The alternative road map describes the separation of lympho-myeloid and myeloid-megakaryocyte-erythroid (myeloid-Mk-E) lineages as the earliest haematopoietic commitment event. However, a number of aspects of this lineage restriction process remain poorly understood. Herein this work identified a lympho-myeloid restricted progenitor in the embryo, which resembles the adult LMPP, and demonstrated that lymphoid lineage restriction is initiated prior to definitive haematopoiesis, much earlier than previously appreciated. In vivo fate mapping showed that lympho-myeloid progenitors significantly contribute to steady state myelopoiesis in the embryo. The early thymic progenitor (ETP) as most primitive cell in the thymus was characterised and demonstrated to sustain B, T and myeloid but not Mk potentials at the single cell level. The ETP therefore largely resembles the cellular properties of lympho-myeloid progenitors in bone marrow and foetal liver, which points to these cells as candidate thymus seeding progenitors (TSP). Furthermore the existence of a putative Mk progenitor was explored within the LSKCD150⁺CD48⁺Gata1^pos compartment of a Gata1 reporter mouse providing the basis for a future prospective characterisation. Finally, this work evaluated the earliest lineage restriction of von Willebrand factor (Vwf)-EGFP⁺ and EGFP^- haematopoietic stem cells (HSCs) through in vitro paired daughter fate mapping. Single Vwf⁺ HSCs showed heterogeneous Mk priming and more frequently sustained Mk potential after cell division. Moreover, analysis of lineage priming between daughter cells revealed the asymmetric expression of key lineage determinants and stem cell regulators, which might be employed as reporters for future fate mapping studies.

Haematological malignancies and their treatment lead to prolonged periods of severe thrombocytopenia (platelet count ≤ 50 x 10⁹/l). Despite the use of prophylactic platelet transfusions, haemorrhage remains an important complication during this thrombocytopenic period. Within a 30 day period up to 70% of patients have clinically significant haemorrhage (World Health Organization (WHO) grade 2 or above bleeding) and up to 10% have severe or life-threatening haemorrhage (WHO grade 3 or 4 bleeding). Hence our current management of these patients to prevent haemorrhage is sub-optimal. The aim of this thesis was to identify clinical and laboratory factors that may predict the risk of haemorrhage in patients with haematological malignancies and severe thrombocytopenia. This was achieved via several different study designs and assessed the effect of clinical and laboratory factors on any or clinically significant haemorrhage and their effect on intracranial haemorrhage. This thesis has demonstrated that there is no consensus on how bleeding is assessed and graded in this patient group. Also it showed that the absolute immature platelet number may be a better alternative to the total platelet count to guide administration of platelet transfusions. Female sex, a previous history of a fungal infection, a high C-reactive protein, a high white cell count, a low platelet count, anaemia, impaired renal function, and recent clinically significant haemorrhage were all found to be independent risk factors for haemorrhage. Patients who were in complete remission from their haematological malignancy had a much lower risk of bleeding.

Acute Upper Gastrointestinal Bleeding (AUGIB) accounts for 7000 deaths in the UK annually and is the single leading indication for transfusion of blood components. A large UK audit in 2007 reported high case fatality and rates of further bleeding. Since many deaths are determined by pre-existing co-morbidity, strategies to improve outcome should be targeted at preventable deaths and therefore focus upon improved control of haemorrhage and prevention of further bleeding, which are investigated in this thesis. Data for the analyses presented originate from the UK national audit of AUGIB, a laboratory study and a cross sectional survey. Five broad themes were investigated including service provision and timing of endoscopy, the use of transcatheter arterial embolisation (TAE) or surgery for refractory bleeding, the impact of coagulopathy on outcome, management of acute variceal haemorrhage (AVH) and haemostatic derangements after AVH, and the use of red blood cells (RBCs). Although there was no evidence of a “weekend effect” for mortality, earlier endoscopy (<12 hours) was associated with improved control of haemorrhage in higher risk patients compared to later endoscopy (>24 hours). TAE was an effective and safe modality for refractory bleeding, but the high post-surgical mortality (29%) raises questions about the appropriateness of case selection for surgery. Coagulopathy after non-variceal haemorrhage was associated with a 5-fold increase in risk-adjusted mortality. Further bleeding after AVH was strikingly high (26%) with notable deficiencies in the use of vasopressors, antibiotics and endotherapy. Global assessments of coagulation demonstrated that thrombin generation after AVH was normal, but clot strength was poor with excessive fibrinolysis. Platelets, fibrinogen and antifibrinolytics improved haemostasis ex vivo but coagulation factor transfusion had no effect. RBC transfusion practice is variable. This work on AUGIB provides new data highlighting areas of sub-optimal care, and informs both current practice and research questions for new interventional trials.

Antarctic fish from the suborder Notothenioidei inhabit what is perhaps the most thermally stable ocean environment on Earth. Evolutionary theory suggests that by specialising for this environment, Antarctic fish have traded-off their ability to respond to variations in temperature, and like their environment, have become extremely stenothermal. However, previous research has revealed that the Antarctic notothenioid fish Pagothenia borchgrevinki is not as thermally limited as evolutionary theory might predict, and is capable of acclimation to 4 ℃ during a one month period. The purpose of the current research was to investigate the physiological mechanisms that underpin this remarkable acclimatory ability. P. borchgrevinki were acclimated for one month to 4 ℃ and changes in oxygen consumption, prolonged swimming ability, cardiovascular function, enzyme activity and haematology were measured. Significant changes in resting oxygen consumption rate and prolonged swimming ability occurred during the acclimation period, and these changes were mediated by adjustments of enzyme activity and specific aspects of the haematology. By monitoring resting oxygen consumption and prolonged swimming ability over a much longer, six month, acclimation period it was confirmed that the adjustments evident during one month at 4 ℃ were sustainable in the long-term, and were not short-term compensatory mechanisms. Interestingly, fish infected with x-cell gill disease did not possess the same ability to acclimate as was demonstrated by healthy P. borchgrevinki. P. borchgrevinki are unusual among the notothenioids, possessing an active, pelagic lifestyle which differs from the sedentary, benthic lifestyle of most other species within the suborder. Therefore, it was hypothesised that the acclimatory ability demonstrated by this species may also be unusual among the notothenioids. To test this hypothesis, the acclimation ability of three sedentary, benthic notothenioids (Trematomus bernacchii, T. hansoni and T. pennellii) was investigated. Results confirmed the hypothesis, with all three species demonstrating very poor survival at 4 ℃ and absolutely no capacity for acclimation. Such results present a disturbing scenario for the future of Antarctic notothenioid fish in Earth?s rapidly warming climate, and highlights the need for continued research combined with immediate action to combat the warming which currently threatens Antarctic marine biodiversity.

TRAP1 is a Heat Shock Protein (HSP) chaperone to retinoblastoma but also associated to the tumor necrosis factor receptor. HSPs are primarily up regulated in cancer. Work in our lab noted a down regulation of TRAP1 in some non-small cell lung cancers compared to normal lung. The first aim of this project was to evaluate the effect of the loss of TRAP1 on cell proliferation using a spheroid model. The presence of TRAP1 in spheroids promoted cell proliferation and a faster onset of hypoxia. This suggests an oncogenic role for TRAP1 since rapid hypoxia development equates to poor prognosis. Micro array analysis showed that TRAP1’s loss was associated with increased transcrpition of the Junctional Mediating and Regulatory protein (JMY). JMY possesses an oncogenic property due to its ability to facilitate cell motility. Additionally it has tumor suppressor activity in promoting p53 activation. The second aim of this project was to produce an anti-JMY antibody and use it to characterize JMY and additionally verify the association between TRAP1 and JMY. JMY was found to be widely expressed in normal tissues and in many types of tumors. In neoplastic tissues, comparing primary versus metastatic tumors, JMY was found to have significantly higher expression in the metastatic compared with the primary tumors. A pilot study showed that nuclear co-expression of JMY and P53 was associated with shorter overall survival suggesting that a possible tumorigenesis mechanism could be via a deregulation/mutation of JMY/p53 or both. Finally, using 3 dimensional constructions, I demonstrated the distinct morphological difference between an angiogenic tumor and a non-angiogenic tumor. Additionally, I showed a characteristic cytoplasmic p53 sequestration in the non-angiogenic phenotype that is absent in the angiogenic phenotype. This could be the mechanism that the non-angiogenic tumor uses to adapt to hypoxia. This would imply that there is a potential for cancers to escape therapy by switching between these 2 phenotypes.

Epidemiological observations have linked increased host iron with malaria susceptibility. At the same time, blood-stage malaria infection is associated with potentially life-threatening anemia. To improve our understanding of these relationships, this work presents an examination of the mechanisms controlling the upregulation of the hormone hepcidin, the master regulator of iron metabolism, in malaria infection. Chapter 2 presents data from a mouse model of malaria infection which indicate that hepcidin upregulation in malaria infection is associated with increased activity of the sons of mothers against decapentaplegic (Smad) signaling pathway. Although the canonical Smad pathway activators, bone morphogenetic proteins (Bmp) are not increased at the message level following infection, activin B, which has been recently shown to increase hepcidin through the Smad signaling pathway in conditions of inflammation and infection, is upregulated in the livers of malaria-infected mice. Chapter 3 shows that both activin B and the closely related protein activin A upregulate hepcidin in vitro and in vivo. Chapter 3 also explores the effects of the activin-binding protein follistatin in both systems and in the same malaria-infected mouse model as presented in Chapter 2. The work presented in Chapter 4 extends these studies to human infections by demonstrating that activin A protein co-increases with hepcidin in human serum during malaria infection. Taken together, these findings are consistent with a novel role for activin proteins in controlling hepcidin upregulation in the context of malaria infection. This work may form a basis for the development of novel therapeutics that speed recovery from malarial anemia by inhibiting activins’ actions. Chapter 5 examines the role of infected red blood cell-derived microparticles in the initial recognition of a P. falciparum malaria infection, and subsequent hepcidin upregulation. Microparticles stimulate production of cytokines from peripheral blood mononuclear cells (PBMC), which also upregulate activin A message in response to both microparticles and whole infected red blood cells. These data are consistent with a model in which malaria-derived stimuli such as microparticles trigger the systemic release of activin proteins, which then act on the liver to upregulate hepcidin. Evidence has shown that cytokine levels at birth are related to malaria risk. In Chapter 6, hepcidin is measured in cord blood samples from participants in a large-scale clinical study in a malaria-endemic area, and shown to be elevated in cord blood from neonates with a clinical history of placental malaria. Cord blood hepcidin is also compared to birth levels of iron markers and other cytokines, and future clinical outcomes. Finally, the contributions of DNA methylation levels to cord hepcidin and cytokine levels are assessed by comparison of CpG methylation, at sites in genes encoding hepcidin and cytokines, to the serum concentrations of the genes’ protein products. Several intriguing associations are noted which indicate a possible novel role for DNA methylation in the determination of birth cytokine and hepcidin levels. Chapter 7 synthesizes the data presented in this thesis, interprets the possible significance of the major findings, and offers suggestions for future work.

Human haematopoiesis resembles a complex hierarchy, however most intermediate stages are only poorly defined. Efforts to characterise human progenitors have been inconsistent and failed to integrate previous knowledge. Furthermore, characterisation of normal progenitors has important implications in acute myeloid leukaemia (AML) biology. We previously established that leukaemic stem cells (LSCs) resemble the immunophenotypic progenitor compartments more closely than the stem cell fraction. Therefore, I set out to characterise human stem and progenitor cells (HSCPs) on phenotypic, molecular and functional level to complete the picture of human haematopoiesis. I purified HSPCs based on their immunophenotype from adult bone marrow (BM) and umbilical cord blood (CB) to investigate steady state and neonatal haematopoiesis. To define differentiation potentials, HSPCs were subjected to functional in vitro assays on bulk and clonal level. Limit dilution assays were used to determine the frequency of cells with multiple differentiation potentials. RNA sequencing revealed underlying lineage priming and specific gene expression signatures. I successfully characterized the incompletely defined Lin^-CD34⁺CD38^-CD45RA⁺ fraction in BM and CB, containing a CD10^lo lymphoid-primed multipotent progenitor (LMPP) with T cell, B cell, NK cell, granulocytic and monocytic differentiation potential, and succeeded in placing it in the haematopoietic hierarchy with relation to similar lympho-myeloid progenitors defined by other groups. This research lays the foundation to characterise early human progenitors with a comprehensive toolkit on a phenotypic, molecular and functional level. Findings from this thesis might provide knowledge about potential targets in LSCs.

The production of adult blood initiates from the haematopoietic stem cell (HSC). This clinically important cell has the capacity to maintain all blood lineages throughout the lifetime of an organism. HSCs emerge de novo from the haemogenic endothelium in the ventral wall of the embryonic dorsal aorta, from where they go on to seed adult sites of haematopoiesis. We have shown that Lmo4a is required for the emergence of HSCs in the zebrafish, and go on to demonstrate that Lmo4a regulates expression of the critical transcription factor, gata2a. Strikingly, both over- and under-expression of gata2a in the dorsal aorta severely diminishes HSC production. The LIM-only domain protein Lmo4 has previously been shown to interact with the known haematopoietic regulator, Ldb1. Together with our collaborators, we have identified novel binding partners of Lmo4 in mouse erythroleukaemic cells. Our functional analysis shows that many of these partners are also necessary for HSC emergence, thus revealing several new potential regulators of HSC formation. Given that these proteins were identified in an in vitro model of definitive erythropoiesis, it is remarkable that they also appear to act together in vivo at the level of HSC formation, and our data suggests that a transcriptional complex containing Lmo4 and these partners may directly repress gata2a. The related protein Lmo2 is also known to bind Ldb1. Together with Scl, Lmo2 is a master regulator of the haemangioblast programme. We have been utilising this activity, together with recent structural studies, to identify functionally important residues in the Lmo2 molecule. As a cell’s transcriptional programme drives both normal and pathological development, and misexpression of both Lmo2 and Lmo4 is involved in a variety of oncogenic states, the work presented in this thesis is likely to inform efforts to develop therapeutically relevant reagents.

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

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-10-04T18:07:36Z No. of bitstreams: 1 2017 - Camila Fl?via Magalh?es Botelho.pdf: 1531668 bytes, checksum: b445ba2353dc7515e478a8e151b24d75 (MD5) / Made available in DSpace on 2017-10-04T18:07:36Z (GMT). No. of bitstreams: 1 2017 - Camila Fl?via Magalh?es Botelho.pdf: 1531668 bytes, checksum: b445ba2353dc7515e478a8e151b24d75 (MD5) Previous issue date: 2017-02-14 / CAPES / Hemotrophic mycoplasmas hemoparasites are pleomorphic, epicellular, gram negative organisms that are located on the surface of erythrocytes of several species. In dogs, hemoplasma infection can cause hemolytic anemia in the acute phase, while in the chronic phase the signs are generally inapparent, and immunosuppression can trigger the acute disease. The present study aimed to detect molecularly the presence of hemotrophic mycoplasma agents in domestic dogs from the urban and rural areas of Aragua?na, Tocantins and, in addition, to correlate hematological alterations and factors associated with natural infection. In the analysis of the results, 2.9% (3/105) of the dogs sampled from the urban area and 24.49% (24/99) from the rural area were positive for Mycoplasma spp. by the Real Time Polymerase Chain Reaction (qPCR). The frequency of hemoplasmas species infection by PCR using species-specific oligonucleotides was 1.9% (2/105) and 13.26% (13/99) for Mycoplasma haemocanis in the urban and rural areas, respectively. No positive dogs were observed for ?Candidatus Mycoplasma turicensis? and ?Candidatus Mycoplasma haematoparvum?/?Candidatus Mycoplasma haemominutum? in both areas studied. Regarding hematological alterations, there was no statistical association with the infection by Mycoplasma spp. Similarly, among the analyzed variables (age, gender, racial pattern, site cleanliness and housing), no statistical correlation was observed in either urban or rural areas. This is the first report of the molecular detection of Mycoplasma haemocanis in dogs from Tocantins and opens up prospects for future studies in this group of hemotrophic agents. / Os hemoparasitos micoplasmas hemotr?ficos s?o organismos pleom?rficos, epicelulares, Gram negativos, que se localizam na superf?cie dos eritr?citos de diversas esp?cies. Em c?es, a infec??o pelos hemoplasmas pode causar anemia hemol?tica na fase aguda, enquanto na doen?a cr?nica os sinais em geral s?o inaparentes, sendo que a imunossupress?o pode desencadear a doen?a aguda. O presente estudo objetivou detectar molecularmente a presen?a de infec??o pelos agentes micoplasmas hemotr?ficos em c?es dom?sticos procedentes de ?reas urbana e rural de Aragua?na, Tocantins e, adicionalmente correlacionar as altera??es hematol?gicas e fatores associados com a infec??o natural. Na an?lise dos resultados obtidos, 2,9% (3/105) dos c?es amostrados da ?rea urbana e 24,49% (24/99) da ?rea rural foram positivos pela Rea??o em Cadeia da Polimerase em Tempo Real (qPCR) para Mycoplasma spp. A frequ?ncia de infec??o das esp?cies de hemoplasmas pela PCR, utilizando oligonucleot?deos esp?cie-espec?ficos, foi de 1,9 % (2/105) e 13,26% (13/99) para Mycoplasma haemocanis na ?rea urbana e rural, respectivamente. N?o foram observados c?es positivos para ?Candidatus Mycoplasma turicensis? e ?Candidatus Mycoplasma haematoparvum?/?Candidatus Mycoplasma haemominutum? em ambas as ?reas estudadas. No tocante ?s altera??es hematol?gicas, n?o houve associa??o estat?stica com a infec??o por Mycoplasma spp. De modo semelhante, dentre as vari?veis analisadas (idade, sexo, padr?o racial, limpeza do local e moradia) n?o foi verificada qualquer correla??o estat?stica com a infec??o pelo agente Mycoplasma spp., seja na ?rea urbana ou na rural. Esta constitui a primeira detec??o molecular de Mycoplasma haemocanis em c?es do Tocantins e abre perspectivas para futuros estudos neste grupo de agentes hemotr?ficos.