O objetivo desta pesquisa foi determinar a influência do uso da plasmaferese sobre o tempo de recuperação clínica e hematológica de caprinos doadores de sangue total ou plasma. Para tanto, foram utilizados 20 caprinos adultos e clinicamente sadios, distribuídos por dois grupos de 10 animais cada, a saber: grupo controle (de animais doadores de sangue total não tratados) e grupo experimental (de animais doadores que foram tratados através da plasmaferese). Os caprinos foram selecionados e monitorados através de exames físicos (funções vitais) e complementares (hemograma, proteínas totais, albumina, globulinas, relação A:G, uréia, creatinina e hemoglobina livre no plasma) realizados nos seguintes momentos: imediatamente antes e após a doação de sangue: 12 h, 24h, 72h, 120h, 360h, 480h, e 720 horas após os procedimentos. Os resultados foram analisados com comparações dentro e entre os dois grupos nos diferentes momentos do estudo. As observações clínicas efetuadas durante o período de até trinta dias após a doação de 20% do volume sangüíneo total, com ou sem a realização da plasmaferese nos animais dos grupos estudados não sofreram variações influenciadas por esses procedimentos. Observou-se significativa variação dos componentes do eritrograma, tendo o grupo experimental apresentado as melhores taxas de recuperação em função do tempo. Com base nos resultados obtidos, a aplicação da técnica da plasmaferese em caprinos mostrou-se eficiente como recurso para a otimização do tempo de recuperação dos valores do hemograma de animais doadores de plasma, não determinando hemólise durante o seu procedimento / The objective of this study was to determine the influence of plasmapheresis on clinical and haematological recovery time of whole blood or plasma donor goats. For this, 20 clinically healthy adult goats were divided into two groups of ten animals each: control group (not-treated whole blood donor animals), and experimental group (donor animals which were treated with plasmapheresis). Goats were selected and evaluated through physical examination (vital functions) and complementary tests (haemogram, total proteins, albumin, globulin, albumin:globulin ratio, urea nitrogen, creatinine, and plasma free haemoglobin), carried out at the following moments: immediately before and after blood donation, 12, 24, 72, 120, 360, 480, and 720 hours after the procedures. Results were analysed comparing animals in and between both groups (at differents moments of the study). The clinical observations made during the period of thirty days after donation of 20% of total blood volume, with or without plasmapheresis in the animals of studied groups, were not influenced by these procedures. The results revealed significant variation of eritrogram components, showing the experimental group to have better recovery rates according to time. Based on the results obtained in the present study, plasmapheresis technique application in goats showed to be efficient as a resource to optimize recovery time of haemogram values of plasma donor animals, and did not cause hemolisis during its procedure

Multipotent haematopoietic stem cells (HSCs) supply the organism with mature blood cells of all lineages throughout adult life. These cells first originate in the dorsal aorta (DA) of the vertebrate embryo, and a multitude of signalling pathways regulate their specification in the embryo. The emergence of HSCs is dependent on appropriate arterial specification and vessel maturation, processes which are heavily dependent on Notch signalling. This arterial involvement of Notch obscures its later roles in HSC specification. The Notch ligand dll4 is crucially involved in arterial development in the mammalian embryo, while zebrafish embryos deficient for dll4 activity only exhibit minor arterial defects at the time of HSC emergence. Here, the zebrafish model has been exploited to reveal the first specific evidence for a role of dll4 in HSC specification. Dll4 is required for the expression of runx1, a transcription factor (TF) required for HSC specification, prior to any observed effects on vascular development. HSCs and all their derivatives are depleted in dll4 morphants. To disentangle the genetic requlatory cascade downstream of dll4 and upstream of runx1, RNA-seq was employed to discover downstream effectors of this signalling. Expression and functional screening of best candidate genes revealed seven genes with novel roles in HSC development. Foxc1b is a dll4 target predominantly mirroring the dll4 phenotype, and is thus likely to be the downstream effector of dll4, upstream of runx1. Interestingly, foxc1b also has a later dll4-independent role in HSC development, remarkably similar to that of cmyb. Taken together I show here for the first time a requirement of dll4 upstream of runx1 in HSC specification, mediated by foxc1b, followed by a later dll4-independent phase in HSC development.

Multipotent haematopoietic stem cells (HSCs) originate in the dorsal aorta (DA) during vertebrate embryogenesis, and after migrating to a permanent niche, give rise to a continuous supply of mature blood cells of all lineages throughout adult life. Previous cell tracing experiments have shown that the cells of the DA migrate here from an early collection of haemangioblasts (bipotential precursors of blood and endothelial cells) which reside in the dorsolateral plate (DLP) mesoderm. Development of HSCs is tightly regulated by a number of key signalling pathways in both the DLP and the DA. In particular, notch signalling is considered an important factor in vascular, arterial and HSC development. Here, the relatively slow development and the spatial separation of definitive haematopoiesis from primitive haematopoiesis in Xenopus laevis has been exploited to reveal the first defect of reduced notch signalling in the Xenopus DA. Two notch inputs to HSC programming have been identified in Xenopus: notch4 and its target genes, esr7 and esr10, are expressed from stage 31, immediately after migrating haemangioblast cells reach the midline of the embryo to form the DA, whilst notch1 is expressed slightly later, from stage 34, and controls expression of two further notch target genes, esr1 and hesr1. Using both morpholino knockdown of these six genes, and chemical inhibition of notch signalling using a specific γ-secretase inhibitor, notch signalling has been demonstrated to be essential for HSC programming but dispensable for earlier haemangioblast and arterial programming. Furthermore, esr1, downstream of both notch1 and notch4, is shown to be responsible for repression of endothelial genes in the DA. Taken together, this demonstrates that a cascade of notch and notch effector genes are essential for the programming of Xenopus HSCs.

Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The production and export of ostrich meat from southern Africa, to especially the European Union, are increasing rapidly due to the healthy nature of ostrich meat. The European Union has very high standards when importing food products, and it is inevitable that more emphasis is being placed on the production of high quality ostrich meat. Another aspect also of concern to consumers, is the welfare of animals prior to slaughter, and this forces producers to look at ways to decrease stress of animals during the ante-mortem period. Research regarding the effect of stress during the ante-mortem period, and as a result, on meat quality, haematology and weight loss in ostriches, is lacking and thus the purpose of this study was to investigate the effect of various transport distances, travel conditions and different birds on these factors. Ante-mortem stress was measured using serum corticosterone levels (ng/ml), heterophil: lymphocyte (H:L) ratio, white blood cell (WBC) count, aspartate aminotransferase (AST) and creatine kinase (CK), as well as the rate and extent of pH decline in the M. gastrocnemius. Special emphasis was also placed on the meat quality parameters drip loss, cooking loss, colour and Warner-Bratzler shear force (kg/1.27 cm diameter). Live weight losses, as well as carcass weight and weight of bruises cut off from each bird were also recorded during various stages of the trials. The effect of transport distance on the meat quality of ostriches was investigated. Ultimate pHu measurements were taken at 24 hours post-mortem. The muscles of the ostriches from the control group (i.e. birds that were not transported prior to slaughter) had a lower mean pHu (5.77 ± 0.053) than birds that travelled 60 (5.93 ± 0.053) and 600 km (6.11 ± 0.053), respectively. Differences in meat drip loss percentage were also observed between the three treatments. The birds in the control group (0.40 ± 0.07 %) had the lowest meat drip loss percentage compared to the birds that travelled 60 km (treatment C) (1.36 ± 0.07 %) and 600 km (treatment B) (0.97 ± 0.07 %), respectively, to a commcerical ostrich abattoir. Ostriches that were transported for 600 km (8.13 ± 1.16 %) had a greater percentage live weight loss during the antemortem period than birds that travelled a distance of 60 km (2.4 ± 2.185 %) to the abattoir, although both groups were deprived of feed for the same period. When the haematology of the groups that travelled different distances was compared at various time intervals in the ante-mortem period, both groups of birds experienced significant increases in WBC, s-AST and s-CK. An increased H:L ratio from pre-transport to post-transport was only evident in the birds that travelled 600 km (treatment B). However, the birds that travelled 60 km were the only group of birds that had significant elevated serum corticosterone levels during the ante-mortem period. The increase in the various blood parameters indicates severe physical stress, which negatively affected meat quality. Another trial also investigated the effect of various farming systems and transport on meat quality and bruising of ostrich carcasses. Ostriches were raised in three different farming systems, i.e. feedlot -, semiintensive - and free range conditions. Other factors that could maybe impact on stress susceptibility, such as road conditions, floorspace and floor type were also investigated. A significant difference in meat pHu was found between ostriches that were raised in a feedlot (5.95 ± 0.018) and semi-intensive (6.04 ± 0.033) environment. The feedlot birds also had the greatest percentage of carcass weight removed due to bruising. The free range birds were the birds that had the lowest floor density per birds and also had the least amount of bruising on their carcasses. Incidently the other two groups (feedlot and semi intensive) were the birds that travelled on the same type of road (mountain pass) in a truck with rubber flooring whilst the free range birds travelled on a straight road in trucks with metal grid floors.The results indicate that the type of farming system didn’t have a significant influence on meat quality of ostriches, but that factors such as road conditions, flooring and bird density did play a significant role in the incidence of bruises and injuries obtained during transport. / AFRIKAANSE OPSOMMING: Die produksie en uitvoer van volstruisvleis vanuit suidelike Afrika, na veral die Europese Unie, is gedurig aan die toeneem as gevolg van die gesonde aard van volstruisvleis. Die Europese Unie het baie hoë standaarde wanneer dit kom by die invoer van voedselprodukte en dit is onvermydelik dat meer klem op die produksie van hoë gehalte volstruisvleis gelê word. ʼn Ander aspek wat ook kommer wek by verbruikers is die welstand van diere voor slagting en hierdie aspek noodsaak produsente om te kyk na maniere om stres te beperk tydens die periode voor slagting. Navorsing rakende die effek van stres tydens die ante-mortem periode, asook vleiskwaliteit, hematologie en gewigsverlies in volstruise as gevolg van vervoer, ontbreek. Die doel van die studie was dus om die invloed van verskillende vervoerafstande, vervoersomstandighede en tipe produksiesisteme op volstruise se stresrespons te ondersoek. Die omvang van ante-mortem stres is bepaal deur die serum-kortikosteroon vlakke (ng/ml), heterofiel: limfosiet (H:L) ratio, witbloedsel (WBS) telling, aspartaat aminotransferase AST en creatien kinase CK, asook die tempo en vlak van pH-daling in die M. gastrocnemius, te meet. Spesiale klem is gelê op die vleisgehalte parameters kookverlies, drupverlies, kleur en Warner-Bratzler-skeurwaardes (kg/1.27 cm deursnee). Gewigsverlies is aangeteken op verskillende stadiums tydens die proewe. Karkasgewigte en die hoeveelheid gewig afgesny van elke volstruiskarkas a.g.v. kneusings is ook bepaal. Die eerste studie het die invloed van vervoerafstand op vleiskwaliteit van slagvolstruise ondersoek. Vleis kwaliteit parameters soos pH, drip verlies, kook verlies, taaiheid en kleur is ondersoek. Die pHu metings is op 24 uur post-mortem geneem. Slagvolstruise in die kontrole groep (d.i. -volstruise wat nie voor slagting vervoer is nie) het ’n laer vleis pHu (5.77 ± 0.05) gehad as voëls wat onderskeidelik 60 km (5.93 ± 0.05) en 600 km (6.11 ± 0.05) ver vervoer is. Verskille in persentasie dripverlies is gesien tussen die vleis van die voëls wat nie vervoer is nie (0.40 ± 0.07 %) en die voëls wat 60 km (1.36 ± 0.07 %) en 600 km ver (0.97 ± 0.07 %) onderskeidelik vervoer is. Volstruise wat vir 600 km (8.13 ± 1.16 %) vervoer is, het ‘n groter persentasie lewende gewig tydens die ante-mortem periode as voëls wat 60 km (2.4 ± 2.19 %) ver vervoer is na die abattoir, verloor, al was beide groepe weerhou van voer vir dieselfde tydperk. Beide groepe wat vervoer is (60 en 600 km) het merkbare toenames in witbloedsel (WBS) tellings, s-AST’s en s-CK’s getoon tydens die ante-mortem periode. Daar is slegs ʼn toename in H:L ratio (ʼn indikator van stres) van voor vervoer tot na vervoer gesien in die voëls wat 600 km vervoer is. Daarteenoor was die voëls wat slegs 60 km vervoer is die enigste voëls wat ʼn toename in korticosteroon vlakke getoon het gedurende die ante-mortem periode. Die toenames is heel moontlik ‘n aanduiding van erge fisiese stres wat ‘n negatiewe effek op vleiskwaliteit het. Die tweede studie het die effek van verskillende produksiesisteme en die stress respons van die verskillende groepe slagvolstuise op vervoer ondersoek. Vleis kwaliteit parameters soos pH, drip verlies, kook verlies en taaiheid is ondersoek. Die hoeveelheid kneusings per volstruis is ook gemeet. Daar was ‘n beduidende verskil (P = 0.009) tussen die pHu van die voerkraal (5.95 ± 0.018) en semi- intensiewe (6.04 ± 0.033) volstruise. Die voerkraal volstruise se vleis het die grootste drip- en kookverliese gehad in vergelyking met die ander twee groepe (semi-intensiewe en ekstensiewe) terwyl die ekstensiewe volstruise die taaiste vleis gehad het. Die voerkraalvoëls het ook die grootste persentasie karkasgewig verloor a.g.v. kneusings wat afgesny is. Die ekstensiewe voëls het die laagste vloer digtheid per volstruis gehad asook die minste kneusings. Die ander twee groepe (voerkraal en semi intensief) was die groepe wat op dieselfde pad vervoer is (bergpas) in vragmotors wat rubber vloere gehad het terwyl die ekstensiewe voêls op ‘n reguit pad vervoer is in ‘n vragmotor met ‘n metaal oppervlakte. Die resultate van die studie is ‘n aanduiding dat die tipe plaassisteem nie ‘n groot impak op die hoeveelheid akute stres ervaar deur die voëls tydens vervoer gehad het of gevolglik op die vleiskwaliteit van die volstruise nie, maar dat faktore soos pad toestand, tipe vloer en voëldigtheid wel ‘n wesenlike rol speel in die voorkoms van kneusings en beserings opgedoen tydens vervoer.

Einleitung: Alpakas gehören zu einer Tiergruppe, die in den vergangenen Jahren im Patientengut der Tierärzte immer häufiger anzutreffen ist. Daher ist es von großer Bedeutung, sich mit der Physiologie und Pathologie dieser Tierart zu beschäftigen. Die Versorgung der Neonaten spielt dabei eine große Rolle. Da Alpakacrias, wenn überhaupt, dann erst sehr spät, klinische Anzeichen einer Erkrankung zeigen, ist es für den Untersucher von großem Nutzen, einen Einblick in den Blutgas- und Säure-Basen-Haushalt, sowie Kenntnis von den hämatologischen und blutchemischen Parametern des Neonaten zu erhalten. Des Weiteren bietet die Blutuntersuchung ein wichtiges Hilfsmittel zur Überprüfung der Versorgung mit kolostralen Antikörpern. Ziele der Untersuchungen: Ein Ziel der Untersuchungen war die Erstellung von bisher nicht vorhandenen Referenzwerten für die venöse Blutgasanalyse, die Hämatologie und für einige blutchemische Parameter bei gesunden, lebensfrischen Alpakacrias innerhalb der ersten drei Lebenstage. Des Weiteren sollte in diesem Zusammenhang die Eignung eines mobil einsetzbaren Blutgasanalysegerätes (epoc®, Fa. Alere GmbH, Köln) für die Tierart Alpaka eruiert werden. Hierfür wurden Doppelbestimmungen der Proben mit einem etablierten stationären Blutgasanalysegerät (ABL90 FLEX®, Fa. Radiometer, Kopenhagen) durchgeführt. Tiere, Material und Methoden: In die Studie wurden 20 gesunde neugeborene Alpakacrias integriert. Die Fohlen stammten vornehmlich aus Stuten, die zur Geburtsüberwachung in die Klinik eingestallt wurden, bei anderen handelte es sich um solche, die innerhalb der ersten Lebensstunden wegen vermeintlich verzögerter Tränkeaufnahme vorgestellt worden waren, was sich aber nicht bestätigte. Alle Alpakafohlen wurden nach dem Gießener Vorsorgeschema II für neonatale Equiden klinisch untersucht. Anschließend erfolgte 3-8 Stunden p. n., 24 Stunden p. n. und 72 Stunden p. n. je eine Blutprobenentnahme. Der erste Analysezeitpunkt wurde bewusst nicht unmittelbar post natum gewählt, da die Etablierung einer stabilen Prägungsphase zwischen Muttertier und Cria nach der ersten Tränkeaufnahme abgewartet werden sollte. Die Blutentnahme erfolgte nach Reinigung und Desinfektion aus der ungestauten V. jugularis externa im Bereich des sechsten Halswirbels. Die Blutgasanalyse wurde innerhalb von 15 Minuten mit den zuvor genannten Blutgasautomaten durchgeführt. Die hämatologischen Parameter wurden mit dem pocH-100 iV (Fa. Sysmex Deutschland GmbH, Norderstedt) bestimmt, die blutchemischen Untersuchungen erfolgten mit dem FUJI DRI CHEM 3500 (Fa. Sysmex Deutschland GmbH, Norderstedt). Insgesamt wurden 55 Blutproben entnommen und analysiert. Pro Analysegerät (epoc®, ABL90 FLEX®, pocH-100 iV, FUJI DRI-CHEM 3500) wurden je 55 Messungen durchgeführt. Die statistische Auswertung erfolgte mit dem Programm IBM SPSS Statistics 22.0. Die Normalverteilung wurde mittels Shapiro-Wilk-Test überprüft. Für den Gerätevergleich (epoc®/ABL90 FLEX®) fand der Wilcoxon-Test Anwendung. Der Vergleich der Zeitpunkte erfolgte über den Friedman-Test für verbundene Stichproben. Des Weiteren wurden für die einzelnen Parameter der Median und die Perzentile, bzw. der Mittelwert und die Standardabweichung bestimmt. Die grafische Darstellung erfolgte mit Boxplots und Bland-Altman-Plots. Ergebnisse: Im Gerätevergleich konnte insgesamt auf eine gute Übereinstimmung der Messwerte geschlossen werden. Signifikante und gleichzeitig klinisch relevante Unterschiede gab es lediglich bei der Bestimmung der Sauerstoffsättigung und des Hämatokrits, was auf unterschiedliche Mess- und Berechnungsmethoden bei den Geräten zurückzuführen ist. So liegen die Hämatokritwerte beim epoc® deutlich unter denen vom ABL90 FLEX®. In diesem Zusammenhang wichtig für die Interpretation der Ergebnisse ist, dass für jedes Messgerät die individuellen Referenzbereiche berücksichtigt werden müssen. Die venöse Blutgasanalyse ergab für gesunde Crias zu Beginn des ersten Lebenstages einen pH-Wert von 7,34 – 7,40, einen Sauerstoffpartialdruck (pO2) von 21,6 – 29,2 mmHg, einen Kohlendioxidpartialdruck (pCO2) von 37,3 – 46,0 mmHg, eine Sauerstoffsättigung (sO2) von 30,5 – 48,0 %, ein aktuelles Bikarbonat (HCO3-) von 21,3 – 25,1 mmol/l, eine Standardbasenabweichung (SBE) von -3,3 – 0,2 mmol/l und einen Laktatgehalt von 1,6 – 3,4 mmol/l. Der pH Wert ähnelte im Verlauf dabei dem von Kälbern und Lämmern gleichen Alters, der pO2 war insgesamt etwas niedrig, aber konstant und ähnelte über den Messzeitraum dem von Kälbern. Es wurden bei den Crias im Vergleich zu Fohlen, Kälbern und Lämmern niedrigere pCO2-Werte festgestellt. Die Sauerstoffsättigung ähnelte der von Equidenfohlen, über den Messzeitraum fällt die Konzentration im Mittel geringfügig ab, bei den anderen Vergleichstierarten steigt sie an. Die Glukosekonzentrationen waren postnatal höher als bei anderen Haustierneonaten (3-8 h p.n.: 4,4 – 8,2; 24 h p.n.: 7,3 – 12,8; 72 h p.n.: 7,3 – 16,2 mmol/l). Laktat kann nicht, wie es beim Equidenfohlen postuliert wird, als Indikator für den Gesundheitszustand eines Alpaka-Crias genutzt werden. Hämatologisch sind die spezielle Form und die hohe Anzahl der Alpakaerythrozyten, die hohe Zahl an Leukozyten und Thrombozyten (speziell bei den Crias), sowie die hohe MCHC zu nennen. Schlussfolgerungen: Es konnten teilweise bisher fehlende Daten zur venösen Blutgasanalyse für die Beurteilung der Stoffwechsellage neugeborener Alpakacrias etabliert werden. Das mobile Blutgasgerät epoc® stellt eine für die Anwendung geeignete Alternative dar.

Acute myeloid leukemia (AML) develops following oncogenic alterations to hematopoietic stem (HSC) and progenitor cells (HSPCs) in the bone marrow, resulting in dysregulated proliferation of immature myeloid progenitors that interferes with normal hematopoiesis. Understanding the mechanisms of HSPC protection against damage and excessive division, and how these pathways are altered during leukemic progression, is vital for establishing effective therapies. Here, we show that autophagy, a lysosomal degradation pathway, is increased in HSPCs using a novel imaging flow cytometry autophagy assay. Loss of hematopoietic autophagy following deletion of key gene Atg5 resulted in increased HSC proliferation, leading to HSC exhaustion and bone marrow failure. Although erythrocyte and lymphocyte populations were negatively impacted by autophagy loss, myeloid cells showing immature characteristics were expanded. Deletion of Atg5 in an AML model resulted in increased proliferation under metabolic stress, dependent on the glycolytic pathway, and aberrant upstream mTOR signaling. Moreover, modulation of Atg5 altered leukemic response to culture with stromal cells. Finally, primary AML cells displayed multiple markers of decreased autophagy. These data suggest a role for autophagy in preserving HSC function, partially through suppression of HSPC proliferation, and indicate that decreased autophagy may benefit AML cells. We postulate that modulation of autophagy could help maintain stem cell function, for example during transplantation, and aid AML therapy in a setting-specific manner.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IEs) is a common property of field isolates associated with severe disease (Pain, Ferguson et al. 2001). Platelet receptors CD36 (Pain, Ferguson et al. 2001), P-Selectin (Wassmer, Taylor et al. 2008) and gC1qR (Biswas, Hafiz et al. 2007) mediate clumping. To characterize the molecular specificities of the clumping phenotype, I cloned clumping parasite line IT/C10 by limiting dilution. I characterized var gene expression in the IT/C10 clones using generic primers for the DBL tag region (Bull, Berriman et al. 2005). Clumping assays were conducted in the presence of specific reagents to delineate host factors hypothesized to contribute to development of the clumping phenotype. Finally, I conducted a clinical study with isolates from children with malaria in Kilifi, Kenya. This study shows that in parasite line IT/C10, platelet-mediated clumping is associated with Itvar30 suggesting a prominent role for the PfEMP-1 encoded by this var gene in development of platelet-mediated clumping. For IT/C10 parasites, platelet activation appears to be involved in platelet-mediated clumping. Platelet P-Selectin appears to mediate clumping using lectin-dependent interactions. To further elucidate the mechanisms that mediate clumping by host platelets, I have used a panel of platelet antagonists to delineate specific platelet activation pathways. Our results show that platelet activation plays an important role in platelet-mediated clumping. Finally, in this study, platelet-mediated clumping was associated with parasitaemia, but not with disease severity.

The role of Notch in cell fate determination and lineage restriction in the bone marrow (BM) is controversial in the field. Recent studies have convincingly shown that Notch is dispensable for haematopoietic stem cell (HSC) regulation in adult haematopoiesis (Maillard et al., 2008). In contrast, Notch signaling has been proposed to be of importance in the regulation of BM megakaryocyte progenitor differentiation, based on dominant negative genetic approaches, identifying a potentially distinct role for Notch in adult BM haematopoiesis (Mercher et al., 2008). Here, I found that by selectively ablating the gene coding the transcription factor recombination signal-binding protein J kappa (RBP-Jk), to which all canonical Notch signaling converges, canonical Notch signaling does not mediate HSC maintenance, neither in steady state nor in conditions of stress. Furthermore, I propose, in contrast with previous studies (Mercher et al., 2008), that canonical Notch signaling plays no role in myeloerythropoiesis cell lineage commitment in the BM. My data also show that key Notch target genes are suppressed by RBP-Jk, as their expression is unaffected in Notch1-deficient BM progenitors, while target genes are upregulated in Rbp-Jk-deleted megakaryocyte and erythroid progenitors. This establishes for the first time in mammalian cells in vivo, that Notch target genes are kept in a suppressed state by RBP-Jk, potentially restricting T cell commitment to the thymus and not to the BM, at the expense of myeloerythropoiesis. Notch signaling and GATA3 are two master regulators in T cell commitment (Han et al., 2002; Ho et al., 2009; Pui et al., 1999; Radtke et al., 1999; Zhu et al., 2004). However, although very well established as being involved in the thymic stages of T cell restriction, there is little evidence of Notch and GATA3 being involved in the migration of a thymus settling progenitor (TSP) from the BM to the thymus or in the establishment of the earliest thymic progenitor (ETP) in the thymus. From this thesis work, I conclude that Notch signaling is essential for the emergence of ETPs in the thymus in a NOTCH1-independent manner. Moreover, I demonstrate, as supported by a very recent published study (Hosoya et al., 2009), that GATA3 is important for the development of the earliest T cell progenitor. GATA1 and GATA2 mediate haematopoietic stem cell maintenance in the BM. GATA1 is required for erythropoiesis, megakaryocytes and eosinophils while GATA2 is important for the proliferation and survival of HSCs. In contrast, a role for GATA3 in the BM has never been established. By using a Gata3-conditional knockout mouse model, I demonstrate that GATA3 is dispensable for HSC maintenance in steady state and following active haematopoietic regeneration as well as for HSC self-renewal in the BM.

Allogeneic haematopoietic stem cell transplantation (alloHSCT) is an established therapy for many haematological disorders. Unfortunately, the new donor-derived immune system may damage host cells (graft-versus-host disease (GvHD)), causing significant morbidity and mortality. Since regulatory T cells (Tregs) can modulate immune responses, it was hypothesised that Treg numbers in the haematopoietic stem cell grafts and/or peripheral blood may influence the development of GvHD and other transplant-related complications. In this project, a prospective observational clinical study of putative Tregs in human alloHSCT was performed in Oxford. Flow cytometry and methylation-specific qPCR assays were developed to quantify putative Tregs and lymphocyte populations within the grafts and post-transplant blood samples. Although low CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell numbers were not associated with increased incidence of GvHD, low proportions of CD25(+)FOXP3(+)CD127(-/dim) cells in the graft (as a percentage of total CD4(+) T cells) were independently associated with poor engraftment, increased non-relapse mortality and inferior overall survival. Similarly, falling CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T-cell counts over the first three months post-transplant were associated with higher non-relapse mortality and inferior overall survival. In view of these novel findings, strategies that increase CD4(+)CD25(+)FOXP3(+)CD127(-/dim) T cells in alloHSCT may improve clinical outcomes. One possible route for increasing Tregs is through cellular therapy. This project therefore tested the hypothesis that CD4(+)CD25(+)FOXP3(+) Tregs can be produced in vitro from conventional CD4(+) T cells. In the presence of TGFβ and Azacitidine, FOXP3 was expressed in the majority of activated CD4(+) T cells. These cells also had a demethylated FOXP3 TSDR enhancer which is specific to natural Tregs. However, most of these cells produced pro-inflammatory cytokines, for example, TNFα. Therefore, under these conditions, FOXP3 expression was not sufficient to produce a Treg phenotype. It is proposed that current focus for generating Tregs for human clinical trials should be directed towards improving isolation and expansion of ex vivo isolated Tregs.

Haematopoietic stem cells (HSC) are strictly regulated by intrinsic regulators and extrinsic signals from the microenvironment. Nov (CCN3), a matricellular protein of the CCN family, has been reported as a suppressor gene in solid tumours and chronic myeloid leukaemia (CML). Recent study identified Nov as a positive regulator in human cord blood CD34+ stem cells. However, the functions of Nov in haematopoiesis and adult HSC remain largely unknown.