核糖體抑活蛋白 (RIPs) 屬於糖苷酶的一種，能從23S或28S核糖體核糖核酸中的sarcin-ricin環(sarcin-ricin loop, SRL)移除一個特定的腺嘌呤，引致核糖體失效。由於核糖體蛋白協助RIP到達SRL，因此它們對RIP的核糖體特認性是極大的重要。雖然各RIPs的份子結構及催化活動非常相似，它們的核糖體特認性和效力存著很大的迥異。此外，現時還未能找出只有少數RIPs能同時抑制原核和真核生物的核糖體的原因。我們試圖從玉米核糖體抑活蛋白 (Maize RIP) 和真核生物的核糖體以及志賀毒素 (Shiga toxin) 和原核生物的核糖體的相互作用的研究中去解釋以上的現象。 / 我們發現Maize RIP提供一個前所未見的區域與核糖體蛋白P2結合，並展示RIPs的結構大大限制了它們與核糖體蛋白的相互作用的性質和強度，從而影響RIPs在核糖體上的效力。另外，我們發現志賀毒素跟細菌的核糖體的相互作用比跟真核生物核糖體的相互作用弱，並可能跟細菌核糖體蛋白L7/L10有交聯。我們在蓖麻毒蛋白 (Ricin) 的碳端 (C-terminus) 加上人類免疫缺陷病毒-(HIV-1) 蛋白酶特認的肽以增加 ricin 對HIV-1蛋白酶的特認性，並希望此研究結果有助於應用相類的策略到其他RIPs上。 / Ribosome-inactivating proteins (RIPs) are N-glycosidases that inactivate ribosome by removing a specific adenine from the sarcin-ricin loop (SRL) of 23S or 28S ribosomal RNA. Ribosomal proteins are critical for determining the ribosome specificity of RIPs as they assist RIPs to get access to the SRL. Ribosome specificity and potency of RIPs are highly varied although their tertiary structures and catalytic depurination are highly alike. Moreover, it is still unsolved why only a few RIPs acquiring the ability to inhibit both prokaryotic and eukaryotic ribosomes. We attempted to elucidate the phenomena by investigating the interactions of maize RIP with eukaryotic ribosome and shiga toxin with prokaryotic ribosome. / Here we showed maize RIP presents a novel docking site to interact with ribosomal protein P2 and demonstrated the structure of RIPs imposes a large constraint on the nature and strength of the interaction with ribosomal protein which in turn affect the potency of RIPs on the ribosome. Shiga toxin was found to interact with prokaryotic ribosome weaker than the eukaryotic ribosome and crosslinked to the bacterial ribosomal protein L7/L10. Additionally, we increased the HIV-1 specificity of ricin A chain by incorporating the HIV-1 protease specific peptide to the C-terminus of the toxin and hope our findings would help to extend similar scheme to other RIPs in the future. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Yuen-Ting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 146-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Contents --- p. iv - viii / Chapter Chapter One --- Introduction of ribosome-inactivating proteins / Chapter 1.1 --- Nomenclature and distribution of ribosome-inactivating proteins --- p.1 / Chapter 1.2 --- Enzymatic activity of ribosome-inactivating proteins and their biological role --- p.2 / Chapter 1.3 --- Structure and catalytic centre of ribosome-inactivating proteins --- p.3 / Chapter 1.4 --- Ribosome specificity of RIPs and their interaction with ribosome --- p.5 / Chapter 1.5 --- Cytotoxicity and antiviral activity of ribosome-inactivating proteins --- p.6 / Chapter 1.6 --- Antiviral activity of RIPs --- p.9 / Chapter 1.7 --- Cellular trafficking of ribosome-inactivating proteins --- p.10 / Chapter 1.8 --- Application and therapeutic use of ribosome-inactivating proteins --- p.10 / Chapter 1.9 --- Evolution of RIPs --- p.11 / Chapter 1.10 --- Other activities of RIPs --- p.12 / Chapter Chapter Two --- Characterization of the interaction between RIPs and rat liver ribosome and its correlation with the potency of RIPs / Chapter 2.1 --- Introduction --- p.12 / Chapter 2.1.1 --- Nature of interaction between RIPs and eukaryotic ribosome --- p.12 / Chapter 2.1.2 --- RIPs interact with specific ribosomal proteins --- p.15 / Chapter 2.1.3 --- RIPs demonstrate different specificity towards ribosomes --- p.16 / Chapter 2.1.4 --- Introduction of maize RIP --- p.20 / Chapter 2.1.5 --- Interaction between maize RIP and ribosome --- p.22 / Chapter 2.2 --- Objectives and significance --- p.22 / Chapter 2.3 --- Materials and Methods / Chapter 2.3.1 --- Cloning and site-directed mutagenesis of RIPs --- p.23 / Chapter 2.3.2 --- Protein expression and purification --- p.23-26 / Chapter 2.3.2.1 --- Maize RIP and variants / Chapter 2.3.2.2 --- His-myc-MOD and His-MOD / Chapter 2.3.2.3 --- Trichosanthin (TCS) / Chapter 2.3.2.4 --- Shiga toxin chain A [E167AE170A] (StxA) / Chapter 2.3.2.5 --- Ricin chain A (RTA) / Chapter 2.3.2.6 --- Pokeweed antiviral protein (PAP) / Chapter 2.3.2.7 --- C-terminal His-tagged MOD, TCS and RTA / Chapter 2.3.2.8 --- His-SUMO-protease / Chapter 2.3.2.9 --- P2 and its variants / Chapter 2.3.2.10 --- Protein concentration and storage / Chapter 2.3.3 --- Purification of rat liver ribosome --- p.26 / Chapter 2.3.4 --- In vitro pull-down assay with ribosome --- p.27 / Chapter 2.3.5 --- On-resin crosslinking and mass spectrometry --- p.27 / Chapter 2.3.6 --- Crosslinking assay and western blotting --- p.28 / Chapter 2.3.7 --- In vitro pull-down assay with P2 --- p.29 / Chapter 2.3.8 --- In vitro pull-down assay with P2 and its variants --- p.29 / Chapter 2.3.9 --- Surface Plasmon Resonance --- p.29 / Chapter 2.3.10 --- N-glycosidase activity assay and quantitative PCR --- p.30 / Chapter 2.3.11 --- Cytotoxicity on 293T --- p.31 / Chapter 2.3.12 --- Cellular uptake of RIPs and western blotting --- p.32 / Chapter 2.4 --- Results / Chapter 2.4.1 --- In vitro pull-down assay with ribosome --- p.32 / Chapter 2.4.2 --- On-resin crosslinking and mass spectrometry of crosslinked proteins --- p.37 / Chapter 2.4.3 --- Crosslinking assay and western blotting --- p.40 / Chapter 2.4.4 --- In vitro pull-down assay with P2 --- p.43 / Chapter 2.4.5 --- Sensorgram of binding between P2 and Maize RIP variants --- p.44 / Chapter 2.4.6 --- N-glycosidase activity of maize RIP variants --- p.45 / Chapter 2.4.7 --- Cytotoxicity of maize RIP variants --- p.48 / Chapter 2.4.8 --- In vitro pull-down assay with P2 and its variants --- p.49 / Chapter 2.4.9 --- Surface Plasmon Resonance of P2 and various RIPs --- p.52 / Chapter 2.4.10 --- N-glycosidase activity assay and quantitative PCR --- p.55 / Chapter 2.4.11 --- Cytotoxicity of RIPs to 293T --- p.57 / Chapter 2.5 --- Discussion --- p.59 / Chapter 2.6 --- Conclusion --- p.72 / Chapter Chapter Three --- Identifying prokaryotic ribosomal protein(s) interacting with shiga toxin / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Background of shiga toxin --- p.74 / Chapter 3.1.2 --- Trafficking and activation of shiga toxin --- p.75 / Chapter 3.1.3 --- Intoxication by Shiga toxin --- p.76 / Chapter 3.1.4 --- Dual specificity on ribosome --- p.77 / Chapter 3.2 --- Objectives and significance --- p.78 / Chapter 3.3 --- Materials and methods / Chapter 3.3.1 --- Cloning of Shiga toxin and ribosomal proteins --- p.79 / Chapter 3.3.2 --- Expression and purification --- p.79-80 / Chapter 3.3.2.1 --- His-SUMO StxA, His-StxA, and His-StxA [E167Q] / Chapter 3.3.2.2 --- Ribosomal proteins / Chapter 3.3.3 --- Isolation of E. coli ribosome and rat liver ribosome --- p.80 / Chapter 3.3.4 --- Pull-down assay of prokaryotic and eukaryotic ribosome --- p.81 / Chapter 3.3.5 --- Size-exclusion chromatography of RIPs and prokaryotic ribosome --- p.81 / Chapter 3.3.6 --- Pull-down assay of StxA with HepG2 and C41 lysate --- p.82 / Chapter 3.3.7 --- Two-dimensional electrophoresis --- p.82 / Chapter 3.3.8 --- Mass spectrometric analysis of pull-down assay --- p.83 / Chapter 3.3.9 --- Crosslinking of StxA with r-proteins --- p.84 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Cloning of wild-type shiga toxin --- p.84 / Chapter 3.4.2 --- Pull-down with prokaryotic and eukaryotic ribosome --- p.85 / Chapter 3.4.3 --- Size-exclusion chromatography of RIPs and prokaryotic ribosome --- p.88 / Chapter 3.3.4 --- Pull-down assay of StxA with HepG2 and C41 lysates --- p.90 / Chapter 3.4.5 --- Crosslinking of StxA with r-proteins --- p.97 / Chapter 3.5 --- Discussion and conclusion --- p.99 / Chapter Chapter Four --- Engineering ricin A chain for increasing its specificity toward Human Immunodeficiency Virus (HIV) / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.1.1 --- Human immunodeficiency virus --- p.104 / Chapter 4.1.2 --- Current drugs for HIV --- p.105 / Chapter 4.1.3 --- Anti-HIV mechanism of RIPs --- p.105 / Chapter 4.1.4 --- Engineering cytotoxic protein into HIV-1 specific toxin --- p.107 / Chapter 4.2 --- Objectives and significance --- p.109 / Chapter 4.3 --- Materials and methods / Chapter 4.3.1 --- Design and cloning of RTA HIV-1 specific variants --- p.109 / Chapter 4.3.2 --- Cloning, expression and purification of ricin variants --- p.112 / Chapter 4.3.3 --- Purification of HIV-1 protease --- p.112 / Chapter 4.3.4 --- HIV-1 protease induced cleavage of RTA variants --- p.113 / Chapter 4.3.5 --- Cytotoxicity on 293T and JAR --- p.114 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Purity check of RTA variants --- p.114 / Chapter 4.4.2 --- HIV-1 protease induced cleavage of RTA variants --- p.115 / Chapter 4.4.3 --- Cytotoxicity on 293T and JAR --- p.119 / Chapter 4.5 --- Discussion --- p.124 / Chapter 4.6 --- Conclusion --- p.126 / Concluding remarks and future prospect --- p.127 / Appendices / Appendix 1 --- p.128 - 132 / Appendix 2 --- p.133 - 134 / Appendix 3 --- p.135 - 138 / Appendix 4 --- p.139 - 145 / Bibliography --- p.146 - 159

In the future, the 25 as internal loop region of Pro-RIP can be modified for the optimized recognition of proteases of other HIV strains. This approach opens a new opportunity for the anti-HIV application of maize RIP and other related type III RIPs. A modified maize RIP may also be applied to target other viruses and pathogens, for examples, hepatitis C and malaria, which are dependent on pathogen-encoded proteases for replication. / In this study, we provide an account on the generation of HIV-1 protease-sensitive maize RIP variants by first incorporating the HIV-1 protease recognition sequences to the internal inactivation region of the Pro-RIP. Among the five variants, three variants were cleaved and activated by HIV-1 protease in vitro and in vivo, resulting in an active two-chain form with N-glycosidase activity comparable to the fully active maize RIP. In addition, the variants inhibited viral replication in human T lymphocytes (C8166) infected by two T-tropic HIV-1 strains, HIV-1IIIB and HIV-1 RF/V82F/I84V, and their cytotoxicity towards uninfected cells was similar to the non-activated precursor (TAT-Pro). In comparison to TAT-Pro, variants TAT-Pro-HIV-MA/CA and Pro-TAT-Pro-HIV-p2/NC had 2- to 70-fold increase in the inhibition of p24 antigen production in the HIV-infected cells with low cytotoxicity towards uninfected C8166 cells. / Maize RIP is classified as a type III RIP. It is synthesized in the endosperm of maize as an inactive precursor (Pro-RIP), which contains a 25-amino acid internal inactivation region. During germination, a two-chain activated form (MOD) is generated by endogenous proteolysis of the internal inactivation region, whereas the two chains (16.5 and 8.5 kDa) are tightly associated without disulfide linkage. Our group has solved the crystal structures of both the Pro-RIP and MOD and found that this internal inactivation region is on the surface of the N-terminal domain in Pro-RIP . The removal of this internal inactivation region increases the inhibition of protein synthesis of rabbit reticulocyte lysate by over 600 folds. The presence of the internal inactivation region has led us to derive a novel strategy to enhance the specificity of maize RIP towards HIV-infected cells while minimizing its cytotoxic effect on normal cells. / Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases which cleave the N-glycosidic bond of adenine-4324 at the alpha-sarcin/ricin (SR) loop of 28S rRNA. The depurination of the SR loop results in the inhibition of protein synthesis by impairing the binding of EF-1 or EF-2 to ribosomes. RIPs are therefore highly cytotoxic and have been used as abortificiant, anti-cancer and anti-HIV agents, either alone or as a component of immunotoxins. Many type I and II RIPs, such as MAP30, GAP30, DAP30, pokeweed antiviral protein (PAP) and ricin, have been reported to possess anti-HIV activity by inhibiting viral replication in vitro and in vivo though the anti-HIV mechanism is still unclear. / Law, Ka Yee. / Adviser: Pang-Chui Shaw. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 124-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The medico-scientific advances made in the treatment of HIV and AIDS, which emerged in the mid 1990s, were significant. The Highly Active Anti-Retroviral Treatments (HAART) or anti- HIV treatments have been positioned as resources that changed the way HIV is now medically and socially constructed. Although HIV remains incurable, it is now constructed as a chronic disease that is treatable, manageable and people are no longer positioned as living with a ‘death sentence’. The research on which this thesis is based explores the subjective lived experiences of people with HIV living in urban Australia in the context of this change. The effects that the treatments have had on corporeality have also changed the ways people are now living with HIV in the post-treatment era. It is an era in which treatments for HIV are taken-for-granted, but issues, doubts and concerns relating to treatment use are firmly embedded in the everyday life of people with HIV. The findings suggest that whilst AIDS-related mortality has decreased since the availability of effective treatments, the notion of ‘quality of life’, as subjectively constituted and defined, is an ongoing negotiation that is predicated on people locating meaningfulness in their everyday lives. Despite the decreased threat of failing health and death, the findings also suggest that people are continuing to be confronted by, and therefore positioned as, having to make sense of complex issues embedded in living with a disease for which there is no cure. / Doctor of Philosophy (PhD)

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (D.P.H.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 25, 2009). Includes bibliographical references.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.99-110

Orientadores : Antonio de Azevedo Barros Filho, Maria Marluce dos Santos Vilela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-02T07:54:41Z (GMT). No. of bitstreams: 1 Merhi_VaniaAparecidaLeandro_D.pdf: 25347132 bytes, checksum: e3ac032c3c9e46f7ddb6bd81c22e8bfd (MD5) Previous issue date: 2002 / Resumo: O objetivo deste trabalho foi estudar o crescimento em peso e estatura, segundo sexo, de crianças infectadas com o vírus da imunodeficiência humana por transmissão vertical, de O a 192 semanas de idade e sua relação com o tratamento anti-retroviral administrado, estimando as velocidades de crescimento em peso e estatura. Em um estudo longitudinal, foram avaliadas 101 crianças infectadas com o HIV e 154 sororreversoras (crianças nascidas de mães infectadas pelo HIV, mas que não se infectaram com o vírus), por dois métodos de análise: o 1° método utilizando todos os intervalos na faixa etária de Oa 192 semanas, sendo adotado um modelo cúbico e no 2° método foi utilizado um modelo quadrático segmentado em duas faixas etárias (de Oa 48 semanas e de 48 a 192 semanas). A comparação dos parâmetros entre os dois grupos foi realizada pelo teste de Mann- Whitney e foram construídas curvas de crescimento. Para a análise da influência do tratamento anti-retroviral no peso e na estatura foi utilizado o método das equações de estimação generalizadas e as medidas para o cálculo da idade foram selecionadas de acordo com uma margem de seis semanas nas idades de: 48, 96, 144 e 192 semanas. No 1° método de análise foi constatado que há diferença significativa de crescimento em peso e em estatura entre os grupos de crianças infectadas e sororreversoras, exceto no período de nascimento, não sendo observada as mesmas diferenças pelo 2° método de análise, o qual mostra um padrão de crescimento similar entre os dois grupos. Quando comparado o crescimento em peso e em estatura de crianças infectadas e sororreversoras com o NCHS, verificou-se um comportamento dentro dos parâmetros de normalidade. Não houve diferença significativa do efeito da monoterapia com AZT, comparada com terapia tripla com inibidor de protease, e da terapia dupla comparada com terapia tripla, sobre o crescimento em peso e em estatura. Não houve diferença do comportamento de crescimento em peso e em estatura nos dois grupos, observando-se que as crianças infectadas acompanham o seu ritmo de crescimento normal, sendo o 2° método de análise melhor do ponto de vista fisiológico, pois avalia cada estágio do crescimento. A alteração do estado nutricional é uma manifestação precoce da infecção pelo HIV, e sua compreensão permite o conhecimento de medidas mais eficazes no controle da doença e seu prognóstico / Abstract: This work aimed to study the relationship between the growth rate, based on weight and height of HIV infected children, and the antiretroviral therapy. The group studied comprised of children ranging from o to 192 weeks of age, sorted by sex, infected with HIV vertically transmitted. In a longitudinal study, it were analyzed 101 infected and 154 non-infected children, being two methods applied to estimate weight and height growth: 1)in the first method,it was used all the intervals in the age range- from 0 to 192 weeks, being a cubic analysis model adopted; 2) in the second method, a quadratic model was used, segmented in two age ranges: from 0 to 48 weeks and from 48 to 192 weeks. To compare the parameters analyzed for both groups studied, it was used the Mann-Whitney test, and growth curves were also built. To analyze the influence of antiretroviral therapy on the children weight an height, it was employed the general estimation equation method, and the measures used for the age calculation were taken every 6 weeks in the following ages: 48, 96, 144 and 192 weeks. By analyzing the data with the fIrst method, it was observed that there is a significant difference of weight and height growth between infected and non-infected children, except from the birth period, however, the same was not observed when it was used the second analysis method, which indicated a similar growth pattem for both groups studied. The results obtained in the comparison of weight and height of both infected and non-infected children to the NCHS were in compliance to the normal patterns. It was not detected a significant difference between monotherapy with AZT and triple therapy with protease inhibitors, and also between double therapy and triple therapy on the weight and height growth rates. No difference was noted between weight and height growth patterns of both groups studied, indicating that infected children present normal growth rate, being the second analysis method better fit to the study, from a physiological point of view, since it enables the assessment of each growth phase. One early symptom of the human immunodeficiency virus infection is the change in the patient nutritional status, and better knowledge on the worsening of the patient nutritional condition enables the choice of more efficient measures to control the disease and the prognosis / Doutorado / Medicina Interna / Doutor em Ciências Médicas

Made available in DSpace on 2014-06-12T18:29:17Z (GMT). No. of bitstreams: 2 arquivo3456_1.pdf: 504446 bytes, checksum: b40073abc9d03ea6670f37598a228199 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Introdução - As estimativas de cobertura do teste anti-HIV em gestantes no Brasil são de 62%, com valores de 41% no Nordeste, obstáculo à profilaxia da transmissão vertical do HIV. O objetivo deste estudo foi identificar fatores de risco para o desconhecimento da sorologia para o HIV em gestantes, determinando a cobertura do teste durante toda a gestação e até a 14ª semana. Métodos Estudo caso-controle realizado em uma maternidade de referência para gestação de alto risco no Nordeste do Brasil onde foram entrevistadas 485 puérperas sobre variáveis biológicas, reprodutivas, sócio-demográficas, assistenciais e de informação em saúde. Resultados - Do total de puérperas 21,65% informaram desconhecer o resultado da sorologia para o HIV durante a gestação (cobertura do teste = 78.35%); 87,42% trazia o cartão da gestante, com registro do teste em apenas 48.58% deles, 22% informaram conhecimento do resultado do teste até a 14ª semana de gestação, ou 15,4% se considerado apenas as que realizaram o teste em laboratório público. Análise multivariada: menos de nove anos de estudos (OR, 2,92; P=0,006), morar no interior do estado (OR, 4,11: P=0,001), coletar o teste no terceiro trimestre de gestação (OR, 11,6: p=0,000) e falta de aconselhamento sobre a importância do teste na profilaxia da transmissão vertical do HIV (OR, 2,31; p=0,022), estiveram associados com o desconhecimento. CONCLUSÕES: a cobertura encontrada para o teste anti-HIV foi elevada, para a região Nordeste, quando comparada com estudos anteriores, porém, acentuadamente baixa (22% e 15,4%), até a 14ª semana de gestação. Escolaridade abaixo de nove anos de estudo, procedência de cidades do interior do estado, coleta do teste no terceiro trimestre de gestação e ausência de aconselhamento estiveram associados com o desconhecimento independente de outros fatores