Enterovirus 71 (EV71) belongs to the Enterovirus genus of the family Picornaviridae and is the major causative agent of hand, foot and mouth disease (HFMD). Although clinical manifestations of HFMD are usually mild and self-limiting, severe HFMD patients suffer from a diverse array of neurological diseases and sometimes these diseases are fatal. HFMD usually occurs in young children and gradually becomes a new threaten in Asia. Unfortunately, effective EV71 vaccine is not available to date and alternative treatments are still in debate. This is partially due to the lack of understanding of EV71 pathogenesis and host immune responses against EV71. Natural killer (NK) cells are key effector cells in host antiviral activities by directly killing viral-infected cells and producing cytokines and chemokines, especially in early phase of viral infection. After enteroviruses infection, NK cells were one of the most abundant cell types in the inflammatory infiltrate, and appeared to limit both enteroviruses replication and virus-induced disease in experimental mice model. However, role of human NK cells during EV71 infection, especially the direct interaction between EV71 and human NK cells, was not studied extensively. Clinical observation manifested that patients with severe EV71 infection have marked diminished NK cells in peripheral blood. Therefore we hypothesized that EV71 might directly target human NK cells as one of its immunoevasion strategies. Here, we demonstrated for the first time that fresh primary human NK cells were susceptible to EV71 infection. By flow cytometry and florescence microscope, EV71 capsid protein VP1 was able to be detected in viral-infected NK cells as soon as 6 hours after infection and peaked at 24 hour after infection. In the same time, EV71 viral RNA was detected by quantitative RT-PCR and the viral copies increased from 6 hour onwards to peak at 12 hours after infection. We further demonstrated the infectious entry of EV71 in human NK cells was depended on clathrin-mediated endocytosis. Next, we illustrated that EV71 infection could trigger NK cells apoptosis as evidenced by increased Annexin V+, PI+, and activated caspase 3+ cells in EV71-treated NK cells. We further proved that the cytotoxicity of NK cells was inhibited by EV71 infection and this inhibition might not be related with down-regulation of NKp46, but may be related to the increased apoptosis. In conclusion, our data suggested that EV71 might directly target and kill NK cells as a strategy to evade human innate immunity, which might facilitate virus replication, transmission and then contribute to viral-related pathogenesis. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

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The molecular mechanisms of apoptosis are evolutionarily-conserved with caspases being the chief executioners of this process. Though key regulators of apoptosis, including caspases, inhibitor of apoptosis (IAP) proteins, and IAP antagonists exist in both mammals and flies, there are reportedly mechanistic differences in the way the apoptotic process is executed. One of the differences pertains to the importance of mitochondrial permeabilization for caspase activation. Herein, we demonstrate that dOmi, a Drosophila homologue of the serine protease Omi/HtrA2, is a developmentallyregulated mitochondrial intermembrane space protein that undergoes processive cleavage in situ to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the pro-apoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. Thus, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis, but also results in the release of a bona fide pro-apoptotic protein. DIAP1, in addition to being regulated by dOmi, is also regulated by RINGdependent autoubiquitination and by the N-end rule degradation (NERD) pathway. Despite decreasing the cellular levels of DIAP1, the NERD pathway enhances its antiapoptotic function through an unknown mechanism(s). Herein, we show for the first time that the NERD pathway facilitates trans-ubiquitination and degradation of IAP antagonist bound to DIAP1. Indeed, Grim is trans-ubiquitinated in an Ubr1-dependent manner and requires its interaction specifically with the BIR1 domain of DIAP1. These results demonstrate that similar to RING domain-dependent ubiquitination, the NERD pathway regulates not only the levels of DIAP1, but also of the levels of IAP antagonists bound to it.

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Several genes involved in the regulation of apoptosis can influence longevity. Although observations in several different systems imply that apoptosis and aging are closely linked, the relationship between the two remains largely unknown. In this study, Drosophila melanogaster was used as a model organism to explore the relationship between aging and apoptosis regulation. Apoptosis was investigated using two apoptotic hallmarks: caspase activity and DNA fragmentation. The results showed that apoptosis occured in adult flies at all ages and the changes in apoptosis associated with aging were linked to physiological age and were tissue-specific. During normal fly aging, apoptosis increased gradually within the muscle and was activated in the fat of old flies. However, neither of the two apoptotic signs were shown in the nervous system. The analysis of the apoptotic response to starvation and oxidative stress suggested that the increased apoptosis in muscle resulted from the accumulation of oxidative damage associated with aging. Once the presence of apoptosis during Drosophila aging was confirmed, the expression of anti-apoptotic genes was manipulated in specific tissues to examine the impact of a localized alternation of apoptosis on aging. The overexpression of anti-apoptotic genes in muscle extended Drosophila mean and maximum life span up to 99% and 65%, respectively. This extension was mediated by apoptosis inhibition using the detection of caspase activity and DNA fragmentation. In addition, the long-lived animals exhibited increased resistance to oxidative stress and preserved flight ability. Overall this study establishes that muscle apoptosis limits life span and participates in sarcopenia. This finding may have applications in the development of interventions to improve the life quality of elderly human. / Thesis (Ph.D, Biology) -- Queen's University, 2008-10-27 00:24:02.204

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a risk factor for a variety of atherosclerotic disorders including coronary heart disease. In the current study, the investigators report that incubation of cultured human umbilical vein endothelial cells (HUVECs) with high concentrations of apolipoprotein(a)(apo(a)/Lp(a)) induces apoptosis and endothelial dysfunction in a dose dependent manner. Apo(a), the component of Lp(a) mediates these effects by inducing externalization of Annexin V, DNA condensation and fragmentation which are the hallmarks of death by apoptosis. The pathway of apo(a)-induced apoptosis is associated with overexpression of Bax, caspase-9, p53 phosphorylation, decreased in Bcl-2 expression and activation of caspase-3. Taken together, the data suggest that elevated concentration of apo(a) induces apoptosis in endothelial cells probably by activating the intrinsic pathway. The data also showed that apo(a) induces increased expression of the growth arrest protein (Gas1), which has been known to induce apoptosis and growth arrest in vitro. In addition the data showed that elevated apo(a)/Lp(a) attenuates endothelial nitric oxide (eNOS) activity and endothelin-1 (ET-1) in a dose and time-dependent manner, particularly with small apo(a) isoforms. In summary, the authors proposed a new signaling pathway by which apo(a)/Lp(a) induce apoptosis and this finding could help explain how apo(a)/Lp(a) mediate atherosclerosis related diseases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-06-20 13:59:48.473

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Loss of germ cells that entered meiosis at different developmental stages was compared. Mice were injected with BrdU at 13.3, 14.3 or 15.3 days post coitum (dpc) and sacrificed either 3 days after BrdU injection or 4 days post partum (dpp). BrdU-labeled germ cells were detected in ovarian sections through double immunofluorescent staining for BrdU and either GCNA-1 or MVH as a germ cell marker. The results show that the loss of germ cells that entered meiosis at 13.3 or 15.3 dpc was excessive compared to the loss of total germ cells. Such preferential elimination was not found for germ cells that entered meiosis at 14.3 dpc. We conclude that oocyte loss during meiotic prophase is influenced by the timing of meiotic onset. / The mechanism of germ cell loss during ovarian development was tested by the presence of markers for apoptosis. Mouse ovaries were isolated at 12.5 dpc, 18.5 dpc and 2 dpp and cultured with doxorubicin (DXR) to induce cell death. Ovarian histological sections were double immunofluorescent stained for GCNA-1 and cleaved caspase-3 or PARP-1. The results suggest that caspase-3 is not activated in germ cells throughout ovarian development whereas PARP-1 is activated in germ cells at 12.5 dpc and 2 dpp but not at 18.5 dpc. Thus, no evidence has yet been provided to support the hypothesis that oocyte death during the meiotic prophase is mediated by apooptosis.

The mechanisms by which Angiopoietin-1 (Ang-1) modulates the survival of human endothelial cells were investigated. Ang-1 inhibited both TNFalpha-induced and serum deprivation-evoked apoptosis, an effect which was associated with attenuation of caspase activation, inhibition of Smac release from the mitochondria, up-regulation of Survivin-1 expression (IAPs member) and a significant activation of the pro-survival PI-3 kinase/AKT pathway. In addition, Ang-1 activated, in a time-dependent fashion, both the anti-apoptotic ERK1/2 and pro-apoptotic p38 MAP kinases. Ang-1-evoked ERK1/2 activation was mediated in part through the PI-3 kinase pathway, whereas both, the PI-3 kinase and ERK1/2 attenuated p38 MAP kinase activation. / We conclude that Ang-1 promotes endothelial cell survival through several pathways including the PI-3 kinase/AKT and ERK1/2 pathways, up-regulation of Survivin-1 as well as inhibition of Smac release and caspase activity. The preferential activation of these anti-apoptotic effects, as opposed to the activation of pro-apoptotic p38 MAP kinase, results in a net survival response.