Haematopoietic stem and progenitor cells (HSPCs) are de novo generated within in the ventral aspects of the embryonic dorsal aorta (DA). Cells of this haemogenic endothelium (HE) will eventually undergo an endothelial to haematopoietic transition (EHT) that involves cell budding out of the aortic wall. Despite the detailed description of the cellular events, the exact haemogenic lineage path and the underlying molecular mechanism that establish full haematopoietic competence are still not entirely understood. The transcription factor Runx1 is critical for the emergence of HSPCs and shows expression in the zebrafish HE as early as 24 hpf. To facilitate a detailed analysis of the transient HE population I generated a TgBAC(runx1P2:Citrine) reporter line under the control of the endogenous runx1 promoter on a bacterial artificial chromosome (BAC). Double-transgenic reporter lines for runx1 and the endothelial marker kdrl allowed us to isolate specifically cells of the DA away from the whole endothelial population, which could be further sub-divided into HE and non-haemogenic cells. Genomewide expression analysis within the respective tissues and upon Runx1 loss of function enabled the identification of HE-specific Runx1-regulated genes. Hereby, the gfi1ab gene appeared as the functional homologue of the murine Gfi1. I show that in zebrafish, EHT is orchestrated through a conserved Runx1-Gfi1-Lsd1 axis. The cellular functions of the remaining Runx1 targets imply that maturation into fully functional HSCs depends on epigenetic regulation due to the up-regulation of de novo DNAmethyltransferases, as well as on factors that allow the developing HSCs to respond to extrinsic cues from haematopoietic niches. Lastly, it became evident that the early HE expresses dll4 at similar levels to the rest of the aortic endothelium, indicating a common lineage path. In the absence of RUNX1 the HE remains essentially arterial and persists as an integrated part of the DA.

Thesis (MPhil)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Little scholarly reflection has been published on the subject of medical research and translation. The aim of this study is to contribute to such literature by investigating the quality of original and retranslated medical questionnaires. The various steps medical researchers follow when translating their questionnaires are considered and discussed. Particular attention is given to questionnaires on AIDS-related topics in South Africa, as well as to the role of translation in ensuring the collection of valid data in medical research. Different translation approaches, which are followed when translating medical texts, and the impact they have on the quality of the research, are discussed. These approaches are the linguistic, text-linguistic and functional approaches. Attention is given to translators as communicators and mediators, as well as to the more general role of the translator. This study hypothesises that the quality of translations of medical research questions is largely inadequate in communicating effectively with the target culture for which they are intended. The retranslation hypothesis stating that retranslations are closer to the source text (ST) than original translations is supported. Afrikaans- and Xhosa-speaking adolescents from two secondary schools in the Cape Peninsula participated in a before-after study. These learners received self-administered medical questions on the two occasions. The first set comprised original translations, while the second set contained retranslations of the ST questions. Evaluation questions were included to assess the quality of these translations. The design, translation approach and quality of the original translations are explained, as is the development of the retranslation and evaluation questionnaires. Translations that do not consider their target audience lead to communication gaps, which have an adverse effect on the validity of data derived from questionnaires that are used in medical research. The results of most of these questions are compared for the two target cultures and are analysed qualitatively and quantitatively. The data are further explored to establish whether and how the translational quality of medical questionnaires can be improved. These aspects and the suggested translation process are discussed while bearing in mind the limitations of a study of this kind. Recommendations are made for possible improvement to the quality of translations of medical questionnaires. Projections for further studies in this direction complete this empirical investigation into translation and medical research. / AFRIKAANSE OPSOMMING: Relatief min akademiese nadenke is gepubliseer oor die onderwerp mediese navorsing en vertaling. Hierdie studie wil 'n bydrae maak tot sodanige literatuur deur 'n ondersoek na die kwaliteit van oorspronklik vertaalde en hervertaalde mediese vraelyste. Die onderskeie stappe wat mediese navorsers ten opsigte van die vertaling van hulle vraelyste volg, word bespreek. Aandag word spesifiek gerig op vraelyste oor vigsverwante temas in Suid-Afrika, asook op die rol van vertaling in die versekering dat geldige data in mediese navorsing ingesamel word. Verskeie benaderings wat gevolg word in die vertaling van mediese tekste en die impak wat hulle het op die kwaliteit van die navorsing word bespreek. Hierdie benaderings is die linguistiese, tekslinguistiese en funksionalistiese benaderings. Aandag word geskenk aan vertalers as kommunikeerders en tussengangers, asook die meer algemene rol van die vertaler. Hierdie studie veronderstel dat die kwaliteit van vertalings van mediese navorsingsvraelyste grootliks onvoldoende is om effektief met die betrokke teikengehoor te kommunikeer. Die hervertalingshipotese wat sê dat hervertalings nader aan die brontaal (Bn as oorspronklike vertalings is, word ondersteun. Afrikaans- en Xhosa-sprekende adolessente van twee sekondêre skole in die Skiereiland het deelgeneem aan 'n voor- en agtemastudie. Hierdie leerders het op beide geleenthede die vraelyste self voltooi. Die eerste stel het oorspronklike vertalings bevat terwyl die tweede stel hervertalings van die BT bevat het. Evalueringsvrae is ingesluit om die kwaliteit van hierdie vertalings te help bepaal. Die ontwerp, vertalingsbenadering en kwaliteit van die oorspronklike vertalings word verduidelik, so ook die ontwikkeling van die hervertaling- en evalueringsvraelyste. Vertalings wat nie die teikengehoor in ag neem nie, lei tot kommunikasiegapings wat die geldigheid van data afkomstig van vraelyste in mediese navorsing nadelig kan raak. Die resultate van die meeste van hierdie vrae word vir die twee teikenkulture vergelyk, en dit word kwalitatief en kwantitatief ontleed. Die data word verder ondersoek om vas te stelof en hoe die kwaliteit van die vertaling van mediese vraelyste verbeter sou kon word. Bogenoemde aspekte en die voorgestelde vertalingsproses word bespreek met inagneming van die beperkings van 'n studie van hierdie aard. Voorstelle word gemaak vir die moontlike verbetering van die vertaling van mediese vraelyste. Vooruitskattings vir verdere studie in hierdie rigting voltooi hierdie empiriese ondersoek na vertaling en mediese navorsing. Ek dra hierdie tesis op aan Mattheus vir sy liefde, geduld en al die tee-aandraery, asook aan Ettienne en Jeanelle wat met minder ondersteuning van my as andersins hulle skoolloopbane moes voltooi het.

This dissertation is a history of the use of World War II-era American conscientious objectors as the subjects of medical research. Under the Selective Training and Service Act of 1940, conscientious objectors had two choices: provide noncombatant service within the military or provide work of national importance under civilian direction under the auspices of a program called Civilian Public Service (CPS). Conscientious objectors who chose assignment to CPS were placed in camps in which the men labored on a work project authorized by the U.S. Selective Service System, the government entity that administered the draft. At the outset of the CPS program, the camps were modeled after the work camps of the Civilian Conservation Corps, a New Deal jobs program. Over time, and largely due to protests that such Civilian Conservation Corps-type forestry and soil conservation work assignments were not the promised work of national importance, other types of CPS camps were developed, with work projects dealing with public health, custodial care for the mentally disabled, or scientific research. In the later, which became commonly known as the guinea pig units, over five hundred conscientious objectors voluntarily participated as research subjects for a diverse assortment of scientific studies, including projects that dealt with infectious diseases, diet, frostbite, psycho-acoustics, and the impacts of temperature extremes and of altitude. In addition to describing the creation and operation of the guinea pig units, this dissertation examines the use of American World War II conscientious objectors as research subjects in light of two specific questions: first, why did these men volunteer to be guinea pigs? And second, was the use of World War II-era conscientious objectors as research subjects in keeping with the ethical standards of the time? This dissertation draws upon a diverse array of sources to answer the question of motivation from the volunteers' perspectives. Likewise, this dissertation relies upon a wide array of sources to piece together what researchers of the day, both military and civilian, would have considered acceptable and unacceptable uses of people in the name of research.

Haematopoietic stem cells (HSCs) maintain the vertebrate blood system throughout life. Exploiting their clinical potential requires a thorough understanding of the natural origins of the HSCs. They first arise from the haemogenic endothelium (HE), located in the main embryonic artery, the dorsal aorta. Our understanding of the genetic mechanisms underlying HE specification remains incomplete, but one of the crucial transcription factors is Gata2. We found that a conserved enhancer of zebrafish gata2a gene (i4 enhancer) is active in vivo specifically in endothelial cells, including the HE. To unravel the function of gata2a in specifying the HSCs, we have targeted the i4 enhancer with CRISPR/Cas9, generating the first reported genomic deletion of an endogenous cis-regulatory region in zebrafish. Deletion of the i4 enhancer leads to a decrease in endothelial gata2a expression and a concomitant transient decrease in the number of HSCs. This is marked by an early decrease in the expression of gata2b, a gata2a paralogue previously shown to be required for the initiation of the haematopoietic programme. Our results suggest non-redundant roles of both zebrafish gata2 paralogues in programming of HSCs, providing insights into different roles of GATA2 throughout the programming of HSCs. We also confirmed the previously reported loss of HSCs upon MO-mediated knockdown of lmo4a, associated with increased gata2a expression in HE. We validated the increase in gata2a levels in TALEN-generated lmo4a mutants. To identify the links between lmo4a, gata2a and the HE programming, we have profiled the transcriptome of lmo4a-deficient endothelial cells, including the HE. Our results suggest that Lmo4a may be a global regulator of the transcriptional programming of the HE. Moreover, Wnt signalling pathway may regulate gata2a downstream of lmo4a. This provides novel insights into the gene regulatory network orchestrating the generation of HSCs in the embryo.

The unfolding of recombinant DNA, from research technique to political issue, is described. As a research technique, recombinant DNA (abbreviated rDNA) has opened up new vistas in biological and other fields of research. But its potential yet unproven hazard has created uneasy feelings toward the technique. The controversial nature of the issue finally launched rDNA into the political sphere, involving scientists, the public at large, and Congress in efforts to control the development of the field. The first group to regulate rDNA was the scientists. The scientific community called for a voluntary moratorium on experiments perceived as potentially dangerous at the time. It was an unprecedented act. The National Institutes of Health subsequently issued guidelines for a safe execution of rDNA experiments to minimize potential dangers to public health and well-being. Efforts of the scientific community to control rDNA was seen, however, as a politics of expertise. Challenges to this "technocratic" approach soon emerged. Vocal members of the public suspected expert decision makers as being biased toward scientific interests, reducing rDNA to a technical issue. They rejected the experts’ tunnel vision and demanded a say in decisions. Public participation in the decision-making process precipitated community debates at locations where rDNA research was ongoing. A democratic approach to decision-making proved to be a viable policy-making mode. The ensuing local and state laws, however, seemed inadequate to cover global consequences of rDNA. In an effort to unify regulations of the field, Congress attempted to legislate on the subject. Resistance from the scientific community, which regard legislative control as rigid and unnecessary, was one of the causes of diminishing congressional interest in the matter. None of the introduced bills was enacted. For complex policy areas with uncertain yet far-reaching scientific and societal consequences -- like rDNA -- this dissertation recommends a policy-making process where scientists, interested lay persons, politicians, public administrators, and other relevant parties participate in structured communications prior to an emerging controversy. To facilitate the process, establishment of National Science Fora is recommended. / Ph. D.

Thesis (DPhil)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Human participant research raises a conflict between medical progress as a societal good and the protection of participants as an individual good. Prior to 1960 the discretionary authority for the protection of participants resided in the hands of individual investigators. However, a wave of research atrocities from Tuskegee in 1932 to the Beecher expose in 1966 stimulated a change to a principle based system of regulation. Research Ethics Committees (RECs) and Institutional Review Boards (IRBs) were henceforth charged with the responsibility of human participant protection. Since 1966, this system of research review was established internationally and at one institution in South Africa. In 1997, placebo-controlled HIV vertical transmission trials in a number of developing countries including South Africa raised unprecedented controversy in research ethics internationally and nationally. In 2000, the fraudulent breast cancer trials conducted by Dr Bezwoda at Baragwanath Hospital drew international attention to research ethics in South Africa. However, the events that called into question the efficiency of the system of ethical review most poignantly were the recent deaths of volunteers in research at centres of excellence in the United States. It was charged that if there were deficiencies in the research ethics review system in developed countries, these were more likely to be present in developing countries. Around the same time the Interim National Health Research Ethics Committee (INHREC) was established in South Africa to explore and regulate the ethical review system in South Africa. Cognisant of these issues, the current study was undertaken to establish the various structural, procedural and substantive ethical challenges facing justifiable and ethical review of research in South Africa. A combination of conceptualphilosophical reflection and empirical research was employed in this dissertation. The empirical work employed both quantitative and qualitative research methodology. The quantitative survey explored the composition of RECs reviewing clinical trials research in South Africa with an emphasis on committee composition and structure as well as the review process. The qualitative research was conducted using semi-structured interviews of ten REC Chairpersons in South Africa to explore complex substantive issues like informed consent, standards of care and participant remuneration, inter alia. While the review system in South Africa is functioning at a reasonable level, there is wide variation from one REC to the next. RECs are geographically distant and function in isolation without opportunity to communicate and share ideas. Amongst institutional RECs, there is a stark contrast between historically disadvantaged institutions and historically advantaged institutions. REC membership, ten years into democracy remains white male dominated. Community representation is inadequate. Most RECs are dominated by scientists and clinicians. The review process is widely variable with delays in review ranging from ten days to ten weeks. Procedural and bureaucratic demands impact on the ability of REC members to engage in debate on important substantive ethics issues like standards of care, informed consent and participant remuneration. Research ethics training and educational needs vary widely across the country. Serious attention must be paid to the way in which RECs are constituted in South Africa. Restructuring of RECs with a view to improving representation in terms of race, gender and religion must be prioritized. There is a need for community representation and non-scientific membership to be explored. RECs in South Africa need to revisit the question of whether they should be conducting both scientific and ethics review or ethics review alone. The review process requires a paradigm shift in emphasis from adverse event reporting to monitoring, from informed consent forms to a culturally relevant informed consent process. Aparadigm shift is indicated to shift the focus from informed consent to a more comprehensive review framework. Policies regarding standards of care and participant remuneration must be clarified and articulated. Although the role of RECs in human participant protection has been questioned, it is clear that in the vast majority of cases, they are fulfilling an important role. Their function could certainly be enhanced. This is being facilitated by training programs and an electronic newsletter. However, responsibility for human participant protection does not reside in the domain of the REC alone. A collective responsibility shared by researchers, institutions, research ethics committees, sponsors and participants is integral to human participant protection and the generation of new, valid and relevant scientific knowledge. / AFRIKAANSE OPSOMMING: Navorsing op menslike subjekte gee aanleiding tot ‘n konflik tussen mediese vooruitgang as ‘n voordeel vir die samelewing en die beskerming van deelnemers as iets waarby die individu direkte belang het. Voor 1960 het die diskresionêre gesag vir die beskerming van deelnemers by die individuele navorsers berus. ‘n Golf van navorsingsvergrype, van Tuskegee in 1932 tot die Beecher onthulling in 1966, het egter veranderinge in die rigting van ‘n stelsel van beginsel-gebaseerde regulasie gestimuleer. Navorsingsetiekkomitees (NEKs) en Institusionele Beoordelings- en toesigrade (IBRs) is gevolglik belas met die verantwoordelikheid om toe te sien dat mense wat deelneem, sover moontlik beskerm word. Sedert 1966 is hierdie stelsel van navorsingshersiening en -toesig internasionaal tot stand gebring – ook, aanvanklik, by een instansie in Suid-Afrika. In 1997 het plasebo-beheerde HIV-vertikale oordrag-proewe in ‘n aantal ontwikkelende lande, insluitend Suid-Afrika, tot ongekende kontroversie op die terrein van navorsingsetiek aanleiding gee, internasionaal en nasionaal. In 2000 het die bedrog met borskankerproewe, uitgevoer deur dr Bezwoda by Baragwanath Hospitaal, internasionale aandag op navorsing in Suid-Afrika gevestig. Hierdie gebeure het egter die effektiwiteit van die stelsel van etiese toesig in Suid-Afrika en elders in die wêreld bevraagteken. Die mees kommerwekkende onlangse insident was die dood van navorsingsvrywilligers by sentra van uitmuntendheid in die Verenigde State. Daar is beweer dat as daar tekortkominge in die navorsingsetiektoesigsisteem in ontwikkelende lande is, daar ‘n groter moontlikheid bestaan dat dit ook (en moontlik meer) in ontwikkelende lande voorkom. Ongeveer dieselfde tyd is die Interim Nasionale Gesondheidsnavorsings-etiekkomitee (INGNEK) [Interim National HealthResearch Ethics Committee (INHREC)] in Suid-Afrika gestig om die etiekoorsigstelsel in Suid-Afrika te ondersoek en te reguleer. Met dit in gedagte is die huidige studie onderneem om die verskillende strukturele-, prosedurele- en substantiewe etiese uitdagings wat regverdigbare en etiese oorsig van en toesig oor navorsing in Suid-Afrika in die gesig staar, vas te stel. Daar is van ‘n kombinasie van konseptuele, filosofiese refleksie en empiriese navorsing in hierdie proefskrif gebruik gemaak. Die empiriese werk maak gebruik van sowel kwantitatiewe as kwalitatiewe navorsingsmetodes. Die kwantitatiewe opname bestudeer die samestelling van NEKs wat toesig hou oor kliniese proewe in Suid-Afrika, met die klem op komiteesamestelling, -struktuur en die toesigproses. Die kwalitatiewe navorsing is gedoen met behulp van van semi-gestruktureerde onderhoude van tien NEK-voorsitters in Suid-Afrika om die komplekse substantiewe aspekte, soos onder andere ingeligte toestemming, standaard van versorging en deelnemervergoeding, te ondersoek. Terwyl die etiek-toesigstelsel in Suid-Afriks op ‘n redelike vlak funksioneer, is daar ‘n groot verskil tussen verskillende NEKs. NEKs is geografies verspreid en funksioneer dikwels in isolasie sonder ‘n geleentheid om te kommunikeer en idees te deel. Ten opsigte van die institusionele NEKs bestaan daar ‘n duidelike kontras tussen histories benadeelde instansies en histories bevoordeelde instansies. NEK-lidmaatskap word, tien jaar na demokrasie, steeds gedomineer deur blanke mans. Gemeenskapsverteenwoordiging is onvoldoende. Die meerderheid NEKs word gedomineer deur wetenskaplikes en klinici. Die toesig- en hersieningsprosesse in die verskillende komitees verskil grootliks, met vertragings wat wissel van 10 dae to 10 weke. Prosedurele- en burokratiese vereistes het ‘n impak op die vermoëns van NEK-lede om by debatte oor belangrike substantiewe etiese aangeleenthede betrokke te raak, soos byvoorbeeld die standaard van versorging, ingeligte toestemming en deelnemervergoeding. Opleiding en opvoedkundige behoeftes verskil wyd oor die land.Ernstige aandag moet geskenk word aan die wyse waarop NEKs in Suid-Afrika saamgestel is. Herstrukturering van NEKs met ‘n visie op verbeterde verteenwoordiging in terme van ras, geslag en geloof is ‘n prioriteitsvereiste. Gemeenskapsverteenwoordiging en lidmaatskap van nie-wetenskaplikes moet verder ondersoek word. NEKs in Suid-Afrika moet die vraag of hulle sowel wetenskaplike- as etiektoesig moet uitvoer, of sl slégs etiektoesig, opnuut ondersoek. Die nasiensproses vereis ‘n paradigmaskuif, vanaf ‘n klem op rapportering van gebeurtenisse, na monitering van ingeligte toestemmingsvorms sowel as na ‘n kultureel toepaslike ingeligte toestemmingsproses. ’n Paradigmaskuif is noodsaaklik ten einde die fokus te verskuif vanaf ingeligte toestemming na ‘n meer omvattende toesig- en nasiensraamwerk. Beleid rakende standaard van versorging en deelnemervergoeding moet verduidelik en geartikuleer word. Alhoewel die rol van NEKs in die beskerming van menslike deelnemers aan navorsing bevraagteken word, is dit duidelik dat NEKs in die meerderheid van gevalle wel ‘n belangrike rol vervul. Hul funksie kan natuurlik uitgebrei word. Dit sal gefasiliteer word deur opleidingsprogramme en ‘n elektroniese nuusbrief. Verantwoordelikheid vir die beskerming van mense wat deelneem aan navorsing berus egter nie uitsluitlik by NEKs nie. ‘n Kollektiewe verantwoordelikheid, gedeel deur navorsers, instellings, navorsingsetiekkomitees, borge en deelnemers is ‘n integrale vereiste vir hierdie beskerming sowel as vir die verwerwing van nuwe, geldige en relevante wetenskaplike kennis.