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Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1979. / Bibliography: leaves 147-156. / Microfiche. / x, 156 leaves ill. (some col.) 29 cm

Thesis (M.S. - Soil, Water and Environmental Science)--University of Arizona. / Includes bibliographical references (leaves [64]-69).

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Enteroviruses affect a broad segment of the population throughout the world and have been suspected to play a major role in waterborne disease for quite some time. The presence of these viruses in drinking water supplies constitutes a major health risk to the population because of their low infectious dose. The monitoring and study of these viruses in the environment have been limited by the current standard detection methodologies. Nucleic acid probe hybridization is a new and effective approach for the study and detection of these viruses in the environment. An important step in the detection of viruses in concentrated water samples by nucleic acid probes is the isolation of the viral genome from the water sample for hybridization. Previously, a series of time consuming organic extract ions was used to isolate viral RNA. This study reports the development of an alternative method for the isolation and preservation of viral RNA in environmental samples. Briefly, the sample is heated in the presence of an RNase inhibitor, and then applied to a hybridization membrane. This procedure has greatly reduced the time and difficulty of the assay while maintaining sensitivity and increasing consistency. This study reports the development and modification of a nonradioactive labeling system for the detection of viruses in water. Nonradioactive labels such as biotin offer several advantages over radioactive labels including unlimited shelf life, reduced cost and time of assay, and elimination of the radiation hazard. However, radioactive labels are generally the more sensitive method of detection. By combining direct and indirect labeling strategies, the sensitivity of this nonradioactive assay has been increased ten-fold. This assay can detect as little as 100 plaque forming units of poliovirus, only one order of magnitude less sensitive than radiolabeled probes. This assay is also ten-fold less sensitive than radiolabeled probes for the detection of enteroviruses in water samples. Nonradioactive probes offer a safe, inexpensive alternative to radiolabeled probes and tissue culture for the detection of viruses in the environment when ultrasensitivity is not required.

近年来肠病毒71亚型（EV71）的大规模流行已成为全世界特别是亚太地区的一个严重的公共卫生问题。EV71感染可以引起腹泻，皮疹，手足口病等等一些自愈性疾病。然而在部分儿童患者中，EV71可能导致严重的神经性疾病。目前，关于EV71感染后宿主细胞的反应机制的报导比较少。在本次研究中，我们运用蛋白组学方法对EV71感染后的人横纹肌瘤细胞的蛋白表达情况进行了分析，最终发现了42个差异表达的蛋白（>2倍的变化，P <0.05），其中21个下调， 21个上调。进一步分析表明，这些蛋白主要参与了细胞内代谢，生物学调控，细胞构建，信息传递和细胞死亡的调控。 接下来我们选择了其中一个变化比较大的蛋白：HSP27，对其功能进行了深入分析。我们的研究结果显示：EV71感染的早期阶段，HSP27在转录和翻译水平上都有明显上调。降低HSP27表达可以减少EV71的复制，过表达HSP27则可以提高病毒复制。通过使用特异的磷酸化蛋白抗体，我们发现HSP27第15位以及78位的丝氨酸有明显的磷酸化修饰，而82位的丝氨酸则没有发生改变。使用p38激酶抑制剂预先处理细胞可以降低HSP27的磷酸化修饰，从而抑制EV71的复制。进一步分析表明，HSP27可以帮助EV71蛋白酶2A对真核翻译起始因子eIF4G的剪切，从而加强病毒自身蛋白的翻译，最终促进了病毒的感染。这项研究结果阐明了宿主细胞EV71的反应机制，有利于我们对病毒致病机制的研究，并为EV71的抗病毒研究提供了一个新的药物靶标。 / The outbreaks of enterovirus 71 (EV71) infections have become a major public health issue worldwide, especially in the Asia-Pacific region. EV71 infection can be asymptomatic or cause diarrhea, rashes, and hand, foot, and mouth disease (HFMD). However, EV71 can also cause severe neurological disease even death. To date, little is known about the molecular mechanisms of the host response to EV71 infection. In this study, the expression patterns of host genes in EV71 infected human rhabdomyosarcoma cells were analyzed by using two-dimensional proteomics assays. In total, 42 protein spots were found to be differentially expressed (>2 fold changes, p<0.05) in three pairs of gels, of which 21 proteins were found to be down-regulated while 21 were up-regulated. Data analysis suggested that proteins associated with metabolic process, biological regulation, cellular component organization, cell communication and death were most modified. HSP27, one of the most altered proteins during EV71 infection, was selected to determine its fundamental roles upon EV71 infection. We show that HSP27 is rapidly up-regulated both at the transcriptional and the translational levels at the early stage of EV71 infection. Depleting cellular HSP27 expression reduced EV71 replication, while over-expression of HSP27 greatly enhanced viral infection. By using the phosphorylated specific antibodies, serine residues 15, 78, but not the 82 were found to be phosphorylated during EV71 infection. The phosphorylation depended on the activation of the mitogen-activated protein kinase p38 signaling pathway. After treating with p38 kinase inhibitors, EV71 replication was coordinately decreased. Further analysis showed that HSP27 affected the protease 2A mediated eIF4G cleavage and assisted the IRES driven translation, thus facilitated the EV71 replication. The findings in this work not only provided a global view of the host responses to EV71 infection, but demonstrated HSP27 to be a valid target for anti-EV71 drug development. / Detailed summary in vernacular field only. / Yi, Lina. / "September 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 94-103). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.iv / Publications --- p.v / Table of Contents --- p.vii / List of Tables and Figures --- p.x / List of Abbreviation --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Enterovirus 71 --- p.2 / Chapter 1.1.1 --- Clinical features --- p.2 / Chapter 1.1.2 --- Molecular epidemiology of EV71 --- p.5 / Chapter 1.1.3 --- The virology of EV71 --- p.8 / Chapter 1.1.4 --- Pathogenesis --- p.18 / Chapter 1.1.5 --- Treatment of EV71 infection --- p.20 / Chapter 1.2 --- The heat shock protein 27 --- p.23 / Chapter 1.2.1 --- Properties of HSP27 --- p.23 / Chapter 1.2.2 --- Functions of Hsp27 --- p.26 / Chapter 1.2.5 --- Phosphorylation of Hsp27 --- p.28 / Chapter 1.2.6 --- Hsp27 and Viral infection --- p.31 / Chapter 1.3 --- Thesis hypothesis and objective --- p.33 / Chapter Chapter 2 --- Materials and Methods --- p.34 / Chapter 2.1 --- Cells and Virus propagation --- p.35 / Chapter 2.2 --- Viral infection --- p.35 / Chapter 2.3 --- 2-DE and image analysis --- p.36 / Chapter 2.4 --- MALDI-TOF-MS --- p.37 / Chapter 2.5 --- Database analysis --- p.38 / Chapter 2.6 --- Bioinformatic analysis --- p.38 / Chapter 2.7 --- Plasmids --- p.39 / Chapter 2.8 --- siRNA synthesis --- p.41 / Chapter 2.9 --- Transfection and cell treatment --- p.41 / Chapter 2.10 --- RNA extraction and cDNA synthesis --- p.41 / Chapter 2.11 --- Real-Time Quantitative PCR --- p.42 / Chapter 2.12 --- Western Blotting analysis --- p.44 / Chapter 2.13 --- Luciferase assays --- p.44 / Chapter 2.14 --- Statistical Analysis --- p.45 / Chapter Chapter 3 --- Proteomic analysis of cellular protein alterations in response to EV71 infection --- p.46 / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Results --- p.48 / Chapter 3.2.1 --- EV71 infection of the RD cells --- p.48 / Chapter 3.2.2 --- 2-DE profiling of EV71 infected and non-infected RD cells --- p.49 / Chapter 3.2.3 --- Identification of differentially expressed proteins --- p.50 / Chapter 3.2.4 --- Functional classification --- p.52 / Chapter 3.2.5 --- GO enrichment analysis --- p.54 / Chapter 3.2.6 --- Protein validation by Western blot --- p.56 / Chapter 3.3 --- Discussion --- p.57 / Chapter Chapter 4 --- HSP27 effects on EV71 infection --- p.62 / Chapter 4.1 --- Introduction --- p.63 / Chapter 4.2 --- Results --- p.64 / Chapter 4.2.1 --- Increased Hsp27 expression in EV71 infected cells --- p.64 / Chapter 4.2.2 --- Suppression of Hsp27 inhibits EV71 replication --- p.65 / Chapter 4.2.3 --- Over-expression of Hsp27 increases EV71 replication --- p.66 / Chapter 4.2.4 --- Hsp27 is rapidly phosphorylated during EV71 infection --- p.67 / Chapter 4.2.5 --- Pathways involved in Hsp27 phosphorylation --- p.68 / Chapter 4.2.6 --- Role of Hsp27 phosphorylation during EV71 infection --- p.68 / Chapter 4.3 --- Discussion --- p.70 / Chapter Chapter 5 --- HSP27 facilitate EV71 IRES driven translation --- p.75 / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- Results --- p.79 / Chapter 5.2.1 --- Hsp27 increase viral IRES activity --- p.79 / Chapter 5.2.2 --- Hsp27 affects EV71 2A mediated eIF4G cleavage --- p.80 / Chapter 5.3 --- Discussion --- p.82 / Chapter Chapter 6 --- Summary and Perspectives --- p.87 / Chapter 6.1 --- Summary --- p.88 / Chapter 6.2 --- Perspectives --- p.89 / Reference --- p.93

Lu, Jing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 119-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Οι εντεροϊοί έχουν ιδιαίτερη σημασία για τη δημόσια υγεία, δεδομένου ότι σχετίζονται με επιδημίες γαστρεντερίτιδας (μη βακτηριογενούς προέλευσης) από την κατανάλωση μολυσμένου νερού. Οι ιοί αυτοί μπορούν να απομονωθούν σε μεγάλες ποσόστητες από κόπρανα και ούρα ανθρώπινης προέλευσης, καθώς και από λύματα και μολυσμένα νερά. Επιπλέον, οι Αδενοϊοί είναι παθογόνοι για τον άνθρωπο και η παρουσία τους σε περιβαλλοντικά δείγματα δύναται να προκαλέσει σημαντικές μολύνσεις. Οι Αδενοϊοί είναι ιοί ανθρώπινης εντερικής προέλευσης που περιέχουν DNA και ορισμένοι ορότυποι τους είτε δεν καλλιεργούνται, είτε καλλιεργούνται δύσκολα στις συνήθεις κυτταρικές σειρές. Γι’ αυτόν το λόγο, η ανίχνευσή τους σε μολυσμένο νερό και ο ρόλος τους ως παραγόντων πρόκλησης γαστρεντερίτιδας έχουν υποτιμηθεί. Οι Ρότα ιοί είναι υπεύθυνοι για οξεία περιστατικά γαστρεντερίτιδας στον άνθρωπο και τα ζώα. Κατόπιν του διπλασιασμού του γενετικού τους υλικού στον γαστρεντερικό σωλήνα οι ιοί αυτοί εκκρίνονται και δύνανται να διασπαρούν στο περιβάλλον και το νερό. Γενικά, οι Ρότα ιοί έχουν εμπλακεί σε περιστατικά γαστρεντερίτιδας σε πολλές χώρες. Η σταθερότητα των ανθρωπίνων Ρότα ιών στο νερό και η ανθεκτικότητά τους σε φυσικοχημικές διαδικασίες εξυγίανσης κατά την επεξεργασία των λυμάτων συντείνουν στην εξάπλωσή τους. Στην παρούσα διατριβή ανιχνεύθηκαν και απομονώθηκαν εντεροϊοί, αδενοϊοί και ρότα ιοί από ακατέργαστα λύματα, τα οποία ελήφθησαν από την είσοδο τεσσάρων σταθμών βιολογικού καθαρισμού (δύο στην Αττική και δύο στην Αχαΐα). Συνολικά ελήφθησαν 118 δείγματα ακατέργαστων λυμάτων κατά την περίοδο Σεπτέμβριο 2000 – Σεπτέμβριο 2003. Η μεθοδολογία αφορούσε στην συμπύκνωση των ιών ακολουθούμενη από RT-nested PCR, προκειμένου να επιτευχθεί αύξηση της ευαισθησίας απομόνωσης των ιών. Μετά την απομόνωση γενετικού υλικού των ιών πραγματοποιήθηκε τυποποίησή τους εφαρμόζοντας nucleotide sequencing analysis. Οι Ρότα ιοί ανιχνεύθηκαν σε 17 δείγματα (14.2%). Τα αποτελέσματα της τυποποίησής τους ήταν rotavirus τύπος G1 (88.2%) και τύπος G2 (11.8%). Οι Αδενοϊοί βρέθηκαν σε 55 δείγματα (45.8%). Η Sequencing ανάλυση είχε ως αποτέλεσμα την παρουσία στα δείγματα αδενοϊών της ομάδας F [τύποι 40 (34.6%) και 41 (63.6%)] και της ομάδας C [τύπος 2 (1.8%)]. Οι Εντεροϊοί ανιχνεύθηκαν σε 30 δείγματα (40%) και η sequencing ανάλυση είχε ως αποτέλεσμα την παρουσία αρκετών τύπων όπως (α) coxsackievirus (τύποι A6 - 3.3%, A9 - 3.3%, A16 - 3.3%, B4 - 16.7%, B5 - 3.3%), (β) echovirus (τύποι 2 -6.7%, 6 - 13.3%, 30 -10%), (γ) εντεροϊοί τύποι 68 - 3.3%, 71 - 13.3% καθώς και porcine εντεροϊός (6.7%), poliovirus 1 (6.7%) και poliovirus 2 (10%). Η μικροβιολογική ποιότητα του νερού επομένως και η ανθρώπινη υγεία επηρεάζονται σημαντικά από την παρουσία μικροοργανισμών εντερικής προέλευσης, οι οποίοι προέρχονται από λύματα που καταλήγουν στο υδάτινο περιβάλλον. Πολλές επιδημίες από ιούς εντερικής προέλευσης έχουν κατά καιρούς συνδυαστεί με το νερό. Οι υδατογενείς επιδημίες γενικά εξαπλώνονται στον πληθυσμό από κατανάλωση μολυσμένου νερού, κολύμβηση σε ακατάλληλα νερά αναψυχής καθώς επίσης μεταδίδονται απο τη σωματική επαφή και την εισπνοή. Τα μη επεξεργασμένα λύματα της μελέτης περιέχουν πολλούς και διαφορετικούς τύπους ιών εντερικής προέλευσης οι οποίοι κατά κύριο λόγο προκαλούν γαστρεντερίτιδα. Επομένως καθίσταται αναγκαία η επξεργασία των λυμάτων στο μέγιστο δυνατό βαθμό στους σταθμούς βιολογικού καθαρισμού. Η Sequencing ανάλυση έδειξε την παρουσία ανθρώπινων Ρότα ιών A (τύποι G1 και G2), οι οποίοι προκαλούν παγκοσμίως διάρροια σε παιδιά καθώς επίσης και την παρουσία αδενοϊών τύπου 40 και 41, οι οποίοι είναι σημαντικοί αιτιολογικοί παράγοντες γαστρεντερίτιδας, κυρίως σε θερμά κλίματα. Από την άλλη πλευρά η ποικιλία των εντεροϊών που ανιχνεύθηκε στα ακατέργαστα λύματα ήταν μεγαλύτερη συγκρινόμενη με την αντίστοιχη των υπολοίπων ιών της μελέτης. Η παρούσα διατριβή αναδεικνύει την αποτελεσματικότητα της μεθόδου «nucleotide sequencing analysis», ως μέσου επιδημιολογικής μελέτης και ανάλυσης της συσχέτισης ιών που εμπλέκονται σε ανθρώπινες ασθένειες κι εκέινων που ανιχνεύονται σε ακατέργαστα λύματα. / Enteroviruses have been associated with outbreaks of waterborne non-bacterial gastroenteritis and are of important concern for public health. Significant numbers of viruses can be isolated from faeces and urine of humans as well as from sewage and polluted waters. Adenoviruses are also pathogenic to humans and their presence in environmental samples (polluted waters) may cause infections. Like rotaviruses, adenoviruses are causative agents of gastroenteritis, are the only human enteric viruses to contain DNA and many serotypes are difficult to culture in regular cell lines For this reason, and because adenoviruses are slow growing, their presence in polluted water and their role as originators of gastroenteritis have probably been underestimated. Rota viruses are responsible for severe gastroenteritis in humans and animals. After replicating in the gastrointestinal tract, these viruses are excreted and may be dispersed in environmental waters. Rota viruses have been implicated in waterborne gastroenteritis outbreaks in many countries. The stability of human rotaviruses in environmental water and their resistance to physicochemical treatment processes in sewage treatment plants may facilitate their transmission. In the present study, enteroviruses, adenoviruses and rota viruses were detected in raw sewage samples from inlets of four biological treatment plants in Greece (two in Athens, two in Patras}. Raw sewage samples (118) were analyzed for the presence of these viruses during the period September 2000 to September 2003. Our approach consisted of a simple concentration of viruses from raw sewage followed by RT-nested PCR in order to increase the sensitivity of virus detection. The viral sequences detected were then characterized by nucleotide sequencing analysis. Rota viruses were detected in 17 samples (14.2%). Sequencing analysis of the positive sewage samples revealed the presence of rotavirus type G1 (88.2%) and type G2 (11.8%). Adenoviruses were found in 55 samples (45.8%). Sequencing analysis of the positive sewage samples revealed the presence of adenovirus group F type 40 (34.6%), type 41 (63.6%) and group C type 2 (1.8%). Enteroviruses were detected in 30 samples (40%) and sequencing analysis of the positive sewage samples revealed the presence of several types such as (a) coxsackievirus types (A6 - 3.3%, A9 - 3.3%, A16 - 3.3%, B4 - 16.7%, B5 - 3.3%), (b) echovirus types (2 -6.7%, 6 - 13.3%, 30 -10%), (c) enterovirus types (68 - 3.3%, 71 - 13.3%) as well as porcine enterovirus (6.7%). poliovirus 1 (6.7%) and poliovirus 2 (10%). Water quality and, therefore human health, may be significantly affected by the presence of pathogenic enteric microorganisms derived from sewage discharged to the aquatic environment. Outbreaks of enteric virus disease have been linked to water at various times and to different causes. Waterborne disease may be transmitted by consumption of polluted drinking water, by immersion in recreational water or by contact with skin or inhalation. Raw sewage was found to be contaminated by different types of enteric viruses that mainly cause gastroenteritis; therefore, it is necessary to use the most efficient water treatment measures in sewage treatment plants. Sequencing analysis showed the presence of human rotavirus A type G1 and G2 which cause childhood diarrhea worldwide and enteric adenoviruses (types 40 and 41) which are important etiological agents of pediatric gastroenteritis, principally in temperate climates. On the other hand, the variety of enteroviruses identified in the raw sewage samples was more extensive compared to the other viruses of the study. The present study demonstrated the efficiency of the nucleotide sequencing analysis for studying epidemiological relationships between strains involved in human infections and those found in raw sewage.