16L1 in the promotion of anti-bacterial autophagy. Lastly, we show that stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that ULK1-signalling to ATG16L1 is a double-edged sword, enhancing function of the wildtype ATG16L1, but promoting degradation of caATG16L1.

Cyclin-Dependent Kinase 11 (CDK11) is a serine/threonine kinase encoded at human locus 1p36.3 by two paralogous genes CDK11A and CDK11B. CDK11 has diverse roles in the regulation of transcription, splicing, apoptosis and mitosis. In proliferating cells, two predominant isoforms are expressed: CDK11p58 and CDK11p110. CDK11p110 is expressed throughout the cell cycle and regulates transcription and splicing. CDK11p58 is expressed at mitosis via IRES-dependent translation; it mediates mitotic progression and faithful chromosome segregation. Loss of Cdk11 in murine models causes early embryonic lethality, demonstrating that CDK11 is essential for normal development. Furthermore, dysregulated CDK11 expression is associated with numerous late-onset disease states, indicating its importance in adult life. In cancer, abnormal expression of CDK11 correlates with poor prognosis in a variety of tumours. Moreover, deletion of the chromosomal region 1p36.3, containing the CDK11 locus, is frequently observed in cancer and has recently been identified in a case of the development disorder, Cornelia de Lange Syndrome (CdLS). This thesis aimed to examine the functions of CDK11 and the impact of their dysregulation in cancer and developmental phenotypes. The initial aim was to investigate the novel role for CDK11 in regulating autophagy in cancer cells; CDK11 depletion causes a marked autophagy phenotype, with accumulation of autophagy protein LC3. I demonstrate that this CDK11-mediated autophagy occurs as a consequence of mitotic dysregulation. Subsequently, I examined the role of autophagy following aberrant mitosis and chromosome missegregation. I show that autophagy is important in the maintenance of aneuploid karyotypes, with loss of autophagy impairing the survival of aneuploid cell populations. I then investigated the effects of CDK11 in regulating cancer cell motility and determined that CDK11 depletion retards cancer cell migration. However, I was unable to identify any failure in cell adhesion or cell polarization to explain this migration phenotype. Subsequently, I interrogated the CDK11 interactome to further characterize the mechanisms through which CDK11 regulates both novel and established functions. This work indicated the involvement of the distinct CDK11 isoforms in pathways that have not previously been reported. This included the interaction of CDK11p110 with ribosomal and spliceosomal proteins during mitosis and the interaction of CDK11p58 with spliceosomal and proteosomal constituents also during mitosis. These findings may provide the foundation for further study. Finally I describe work undertaken to sequence the CDK11 locus in a cohort of CdLS patients, with no known causative genetic mutation, to investigate CDK11A/CDK11B as candidate disease-associated genes. Although no causative mutation in CDK11A or CDK11B was identifying, sequencing of this region indicated NCBI and UCSC genome assemblies of this locus were inaccurate due to the genomic duplication. This has been confirmed by others and corrected in the most recent genome assemblies.

La mitophagie, la dégradation sélective des mitochondries par autophagie, est impliquée dans l’élimination des mitochondries endommagées ou superflues et requiert des régulateurs et protéines spécifiques. Chez la levure, Atg32, localisée dans la membrane externe mitochondriale, interagit avec Atg8, et permet le recrutement des mitochondries et leur séquestration à l’intérieur des autophagosomes. Atg8 est conjuguée à de la phosphatidyléthanolamine et est ainsi ancrée aux membranes du phagophore et des autophagosomes. Chez la levure, plusieurs voies de synthèse de PE existent mais leur contribution dans l’autophagie et la mitophagie est inconnue. Dans le premier chapitre, nous avons étudié la contribution des différentes enzymes de synthèse de PE, dans l’induction de l’autophagie et la mitophagie et nous avons démontré que Psd1, la phosphatidylsérine décarboxylase mitochondriale, est impliquée dans la mitophagie seulement en condition de carence azotée alors que Psd2, localisée dans les membranes vacuolaires, endosomales et de l’appareil de Golgi, est nécessaire en phase stationnaire de croissance. Dans le second chapitre, la relation entre Atg32, la mitophagie et le protéasome a été étudiée. Nous avons démontré que l’activité du promoteur d’ATG32 et la quantité de protéine Atg32 exprimée sont inversement régulées. En phase stationnaire de croissance, l’inhibition du protéasome empêche la diminution de l’expression d’Atg32 et la mitophagie est stimulée. Nos données montrent ainsi que la quantité d’Atg32 est reliée à l’activité du protéasome et que cette protéine pourrait être ubiquitinylée. Dans le troisième chapitre, nous nous sommes intéressés au rôle potentiel de Dep1, un composant du complexe nucléaire Rpd3 d’histones déacétylases, dans la mitophagie. Dans nos conditions, Dep1 semble être mitochondriale et elle est impliquée dans la régulation de la mitophagie. BRMS1L (Breast Cancer Metastasis suppressor 1-like) est l’homologue de Dep1 chez les mammifères. Cette protéine possède un rôle anti-métastatique dans des lignées de cancer du sein. Nous avons trouvé que l’expression de BRMS1L augmente en présence de stimuli pro-mitophagie. / Mitophagy, the selective degradation of mitochondria by autophagy, is implicated in the clearance of superfluous or damaged mitochondria and requires specific proteins and regulators. In yeast, Atg32, an outer mitochondrial membrane protein, interacts with Atg8, promoting mitochondria recruitment to the phagophore and their sequestration within autophagosomes. Atg8 is anchored to the phagophore and autophagosome membranes thanks to phosphatidylethanolamine (PE). In yeast, several PE synthesis pathways have been characterized, but their contribution to autophagy and mitophagy is unknown. In the first chapter, we investigated the contribution of the different enzymes responsible for PE synthesis in autophagy and mitophagy and we demonstrated that Psd1, the mitochondrial phosphatidylserine decarboxylase, is involved in mitophagy induction only in nitrogen starvation, whereas Psd2, located in vacuole/Golgi apparatus/endosome membranes, is required preferentially for mitophagy induction in stationary phase of growth. In the second chapter, we were interested in the relationship between Atg32, mitophagy and the proteasome. We demonstrated that ATG32 promoter activity and protein expression are inversely regulated. During stationary phase of growth, proteasome inhibition abolishes the decrease in Atg32 expression and mitophagy is enhanced. Our data indicate that Atg32 protein is regulated by the proteasome activity and could be ubiquitinated. In the third chapter, we investigated the involvement of Dep1, a member of the nuclear Rpd3L histone deacetylase complex, in mitophagy. In our conditions, Dep1 seems to be located in mitochondria and is a novel effector of mitophagy both in nitrogen starvation and stationary phase of growth. BRMS1L (Breast Cancer Metastasis suppressor 1-like) is the mammalian homolog of Dep1 and has been described in breast cancer metastasis suppression. We found that BRMS1L protein expression increases upon pro-mitophagy stimuli.

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2) are related innate immune receptors responsible for initiating a response to bacterial infection. They belong to a class of receptors known as Pattern Recognition Receptors (PRRs), which are germline encoded immune receptors that mediate various innate immune responses. These receptors recognize conserved microbial motifs known as Pathogen-Associated Molecular Patterns (PAMPs). The PRR-PAMP paradigm forms the bedrock of how innate immunity is understood today. As two of the first intracellular PRRs discovered, NOD1 and NOD2 came to define an entire subclass of PRRs, the NOD-like receptors (NLRs). PRRs relay their signals through protein:protein interaction motifs that typically adopt a characteristic Death Domain (DD) fold. NOD1 and NOD2 signal through their respective CAspase Recruitment Domains (CARDs), which are part of a DD subfamily. The CARDs of NOD1 and NOD2 interact with multiple downstream effectors and are thus situated at a key point for regulation and coordination of NOD1 and NOD2 signaling. To better understand this regulation, I structurally and functionally characterized interactions made by the CARDs of NOD1 and NOD2. Receptor Interacting Protein kinase 2 (RIP2) is an effector of both NOD1 and NOD2 that activates the NF-ΚB pathway to elicit an inflammatory response. I discovered a new binding interaction between the CARDs of NOD1 and NOD2 and ubiquitin. Furthermore, I elucidated a role for this interaction by showing that ubiquitin binds NOD1 and NOD2 CARDs competitively with the CARD of RIP2. Through biophysical and biochemical investigation, I identified mutants of NOD1 CARD that did not bind ubiquitin and were thus insensitive to its competitive effect on RIP2 binding. Utilizing this mutant in functional studies defined ubiquitin as a negative regulator of NOD1 signaling. Characterizing NOD1 allowed rational design of mutations that uncovered a similar role for ubiquitin in the NOD2 pathway. This introduces the potential for broader application of these findings in other DD-mediated pathways. NOD1 and NOD2 also bind the autophagy protein ATG16L. I investigated the molecular mechanisms of this interaction and found that NOD1 and NOD2 bind ATG16L through their CARDs. I also found that the domain on ATG16L responsible for binding NOD1 and NOD2 is the C-terminal WD40 Β-propeller. Furthermore, the CARD:Β-propeller interaction is sufficient to mediate interaction between NOD1 or NOD2 and ATG16L. The finding that the ATG16L Β-propeller also binds ubiquitin leaves open the possibility that ubiquitin regulates pathway selection by NOD1 and NOD2. Together, these studies advance our understanding of NOD1 and NOD2 signaling and lay the groundwork for further mechanistic investigations into coordination of inflammatory and autophagic signaling pathways by the immune system in general.

A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.

The amnioserosa (AS) is an extraembryonic tissue that undergoes programmed cell death (PCD) during the normal course of Drosophila embryogenesis. AS degeneration involves morphological evidence of autophagy as well as caspase activation, but the relationship between these two processes is not well defined. While the bulk of the AS tissue dies at the conclusion of the morphogenetic process of dorsal closure (DC), approximately 10% of AS cells are actively extruded from the epithelium during DC. Using live imaging confocal microscopy and various fluorescent protein sensors, I have been able to observe caspase activation as well as autophagy upregulation in the context of epithelial extrusion events as well as overall AS degeneration. The data show that epithelial extrusion events are caspase-dependent but are also associated with localized onset of autophagy. Furthermore, extensive characterization of loss of function mutants of the key Drosophila regulator Atg1 kinase indicates that autophagy is not required for the normal degeneration of AS, contrary to earlier studies. This thesis also introduces new relationships between caspase activation and autophagic cell death. In addition, new data suggest that the InR/TOR and EGFR/Ras/MAPK signaling pathways interact with the pro-apoptotic protein Head involution defective (Hid) and Atg1 kinase to regulate the progression of programmed cell death in the AS.

A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.

Microalgae are an important potential feedstock for biodiesel production. Understanding the physiology of lipid biosynthesis in microalgae is pivotal to microalgal aquaculture management. A freshwater green microalga strain, UTEX 2219-4, was isolated from UTEX 2219 which was reported containing two strains. Its ITS sequences are closely related to those in the family of Scenedesmaceae in the GenBank. Nitrogen starvation, salt stress and osmotic stress greatly enhanced lipid biosynthesis in this strain, while combination of nitrogen deficiency and osmotic stress had the most dramatic effect. Chloroplast was condensed and photosynthesis efficiency declined about 50% after 3 days of nitrogen starvation. Chlorophyll degradation followed the same trend but was more severe than the reduction of photosynthesis efficiency. Oil body formation was not observed in the cells kept in the dark under nitrogen starvation, suggesting photosynthesis rather than autophagy is the major player in oil body formation. Under non-saturation levels of light intensities coupled with nitrogen starvation, the oil body formation under 150 £gmol/m2s light intensity was more efficient than that under 75 £gmol/m2s. DCMU blocked photosynthesis as well as oil body formation, supporting that the energy for oil body formation was mostly from photosynthesis rather than autophagy during nitrogen starvation.

Autophagy is an important cellular process in maintenance of protein homeostasis. Emerging evidence indicates differential roles of autophagy in cellular function under different pathophysiologic conditions. In some circumstance, autophagy results in cell survival, wheras in other situations it results in cell death. Endotoxin affects neurons in the nucleus tractus solitarii (NTS), baroreceptor afferent terminal site in the brain stem, resulting in cardiovascular depression. The aim of this study was to examine whether modulation of autophagic activity in NTS and other brain regions subserving cardiovascular regulation are associated with cardiovascular depression during experimental endotoxemia. Adult male Sprague-Dawley rats received continuously intraperitoneal infusion via osmotic minipump of lipopolysaccharide (LPS, 2.5 mg/kg/day) or normal saline (NS). Body weight (BW) and systolic blood pressure (SBP) were recorded in animals on days 1, 2, 3, 5, 7, 10, and 14 after LPS treatment. Western bolotting was used to assess the expression of autophagic activity marker, microtubule-associated protein 1 light chain 3 (LC3). Rapamycin (0.55 mg/Kg/day), chemical reported to activate autophagy, was infused continuously into the lateral ventricle of the endotoxemic rats for 7 days via osmotic minipump. Both BW and SBP of rats were decreased in the initial 5 days, followed by a gradual return to baseline after LPS treatment. There was a trend in the decrease in autophagic activity (using the ratio of LC3-¢º/LC3-I as an experimental index) at NTS. However, there is no apparent association between the change in autophagic activity at NTS and the LPS-induced cardiovascular depression. In addition, there was no obvious change in the autophagic activity at RVLM, hypothalamus and hippocampus. Intracranial infusion of rapamycin, a mTOR inhibitor that maintains cellular autophagic activity, resulted in a further enhancement of cardiovascular depression induced by LPS. These results suggest that continuously intraperitoneal infusion via osmotic minipump of LPS result in decreases of body weight and systolic blood pressure. However, the present study provides no direct evidence to support for a cause-and-effect role of autophage at NTS, RVLM, hypothalamus as well as hippocampus in the LPS-induced cardiovascular depression during experimental endotoxemia.

Hepatitis B virus (HBV) is a significant global threat to human health due to its ability to cause chronic infections that can lead to hepatocellular carcinoma (HCC). While the process by which HBV increases the risk of HCC is unclear, evidence suggests that the hepatitis B X protein (HBx) may be a contributing factor. Cellular senescence is an important barrier to tumorigenesis, blocking the proliferation of cells that harbor excessive DNA damage or contain activated oncogenes. Autophagy is a non-proteasomal degradative pathway used by cells to recycle cytoplasmic contents under periods of nutrient starvation. This pathway is induced in response to a wide range of cellular stress factors, and has also been characterized as an effector mechanism for the establishment of cellular senescence. In this study, retroviral transduction of HepG2 cells with HBx resulted in the induction of cellular senescence and autophagy. The mechanism by which HBx can induce senescence is unclear. However, an increase in the accumulation of DNA damage was observed. HBx did not modulate the levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, or Mcl-1, which can inhibit autophagy through interactions with the autophagy regulator Beclin 1. As well, the activity and phosphorylation status of JNK/SAPK, an inducer of autophagy via Bcl-2 phosphorylation, was unchanged. These results suggest that senescence may act as a barrier to HBx-induced oncogenesis, and may offer some explanation as to why HBx does not function as a more potent oncogene. Also, we propose that HBx modulates autophagy through a mechanism other than Bcl-2 phosphorylation or expression over the time course of this study.