Thesis (M. Arch.)--Massachusetts Institute of Technology, Dept. of Architecture, 1995. / Includes bibliographical references (p. 89-92). / With the much-commented upon shifts in the global economy and the problematizing of traditional social, economic, cultural, and political relationships, the role of architecture as a "public" practice has fallen under scrutiny. The "privatization of public space", resulting from the overt merging of "public" concerns (typically represented through the State) and "private" interests (in our society, usually defined by groups with significant power over investment decisions) is often taken as symptomatic of a general trend towards the breakdown of democracy itself. Here, however, it is important to differentiate between what can be termed the "public sphere", or the abstract discursive arena where thought is (re)produced and debated, and "public space", or the actual built spaces of personal encounter among strangers. Certainly, since the Enlightenment when the terms first acquired widespread currency, the latter has depended upon some conception of the former. / Eric Chia-Kong Mar. / M.Arch.

Thesis (doctoral)--Stockholm University, 2002. / Includes bibliographical references.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Political Science, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND DEWEY / Bibliography: leaves 67-71. / by Nancy Elaine Wright. / M.S.

Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, <em>Anguilla rostrata</em>. <br> <br> RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders. <br><br> HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues. <br><br> Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked <em>Mx</em> induction, which is the first demonstration of PKR mediating Mx expression. <br><br> The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication. <br><br> The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, <em>Anguilla rostrata</em>. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis. <br><br> This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, <em>Anguilla rostrata</em>. <br> <br> RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders. <br><br> HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues. <br><br> Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked <em>Mx</em> induction, which is the first demonstration of PKR mediating Mx expression. <br><br> The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication. <br><br> The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, <em>Anguilla rostrata</em>. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis. <br><br> This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19957号 / 理博第4224号 / 新制||理||1607(附属図書館) / 33053 / 京都大学大学院理学研究科化学専攻 / (主査)教授 北川 宏, 教授 島川 祐一, 教授 竹腰 清乃理 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

O processo de cloração da água pode levar à formação de compostos tóxicos como o 3-cloro-4-(diclorometil)-5-hidroxi-2[5H]-furanona (MX). Embora tenha sido detectado em baixas concentrações (ng/L), ele representa cerca de 50% da atividade mutagênica das águas potáveis, previamente tratadas com cloro. Neste estudo, foi desenvolvido um método quantitativo para a análise de traços de MX em águas cloradas. As técnicas de extração em fase sólida (SPE), microextração em fase sólida (SPME) e a extração líquido-líquido (LLE) foram avaliadas, sendo que esta última se mostrou mais eficiente para a análise de MX em água, com recuperação de 50% para concentrações &#8804; 50 ng/L. A determinação do MX foi realizada por cromatografia a gás acoplada a espectrometria de massas (GC/MS). O método se baseia na derivatização online do MX usando o reagente bis(trimetilsilil)trifluoroacetamida (BSTFA), um procedimento experimental simples e rápido. A precisão elevada e a detecção em concentrações baixas possibilitou a aplicação do método em amostras reais. A confiabilidade dos resultados, se deve ao uso do espectrômetro de massas, que permitiu a identificação e quantificação precisa do MX em águas. Uma pré-concentração de 2000 vezes do extrato foi necessária, visto que o limite de detecção instrumental obtido foi 3 &#181;g/L. O método otimizado foi aplicado em amostras de água provenientes de redes de abastecimento na cidade de São Paulo, coletadas antes e após o processo de cloração. As concentrações de MX nessas águas cloradas variaram de 3 a 22 ng/L, valores comparáveis aqueles encontrados em outros países. / A by-product of chlorination in water is the 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), a toxic compound which has been the subject of recent studies. Although MX has been found at low concentrations (ng/L), it accounts for approximately 50% of the total mutagenicity in drinking water. In this study, a method was developed to analyze MX at trace leveI in chlorinated waters. The MX extraction efficiency was evaluated using three different techniques: solid phase extraction (SPE), solid phase microextraction (SPME) and liquid-liquid extraction (LLE). The LLE technique provided higher recovery with value of 50% at concentrations &#8804; 50 ng/L. A simple and fast procedure involving on-line based trimethylsilyl derivatization for the quantitative analysis of MX by gas chromatography with mass spectrometry (GCIMS) is proposed. Good precision and low limit of detection of the method allowed its application in real water samples. The reliable MX identification and quantification were obtained due to the use of the mass spectrometry. Since the instrumental limit of detection was equal to 3.0 &#181;g/L, the samples had to be concentrated to 2000 times the original concentration. The optimized method was applied to water samples collected before and after chlorination process in water suppliers located in São Paulo City, Brazil. Concentrations of MX in those samples ranged from 3 to 22 ng/L which are similar to those detected in other countries.

O processo de cloração da água pode levar à formação de compostos tóxicos como o 3-cloro-4-(diclorometil)-5-hidroxi-2[5H]-furanona (MX). Embora tenha sido detectado em baixas concentrações (ng/L), ele representa cerca de 50% da atividade mutagênica das águas potáveis, previamente tratadas com cloro. Neste estudo, foi desenvolvido um método quantitativo para a análise de traços de MX em águas cloradas. As técnicas de extração em fase sólida (SPE), microextração em fase sólida (SPME) e a extração líquido-líquido (LLE) foram avaliadas, sendo que esta última se mostrou mais eficiente para a análise de MX em água, com recuperação de 50% para concentrações &#8804; 50 ng/L. A determinação do MX foi realizada por cromatografia a gás acoplada a espectrometria de massas (GC/MS). O método se baseia na derivatização online do MX usando o reagente bis(trimetilsilil)trifluoroacetamida (BSTFA), um procedimento experimental simples e rápido. A precisão elevada e a detecção em concentrações baixas possibilitou a aplicação do método em amostras reais. A confiabilidade dos resultados, se deve ao uso do espectrômetro de massas, que permitiu a identificação e quantificação precisa do MX em águas. Uma pré-concentração de 2000 vezes do extrato foi necessária, visto que o limite de detecção instrumental obtido foi 3 &#181;g/L. O método otimizado foi aplicado em amostras de água provenientes de redes de abastecimento na cidade de São Paulo, coletadas antes e após o processo de cloração. As concentrações de MX nessas águas cloradas variaram de 3 a 22 ng/L, valores comparáveis aqueles encontrados em outros países. / A by-product of chlorination in water is the 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), a toxic compound which has been the subject of recent studies. Although MX has been found at low concentrations (ng/L), it accounts for approximately 50% of the total mutagenicity in drinking water. In this study, a method was developed to analyze MX at trace leveI in chlorinated waters. The MX extraction efficiency was evaluated using three different techniques: solid phase extraction (SPE), solid phase microextraction (SPME) and liquid-liquid extraction (LLE). The LLE technique provided higher recovery with value of 50% at concentrations &#8804; 50 ng/L. A simple and fast procedure involving on-line based trimethylsilyl derivatization for the quantitative analysis of MX by gas chromatography with mass spectrometry (GCIMS) is proposed. Good precision and low limit of detection of the method allowed its application in real water samples. The reliable MX identification and quantification were obtained due to the use of the mass spectrometry. Since the instrumental limit of detection was equal to 3.0 &#181;g/L, the samples had to be concentrated to 2000 times the original concentration. The optimized method was applied to water samples collected before and after chlorination process in water suppliers located in São Paulo City, Brazil. Concentrations of MX in those samples ranged from 3 to 22 ng/L which are similar to those detected in other countries.

No description available.

Dissertation (Ph.D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.