Drugs for treating rare diseases had been neglected by the pharmaceutical industry for a long time, due to the complex and costly drug R&D process as well as a small unprofitable market. Since its introduction in 1983, the Orphan Drug Act (ODA) has sought to prompt the innovation of drugs for minority diseases by reducing the regulatory and economic barriers. The incentives of the ODA have been effected through market protection, tax credit, fee waiver and grants to increase the accessibility of orphan products for the public. The number of orphan drugs available in the market has risen sharply from just ten in the decade before 1983 to over 400 since 1983. This increase implies a substantial improvement of the healthcare of patients suffering rare diseases and a success of the orphan drug legislation with the aim to motivate the development and manufacture of products that have low commercial potentials. Although it is evident that the ODA has successfully stimulated drug companies to develop numerous orphan products, treatments are very expensive. The sales of blockbuster orphan drugs have provided drug companies with unusually highprofit margins and limited patient access to treatments. The dilemma presented by the ODA reflects many of the issues currently faced by policymakers. In this thesis, we have analyzed the long-term evolution of the biopharmaceutical industry. In particular, we have examined drug discovery in the period of random screening, rational design and network collaboration, and explored the influence of the ODA. We have taken the theory of the sectoral system of innovation, and combined it with the complex adaptive model of innovation, and found that the complex version of that theory is capable of explaining the comprehensive drug innovation system. A Multi-agent Based Model has been introduced to identify and analyze the dynamics of bio-pharmaceutical innovation. The model has explored the roles of the main players in the sector and the influence of their relationships embedded in the process of orphan drug innovation. Through this model, we have investigated the mechanisms of how the incentives stimulate orphan drug innovation during the period from 1983- 2012. Moreover, the model has been applied to solve the dilemma of the ODA through analyzing how to achieve the best trade-off between orphan drug developments. Drawing upon the results of the simulation, we provide a sound basis for adjusting the ODA incentives to strikes an appropriate balance between stimulating orphan drug innovation and providing benefits to society, propose some resolutions to the ODA, while also to motivate orphan drug development in a financial way. The Advice for other countries planning to enact the orphan drug legislation and directions for further research suggested by this model have been put forward.

The author seeks to compare the Chinese and English editions of Wu Zhuoliu’s (1900-1976) Orphan of Asia (1956). Through the analysis of several characters and the political ambiguity within the text, the author first attempts to compare the two target translations of the original Japanese text. In addition to the close reading of the novel(s), the author employs paratextual analysis of the Chinese and English versions of the story in order to challenge the publishing practices of translation. The re-reading of translations thus includes an investigation of the content of the story as well as the packaging of the text. The objectives of this project include adding to the research completed on Wu’s canonical text, in translation studies, and in paratextual studies.

The author seeks to compare the Chinese and English editions of Wu Zhuoliu’s (1900-1976) Orphan of Asia (1956). Through the analysis of several characters and the political ambiguity within the text, the author first attempts to compare the two target translations of the original Japanese text. In addition to the close reading of the novel(s), the author employs paratextual analysis of the Chinese and English versions of the story in order to challenge the publishing practices of translation. The re-reading of translations thus includes an investigation of the content of the story as well as the packaging of the text. The objectives of this project include adding to the research completed on Wu’s canonical text, in translation studies, and in paratextual studies.

In this study an attempt was firstly made to define, describe and explicate the phenomenon of HIV/AIDS providing a basis for understanding the multidimensional nature, key characteristics and impact of HIV/AIDS in terms of its background, the current status as well as the future of the epidemic. Literature concerning HIV/AIDS in general, global and in particular the South African situation was discussed. Secondly the concept AIDS orphans was investigated after which grounding, description and explanation of the problems and needs of AIDS orphans were presented in order to give a clear picture of challenges faced by these children. Problems of orphan-hood such as legal and ethical issues, socio-emotional issues, educational issues, financial issues and child-headed households were identified. The study focused on early adolescent AIDS orphans therefore adolescence, as a life phase with specific emphasis on early adolescence was reviewed. Hereafter, the researcher presented a newly self-developed life skills programme for early adolescent AIDS orphans (i.e. AIDS ORPHANS LIFE SKILLS PROGRAMME) followed by all the empirical research findings, a general summary, conclusions and recommendations. The broad aim of the study was to develop and empirically test the effectiveness of a life-skills programme for early adolescent AIDS orphans. Two research questions and a hypothesis were formulated for the study. The research questions included: (a) what is the nature and prevalence of socio-emotional needs and problems of early adolescent AIDS orphans? (b) What are the life skills needed by early adolescent AIDS orphans? Accordingly the hypothesis of the study read: If early adolescent AIDS orphans undergo a life-skills programme then their skills will be enhanced in order to cope better with their socio-emotional needs and problems. In the context of applied research the type of research conducted in this study was intervention research. This type of research was relevant for this particular study because it is a problem-solving process seeking an effective intervention programme for the promotion of life skills for early adolescent AIDS orphans. In view of the fact that the AIDS orphan situation is a crises for the whole nation innovative preventative positive educational programmes for children orphaned by AIDS are deemed pivotal. The focus of this research study was two-folded using a combination of quantitative and qualitative methods. The first phase of the study was qualitative and explorative in nature. The aim of the researcher was to have a broader understanding of the phenomenon HIV/AIDS, the socio-emotional needs and problems of and life skills needed by early adolescent AIDS orphans in South Africa. The focus of the second phase was to develop a life skills programme for early adolescent AIDS orphans, based on the information collected in the first phase of the study and then to empirically test the effectiveness of the newly developed life skills programme. The researcher used semi-structured interviews with a schedule to collect qualitative data during the first phase of the research. During the second phase, the researcher utilised a self-constructed group administered questionnaire to collect quantitative data before and after implementation of the life skills programme (pre-test and post-test). In order to explore the socio-emotional needs and problems of and life skills needed by early adolescent AIDS orphans, a phenomenological design seemed appropriate. The research design was selected to reach the first three objectives of the study, namely: To conceptualise theoretically the phenomenon of HIV/AIDS and AIDS orphans, the specific characteristics, needs and problems of early adolescents as well as life skills for early adolescents; a) To explore and identify the nature and prevalence of socio-emotional needs and problems of early adolescent AIDS orphans; b) To explore and identify the life skills which AIDS orphans, in their early adolescent phase need to improve their coping capabilities; Qualitative data through semi-structured interviews with a schedule was collected. The sample thus included 40 respondents i.e. 10 social workers, 10 caregivers and 20 AIDS orphans. The empirical research findings based on the first part of the study confirmed that HIV/AIDS has forced vast numbers of children into precarious circumstances, putting them at high risk of becoming infected with HIV. AIDS orphans are especially vulnerable to HIV infection for a host of social and economic reasons including poverty, sexual exploitation, violence, and lack of access to HIV information and prevention services. The consequence of this is that children are often socially isolated and deprived of basic social services. The findings further confirmed that there are currently no life skills programmes specifically designed for early adolescent AIDS orphans in South Africa. Deficiencies in life skills contribute to the vulnerability and exploitation of these children. Life skills were viewed as crucial in improving the quality of life of AIDS orphans. Life skills can enable adolescents to develop sound and positive view of life. The researcher also applied the comparison group pretest-posttest design (i.e. a quasi-experimental comparison group pretest-posttest design) with respondents to reach the last three objectives of the study, namely: a) To develop a life-skills programme for early adolescent AIDS orphans; b) To empirically test the effectiveness of the developed life skills programme for early adolescent AIDS orphans; and c) To suggest practical recommendations for further utilisation of the newly developed life skills programme for early adolescent AIDS orphans. The researcher developed a life skills programme for early adolescent AIDS orphans namely AIDS Orphans Life Skills Programme. The evaluation of the self-developed life skills programme for early adolescent AIDS orphans was done by a self-constructed group administered questionnaire in the pre-test i.e. before implementation of AIDS orphans life skill programme, and post-test with both the experimental (30 respondents) and comparison group (30 respondents). The sample thus included a total of 60 early adolescent AIDS orphans and the empirical data was collected to include 2 measurements once before and once after the intervention (AIDS orphans life skills programme). The findings confirmed that there was a statistical significance difference in the experimental groups life skills (i.e. sense of identity and self-esteem, communication, assertiveness, self-awareness, coping and stress management, decision making, problem solving, conflict management and a healthy life style) with a 95% chance that the results were due to AIDS Orphans Life Skills. There was not statistical difference in the experimental groups critical and creative thinking skills. Nine out of ten key elements of AIDS orphans life skills programme were thus successful in that they promoted life skills amongst early adolescent AIDS orphans. AIDS orphans life skills programme is perceived as having had the impact that was hoped for. / Thesis (DPhil (Social Work))--University of Pretoria, 2007. / Social Work / unrestricted

L'immortalité cellulaire est toujours accompagnée par l'activation du mécanisme de maintien des télomères. Dans la plupart des cancers humains, ce rôle est assuré par l'enzyme télomérase. Cependant, dans 15 % des tumeurs, la télomérase n'est pas activée et les télomères sont maintenus par l'allongement alternatif des télomères (ALT), voie qui implique la recombinaison des télomères. ALT est plus fréquent dans les tumeurs provenant de tissus mésenchymateux (sarcomes), representant 40-60 % des cas, que dans les tumeurs épithéliales. Comprendre le mécanisme ALT est primordial dans les thérapies anti-cancéreuses puisque certaines drogues inhibant la télomérase conduisent souvent à l'activation de l'ALT.La voie ALT est définie par de caractéristiques typiques des télomères. Dans les cellules ALT, les recombinaisons aberrantes d'ADN ne se limitent pas aux télomères puisque les génomes sont souvent fortement réarrangés. Les liens de ces caractéristiques génomiques anormales et la maintenance des télomères atypique ne sont pas connues, mais l'instabilité du génome contribue certainement à la transformation. Notre équipe a montré que les récepteurs orphelins appartenant aux familles NR2C/F ont été trouvés enrichies dans les télomères des lignées cellulaires ALT. Nous avons proposé que ces facteurs puissent être recrutés aux télomères par liaison directe à la séquence répétée GGGTCA, un site de liaison à haute affinité pour ces protéines. Mon projet vise à comprendre (i) leur mécanisme de liaison et (ii) leur rôle, dans le processus d'ALT.Dans cette étude nous montrons que dans les sarcomes primaires humains, les télomères d'ALT sont souvent liés par des récepteurs nucléaires orphelins des sous-familles NR2C/F, en particulier dans les tumeurs au stade avancé. Ceci suggère un rôle actif de ces facteurs dans la progression tumorale ALT. En utilisant la technique de ChIP-sequencing, nous avons montré que les protéines NR2C/F se lient à une répétition directe amplifiée (DR0) aux télomères, et pas de manière significative à toute autre combinaison de motif GGGTCA. Nous avons également analysé la distribution sur tout le génome de NR2C2/F2 et TRF2, une protéine de liaison des télomères, dans des cellules ALT (-) et ALT (+). Bien qu'il n'y ait que peu de sites génomiques liés par TRF2 dans les cellules ALT (-), nous avons été surpris d'identifier plusieurs centaines de régions liées par TRF2 dans les cellules ALT (+). Plus surprenant, la grande majorité de ces régions spécifiques TRF2 ALT chevauche des sites endogènes de NR2C2/F2. Étant donné que ces sites ne contiennent généralement pas les répétitions des télomères, TRF2 est probablement recruté de façon indirecte. Conformément à cette interprétation, nous montrons que les facteurs NR2C/F entrainent un rapprochement des loci et sont responsables du regroupement atypique des télomeres dans ALT. De plus, un sous-ensemble de ces régions génomiques uniques a des additions hétérogènes des séquences télomeriques ALT, suggérant un rôle dans le recrutement des télomères par des protéines NR2C/F mais aussi une fonction de ciblage de recombinaison génomique. Systématiquement, nous trouvons que ces réarrangements des télomères/génome sont situés à proximité des motifs GGGTCA endogènes. Le caryotype spectral des lignées cellulaires ATL montre que les sites télomériques interstitielles sont fréquemment localisés aux niveaux des sites de translocations/réarrangements entre deux ou plusieurs chromosomes, ce qui est également observé dans les données de ChIPseq. Ces résultats suggèrent que les réarrangements entres les télomères et le génome pourraient participer à la formation d'un caryotype complexe ce qui caractérise environ 50% des sarcomes. De plus, l'addition de sites télomériques interstitielles dans le génome est spécifique des cellules ALT et est favorisée par les dommages de l'ADN. / Cellular immortality is always accompanied by the activation of telomere maintenance mechanism. In most human cancers this role is fulfilled by the telomerase enzyme. However in 15% of tumors, telomerase is not activated and telomeres are maintained by an Alternative Lengthening of Telomeres (ALT) pathway that involves telomere-telomere recombination. Interestingly ALT is more prevalent in tumors originating from mesenchymal tissues (sarcomas), where it is present in 40-60% of cases, than in epithelial tumors. Understanding ALT maintenance is critical since inhibiting telomerase in tumors leads to the activation of ALT. The ALT pathway is operationally defined by typical telomere hallmarks. In ALT cells, aberrant DNA transactions are not restricted to telomeres since genomes are often highly rearranged. Whether these abnormal genomic features are linked to atypical telomere maintenance is not known, but genome instability is certainly contributing to transformation. We have previously shown that orphan receptors of the NR2C/F families were enriched at telomeres in ALT cell lines. We proposed that these factors could be recruited to telomeres through direct binding to the GGGTCA variant repeat, a high affinity binding site for these proteins. My project is aimed at understanding (i) their mechanism of binding and (ii) their role, if any, in the ALT process.We show that in human primary sarcomas, ALT telomeres are often bound by orphan nuclear receptors of the NR2C/F subfamilies, particularly in more advanced-stage tumors. This suggests an active role for these factors in ALT tumor progression. Using ChIP-sequencing, we show that NR2C/F proteins bind to an amplified direct repeat (DR0) at telomeres, and not significantly to any other GGGTCA motif combination. We also analyzed the genome wide distribution of NR2C2/F2 and TRF2, a telomere binding protein, in ALT(-) and in ALT(+) cells. While there are only few genomic sites bound by TRF2 in ALT(-) cells, we were surprised to identify several hundred regions bound by TRF2 in ALT(+) cells. More surprisingly, the great majority of these ALT specific TRF2 regions overlap with endogenous NR2C2/F2 sites. Since these sites usually do not contain telomere repeats, TRF2 is likely indirectly recruited. Consistent with this interpretation, we show that NR2C/F factors drive locus proximity. Moreover, a subset of these unique genomic regions harbor heterogeneous ALT telomere sequence additions, not only suggesting a telomere recruitment role for NR2C/F proteins but also a recombination targeting function in the genome. Consistently, we find these telomere/genome rearrangements are located close to endogenous GGGTCA motifs. Next, we wanted to evaluate a role of these rearrangements in formation of complex karyotype which characterize approximately 50% of sarcomas. We found by spectral karyotyping that interstitial telomeric sites are frequently located at translocation/ rearrangements sites between two or more chromosomes, which we could also observe in our ChIPseq data. Furthermore, we demonstrate that addition of interstitial telomeric sites to the genome is enhanced by DNA damage and specific for ALT genome. Therefore we conclude that NR2C/F factors target telomere proximity to defined NR2C/F regions which enables telomere-genome rearrangements under DNA damage condition. This contributes not only to efficient telomere recombination, but also it drives further genomic instability at selected NR2C/F sites.We believe we identified a new mechanism of telomere dysfunction potentially driving targeted genome instability and mediated by NR2C/F proteins in ALT cells which probably underlie complexity of sarcomas genome. Understanding the ALT mechanism allows designing NR2C/F-targeted therapies in treatment of ALT tumors and therapies for patients treated with anti-telomerase drugs to prevent ALT appearance.

In the first few decades of the nineteenth century, Americans and immigrants moved to New Orleans hoping to take advantage of the opportunities the city offered. Many American citizens moved from cities like Boston, New York, and Philadelphia. Recognizing the lack of social welfare programs and assistance given to the poor, a group of women established the Female Orphan Society. From its creation, the Female Orphan Society worked in providing aid to indigent mothers and their children through providing religious, vocational, and educational training. In a short time, the FOS emerged as the only private, Protestant female refuge for immigrant families and their children in New Orleans. This involvement elevated the role of the asylum in the city and heightened the influence of an institution run by southern, upper-class white women.

Le statut social et juridique de l’orphelin a accompagné, au cours des cinquante dernières années, les mutations, souvent violentes, subies par les sociétés d’Afrique centrale. En raison du nombre des orphelins, les problèmes individuels d’intégration sociale, deviennent également une préoccupation majeure. L’écriture romanesque pose avec insistance, avec les moyens narratifs et esthétiques qui lui sont propres, la question de la place des orphelins dans l’ordre social. Toutes les ressources d’un imaginaire narratif sous-jacent (enfants-sorciers, enfants des la rue, enfants-soldat, etc.) sont convoquées pour alimenter le débat. Il s’agira d’étudier comment la figuration narrative ce personnage engage un type d’écriture liée à la critique des institutions étatiques. Cette problématique soulève un nombre d’interrogations portant sur le lien entre le récit et les modèles d’organisation sociopolitique en Afrique centrale. Les sociétés africaines se racontent et se régulent par le roman et le personnage de l’orphelin est un acteur décisif dans Johnny chien méchant, L’enfant aux larmes de sang, Allah n’est pas obligé, Et Dieu seul sait comment je dors, Les Larmes de Tsiana, Tarmac des hirondelles et Histoire d’un enfant trouvé. / The social and legal status of orphan accompanied during the last fifty years, mutations, often violent, sustained by the Central African societies. Due to the number of orphans, individual issues of social integration, also become a major concern. The novel writing poses with insistence, with the narrative and aesthetic resources of its own, the question of the place of orphans in the social order. All resources of a narrative imagination underlying (child witches, street children, child soldiers, etc.) Are convened to the debate. It will explore how the narrative figuration this character commits a type of writing related to the criticism of state institutions. This issue raises a number of questions on the relationship between narrative and sociopolitical organization models in Central Africa. African societies are counted and regulate the novel and the character of the orphan is a key player in L’Enfant aux larmes de sang , Allah n’est pas obligé, Les Larmes de Tsiana, Tarmac des hirondelles and Histoire d’un enfant trouvé.

研究背景與研究目的 / 細胞衰老是指細胞進入不可逆的永久化的生長停滯狀態。目前，細胞衰老作為重要的抑癌機制受到廣泛認可，对其相關信號通路的研究為腫瘤的靶向治療提供了新的依據和策略。TLX核受體基因属于核受體亞家族2組E成員1，是一種孤兒受體。雞和老鼠TLX基因最初作為果蠅末端/间隙基(tailless) 的同源基因而被發現，而人TLX 基因是在檢索惡性淋巴癌中的抑癌細胞而從人胚胎的腦cDNA文庫中克隆出來的。TLX基因敲除的轉基因老鼠的研究表明TLX基因對維持胚胎腦和成体腦神經幹細胞的分裂增殖起重要作用。最近的研究發現，TLX在臨床神經胶质瘤組織中高表達。並且，在轉基因鼠中，TLX的高表達会引起神經幹細胞的大量增殖而形成腦腫瘤，提示TLX可能參與腦腫瘤的發生和發展。但是，TLX对包括前列腺癌在内的人類惡性腫瘤的發生發展中所起的功能及作用機制尚不清楚。表達譜研究發現，TLX在前列腺細胞中的表達水平高於永生化的正常前列腺上皮細胞的表達，並且，TLX在臨床惡性程度高的前列腺癌中呈高表達趨勢，預示TLX可能參與促進前列腺癌的惡性進展。因此本研究的主要目的是TLX在前列腺癌細胞中的功能研究。 / 研究材料與方法 / 為了研究TLX對前列腺癌細胞生長的影響以及相關機制，本論文主要採用以下方法：1）運用免疫組化的方法檢測TLX在臨床前列腺癌組織中的表達，並應用實時螢光定量PCR方法檢測TLX在永生化的非惡性前列腺上皮細胞以及前列腺癌細胞株中的表達；2）根據不同的p53表達狀態選擇雄激素依賴（LNCaP）和雄激素非依賴（PC-3, DU145）的前列腺癌細胞株，分別采用慢病毒感染和逆轉錄病毒感染的方法建立TLX-敲除和TLX-過表達的細胞株，並研究這些穩轉細胞系離體和在體的生長表型（包括檢測細胞生長，細胞週期，細胞衰老，細胞的遷移和侵染，化療藥物抗性，缺氧耐受性以及體內成瘤能力）；3）採用檢測β-半乳糖苷酶活性的方法檢測TLX穩轉系細胞在衰老因素誘導和非誘導狀態下TLX缺失和高表達對細胞衰老的影響；4）采用免疫印跡（western blot）的方法檢測TLX穩轉系細胞中參與細胞衰老的關鍵蛋白的表達情況；5）利用雙螢光素酶報告基因方法和染色質免疫沉澱技術，研究TLX對靶基因的調控；6）構建TLX缺失變異體（△ZF1 和 △LBD-AF2），在前列腺細胞系和非前列腺細胞系中外源性表達相應的變異體進一步驗證TLX的功能。 / 結果 / 本論文研究結果總結如下：1）TLX在前列腺癌細株中和惡性程度高的前列腺癌組織中高表達；2）在前列腺癌中進行TLX基因敲除能顯著抑制細胞體外和體內的生長並誘導前列腺癌細胞的衰老；3）相反，TLX的過表達能促進前列腺癌細胞體外和體內的惡性生長，包括促進細胞的錨定和非錨定性生長、促進細胞的遷移與侵染、增強細胞缺氧耐受、對化療藥物抗性、以及增強細胞異位移植瘤的成瘤能力；4）TLX 的高表達抑制了前列腺癌細胞衰老，並保護細胞免受多柔比星誘導的細胞衰老以及持續性激活的癌基因H-RAS（H-RAS{U+1D33}¹²{U+2C7D}）誘導的細胞衰老；5）TLX可以結合到p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹基因（其後縮寫為p21）的啟動子序列並抑制p21的啟動子的轉錄活性，並且在TLX-過表達細胞中外源性高表達p21能誘導前列腺癌細胞重新進入衰老狀態；6）TLX也能結合到SIRT1基因的啟動子序列並激活SIRT1的轉錄活性，在TLX-過表達細胞中對SIRT1進行基因沉默能誘導這些細胞的再次衰老；7）TLX介導的衰老抑制效應以及對其靶基因的轉錄調控作用需要完整的DNA-結合域以及配體結合域，對TLX兩個區域的缺失變異影響TLX在前列腺細胞和非前列腺細胞中的生理功能及轉錄調控活性。 / 結論 / 本論文的研究結果提示TLX通過抑制前列腺癌細胞的衰老在前列腺癌發生發展過程中起重要作用，並且這種衰老抑制作用是通過介導p21基因的轉錄抑制以及對SIRT1基因的轉錄激活而實現的。此研究首次證實了TLX在前列腺癌中高表達，並且TLX能夠抑制前列腺癌細胞的衰老從而促進前列腺癌的發生發展，提示TLX有可能成為前列腺癌治療潛在的重要靶點。 / Background and aims of the study / Cellular senescence represents an irreversible form of permanent cell-cycle arrest and it acts a key process of tumor suppression, while targeting to pathways involved in this process can provide potential and promising therapeutic strategies to cancer treatments. TLX belongs to the NR2E1 orphan nuclear receptor subfamily. The chicken and mouse TLX genes were initially isolated as a vertebrate homolog to the Drosophila terminal-gap gene tailless (tll), while the human TLX was cloned from a fetal brain cDNA library in a search for putative tumor suppressor genes in lymphoid malignancies. Functional studies in transgenic mouse model of TLX-knockdown show that TLX plays important regulatory roles in the maintenance and self-renewal control of both embryonic and adult neural stem cells. Recent studies of transgenic mice with TLX overexpression combined with its expression studies in human clinical gliomas revealed that TLX is overexpressed in primary human glioblastomas and its dysregulation may contribute to the initiation and development of some brain tumors. However, the exact functional contributions of TLX and the involved mechanism(s) in human malignancies, including prostate cancer, are still far from clear. In an expression profile study, it was demonstrated that TLX exhibited an up-regulated expression pattern in many prostate cancer cell lines and also the high-grade clinical prostate cancer, suggesting that TLX might play a positive regulatory role in the advanced progression of prostate cancer. The overall aim of this study was to elucidate the functional role of TLX in prostate cancer cell growth. / Materials and methods / In order to elucidate the functional roles of TLX in prostate cancer growth and the involved mechanisms, the following experiments were conducted: 1) To investigate and determine the expression pattern of TLX in clinical prostatic tissues by immunohistochemistry, and to survey the expression profile of TLX in a panel of prostatic immortalized epithelial and prostate cancer cell lines by quantitative real-time PCR analysis; 2) To generate stable TLX-knockdown prostate cancer cells by lentiviral transduction and TLX-stable expressing cells by retroviral transduction in both hormone-sensitive (LNCaP) and -insensitive (DU145 and PC-3) prostate cancer lines with different expression status of p53; and to conduct growth phenotype characterization studies (including cell growth, cell cycle, cellular senescence, cell migration and invasion, resistance to chemotherapy drugs, hypoxic cell growth assays, and tumorigenesis) on these TLX-transfectants in vitro and in vivo; 3) To characterize cellular senescence phenotype of TLX-infectants by senescence-associated β-galactosidase (SA-β-Gal) staining method with or without senescence inducers; 4) To investigate the expression status of markers involved in cellular senescence in TLX-infectants by immunoblotting; 5) To demonstrate the transcriptional regulation targets of TLX by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay; 6) To confirm the cellular function of TLX in prostatic and non-prostatic cells expressing different TLX deletion mutants (△ZF1 and △LBD-AF2). / Results / Results obtained in this study are summarized as follows: 1) TLX displayed an increased expression pattern in many prostate cancer cell lines and also high-grade (Gleason score ≥ 7) prostate cancer tissues; 2) Depletion of TLX mRNA by RNA interference dramatically suppressed in vitro and in vivo tumor cell growth and triggered cellular senescence (SA-β-Gal histochemical marker) in prostate cancer cells; 3) On the contrary, TLX overexpression significantly enhanced multiple advanced malignant growth capacities (including enhanced anchorage-dependent and -independent cell growth, cell migration and invasion, hypoxia adaptation, resistance to chemotherapy drug Doxorubicin as well as in vivo tumorigenicity) in prostate cancer cells; 4) TLX overexpression significantly suppressed cellular senescence and protected cells against doxorubicin-induced or oncogenic H-RAS (H-RAS{U+1D33}¹²{U+2C7D})- induced senescence; 5) TLX could directly bind to p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹ gene (hereafter p21) promoter and repress the transcriptional activity of p21 promoter, while ectopic restoration of p21 expression in TLX-overexpressed cells could rescue cellular senescence with enhanced SA-β-Gal staining; 6) protein deacetylase SIRT1 gene was also activated by TLX through its direct transcriptional regulation, while knockdown of SIRT1 in TLX-overexpressed cells could rescue cellular senescence; 7) TLX-induced suppression of cellular senescence and also its direct gene regulation would require an intact DBD and LBD domain, as truncated deletion of DBD or LBD domain could both abolish the cellular function and transcriptional activity of TLX in prostatic and non-prostatic cells. / Conclusions / The results obtained in this study suggested that TLX could play a positive growth regulatory or tumor-promoting role in prostate cancer development by its suppression of cellular senescence and this senescence suppression was mediated via its direct transcriptional regulation of both p21 (repression) and SIRT1 (transactivation) genes. Moreover, this study also showed for the first time that TLX, which was overexpressed in prostate cancer tissues, might function to suppress premature senescence in prostate cancer progression and also targeting to TLX could be a potential therapeutic approach for prostate cancer treatment. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wu, Dinglan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 135-151). / Abstract also in Chinese. / Thesis /Assessment Committee --- p.I / ABSTRACT --- p.II / 摘 要 --- p.VI / ACKNOWLEDGEMENT --- p.IX / PUBLICATIONS RELATED TO THIS THESIS --- p.XI / CONTENTS --- p.XII / ABBREVIATION --- p.XV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Prostate cancer --- p.2 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Nature history --- p.4 / Chapter 1.1.3 --- Androgen Axis prostate cancer --- p.7 / Chapter 1.1.3.1 --- Androgen receptor --- p.7 / Chapter 1.1.3.2 --- Function of the androgen receptor in prostate cancer --- p.7 / Chapter 1.1.3.3 --- Mechanisms of CRPC progression --- p.8 / Chapter 1.1.3.4 --- Androgen receptor pathway-directed therapies --- p.10 / Chapter 1.1.4 --- Treatment of prostate cancer --- p.11 / Chapter 1.2 --- Cellular senescence --- p.13 / Chapter 1.2.1 --- What is senescence --- p.13 / Chapter 1.2.1.1 --- Replicative cellular senescence --- p.13 / Chapter 1.2.1.2 --- Oncogene induced senescence (OIS) --- p.15 / Chapter 1.2.1.3 --- Tumor suppressor loss-induced senescence --- p.17 / Chapter 1.2.2 --- Establishment of cellular senescence --- p.19 / Chapter 1.2.3 --- The p16/pRb and ARF/p53/p21 pathway of senescence induction --- p.21 / Chapter 1.2.3.1 --- p16/pRb senescence pathway --- p.22 / Chapter 1.2.3.2 --- ARF/p53/p21 senescence pathway --- p.23 / Chapter 1.2.4 --- Markers of senescence --- p.24 / Chapter 1.2.4.1 --- Cell cycle arrest and morphology --- p.24 / Chapter 1.2.4.2 --- Senescence-associated β-galactosidase --- p.25 / Chapter 1.2.4.3 --- p16/pRb and p53/p21 pathways --- p.26 / Chapter 1.2.4.4 --- γ-H2AX staining as a marker for DNA damage --- p.27 / Chapter 1.2.4.5 --- Senescence-associated heterochromatin foci (SAHF) --- p.27 / Chapter 1.2.5 --- Pro-senescence therapy for cancer treatment --- p.29 / Chapter 1.2.5.1 --- Why pro-senescence therapy --- p.29 / Chapter 1.2.5.2 --- Critical factors of pro-senescence therapy --- p.31 / Chapter 1.2.5.3 --- Strategies of senescence induction --- p.32 / Chapter 1.2.5.4 --- Targeting to senescence-associated secretory phenotype (SASP) --- p.38 / Chapter 1.2.6 --- Future direction --- p.40 / Chapter 1.3 --- TLX --- p.41 / Chapter 1.3.1 --- Nuclear receptor --- p.41 / Chapter 1.3.2 --- Identification of tailless/TLX --- p.42 / Chapter 1.3.3 --- Tailless in drosophila --- p.43 / Chapter 1.3.4 --- Functional role of tll/TLX --- p.45 / Chapter 1.3.4.1 --- Role of tll/TLX in brain development --- p.45 / Chapter 1.3.4.2 --- Role of tll/TLX in visual system developments --- p.46 / Chapter 1.3.4.3 --- Role of TLX in neural stem cell self-renewal --- p.47 / Chapter 1.3.5 --- Target genes of TLX --- p.49 / Chapter 1.3.6 --- Transcriptional regulation of tll/TLX --- p.51 / Chapter 1.3.7 --- TLX in cancer --- p.52 / Chapter CHAPTER 2 --- STUDY AIMS --- p.54 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.57 / Chapter 3.1 --- Human prostatic tissues and Immunohistochemistry --- p.58 / Chapter 3.2 --- Cell lines and cell cultures --- p.59 / Chapter 3.3 --- Antibody and reagents --- p.63 / Chapter 3.3.1 --- Generation of rabbit anti-TLX polyclonal antibody --- p.63 / Chapter 3.3.2 --- Commercial antibody --- p.64 / Chapter 3.4 --- RNA isolation and Reverse transcriptional-PCR --- p.65 / Chapter 3.4.1 --- RNA isolation --- p.65 / Chapter 3.4.2 --- Reverse transcription reaction (RT) --- p.66 / Chapter 3.4.3 --- Polymerase Chain Reaction (PCR) --- p.66 / Chapter 3.5 --- Western blotting --- p.68 / Chapter 3.5.1 --- Protein extraction --- p.68 / Chapter 3.5.2 --- Electrophoresis, Protein blotting and Colorimetric detection --- p.69 / Chapter 3.6 --- Plasmids construction --- p.70 / Chapter 3.6.1 --- PCR for sub-cloning --- p.70 / Chapter 3.6.2 --- PCR for mutant generation --- p.71 / Chapter 3.6.3 --- Restriction enzymes digestion and ligation --- p.72 / Chapter 3.7 --- Retroviral, lentiviral transduction and generation of TLX-stable cells --- p.73 / Chapter 3.8 --- RNA interference --- p.75 / Chapter 3.9 --- In vitro cell growth assay --- p.76 / Chapter 3.9.1 --- Cell counting --- p.76 / Chapter 3.9.2 --- MTT assay --- p.76 / Chapter 3.9.3 --- Soft agar assay for anchorage independent growth --- p.77 / Chapter 3.10 --- Cell cycle assay --- p.77 / Chapter 3.11 --- Cell invasion assay --- p.78 / Chapter 3.12 --- In vivo tumor growth assay --- p.78 / Chapter 3.13 --- In vitro and in vivo SA-β-Gal staining --- p.79 / Chapter 3.14 --- In vitro treatment with doxorubicin --- p.80 / Chapter 3.15 --- Transient Transfection and Luciferase Reporter Assay --- p.81 / Chapter 3.16 --- Chromatin immunoprecipitation (ChIP) assay --- p.82 / Chapter 3.16.1 --- Cross-linking and harvesting cells --- p.82 / Chapter 3.16.2 --- Cell lysis --- p.83 / Chapter 3.16.3 --- Sonication --- p.83 / Chapter 3.16.4 --- Immunoprecipitation --- p.83 / Chapter 3.16.5 --- Washing --- p.84 / Chapter 3.16.6 --- Elution --- p.85 / Chapter 3.16.7 --- Reverse cross-linking and DNA purification --- p.85 / Chapter 3.16.8 --- PCR --- p.86 / Chapter 3.17 --- Statistical analysis --- p.86 / Chapter CHAPTER 4 --- RESULTS --- p.87 / Chapter 4.1 --- TLX is up-regulated in prostate carcinoma and prostate cancer cell lines --- p.88 / Chapter 4.2 --- Knockdown of TLX suppresses in vitro cell growth and triggers cellular senescence in prostate cancer cells --- p.93 / Chapter 4.3 --- Knockdown of TLX inhibits in vivo tumor growth and induces cellular senescence of prostate cancer cells --- p.97 / Chapter 4.4 --- Ectopic expression of TLX enhances in vitro cell growth and multiple advanced malignant phenotypes in prostate cancer cells --- p.100 / Chapter 4.5 --- Ectopic expression of TLX suppresses cellular senescence in prostate cancer cells --- p.105 / Chapter 4.6 --- TLX suppresses cellular senescence via its direct transcriptional repression of p21{U+1D42}{U+1D2C}{U+A7F1}¹/{U+A7F0}{U+1D35}{U+1D3E}¹ gene --- p.110 / Chapter 4.7 --- TLX also suppresses cellular senescence via its transcriptional regulation of SIRT1 gene --- p.116 / Chapter CHAPTER 5 --- DISCUSSION --- p.121 / Chapter CHAPTER 6 --- SUMMARY --- p.131 / REFERENCES --- p.135

Doctor of Philosophy / This thesis is concerned with an examination and re-assessment of the establishment, operation and management of the Female and Male Orphan Schools, in the first half of the nineteenth century in New South Wales. The chaplains and governors in the early penal settlement were faced with a dilemma, as they beheld the number of children who were ‘orphaned’, neglected, abandoned and destitute. In order to understand the reasons why these children were in necessitous circumstances, the thesis seeks to examine the situations of the convict women, who were the mothers of these children. Governors Philip Gidley King and Lachlan Macquarie respectively in 1801 and 1819 established the Schools, which provided elementary education, training and residential care within a religious setting. Researching the motives underlying the actions of these men has been an important part of the thesis. An examination of the social backgrounds of some of the children admitted to these Schools has been undertaken, in order to provide a greater understanding of the conditions under which the children were living prior to their admissions. Information about family situations, and the social problems encountered by parents that led them to place their children in the Schools, have been explored. The avenues open to the girls and boys when they left the Schools, has formed part of the study. Some children were able to be reunited with family members, but the majority of them were apprenticed. A study of the nature of these apprenticeships, has led to a greater understanding of employment opportunities for girls and boys at that time. In 1850 the Schools were amalgamated into the Protestant Orphan School at Parramatta. By examining the governance and operation of the Schools during their last two decades as separate entities, we have more knowledge about and understanding of these two colonial institutions. It is the conclusion of this thesis that some of the harsher judgements of revisionist social historians need to be modified. It was the perception that more social disorder would occur if action was not taken to ‘rescue’ the ‘orphaned’ children, usually of convict parentage. However genuine charity, philanthropy and concern was displayed for the children in grave physical and moral danger. The goals of the founders were not always reached in the Orphan Schools, nevertheless they performed an invaluable service in the lives of many children.

For large regions of many proteins, and even entire proteins, no homology to known domains or proteins can be detected. These sequences are often referred to as orphans. Surprisingly, it has been reported that the large number of orphans is sustained in spite of a rapid increase of available genomic sequences. However, it is believed that de novo creation of coding sequences is rare in comparison to mechanisms such as domain shuffling and gene duplication; hence, most sequences should have homologs in other genomes. To investigate this, the sequences of 19 complete fungi genomes were compared. By using the phylogenetic relationship between these genomes, we could identify potentially de novo created orphans in Saccharomyces cerevisiae. We found that only a small fraction, &lt;2%, of the S. cerevisiae proteome is orphan, which confirms that de novo creation of coding sequences is indeed rare. Furthermore, we found it necessary to compare the most closely related species to distinguish between de novo created sequences and rapidly evolving sequences where homologs are present but cannot be detected. Next, the orphan proteins (OPs) and orphan domains (ODs) were characterized. First, it was observed that both OPs and ODs are short. In addition, at least some of the OPs have been shown to be functional in experimental assays, showing that they are not pseudogenes. Furthermore, in contrast to what has been reported before and what is seen for older orphans, S. cerevisiae specific ODs and proteins are not more disordered than other proteins. This might indicate that many of the older, and earlier classified, orphans indeed are fast-evolving sequences. Finally, &gt;90% of the detected ODs are located at the protein termini, which suggests that these orphans could have been created by mutations that have affected the start or stop codons. / <p>authorCount :2</p>