Approximately 15% of human cancers utilize a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT) to maintain the lengths of their telomeres. The Fanconi anaemia protein FANCJ localizes to telomeric foci in human ALT cells, but not in telomerase-positive or primary cells. Telomere-associated FANCJ frequently localizes with FANCD2 and BRCA1, and primarily localizes to ALT-associated PML nuclear bodies. Depletion of FANCJ in human ALT cells causes the loss of BRCA1 at telomeric foci and a decrease in telomeric repeat DNA content primarily as a result of the loss of the brightest telomeric repeat DNA foci. In contrast, depletion of the FANCD2 results in increased telomeric repeat DNA synthesis and this is suppressed upon the codepletion of FANCJ. Together, data from this study suggest that FANCJ is required for telomeric repeat DNA synthesis in human ALT cells, which may or may not be dependent on BRCA1, and FANCD2 restrains this synthesis.

No abstract available. / Thesis (Ph.D.)-University of Natal, Durban, 1973.

The life cycle of the malaria parasite is very complex and provides a number of potential targets for vaccination. In this thesis, data on two plasmodial asexual blood stage antigens (RAP-2 and Pf332) are presented. A partial aim of the work presented herein was to investigate the mechanisms responsible for the destruction of erythroid cells in anaemia, and more specifically to define the role of the rhoptry associated protein (RAP)-2 and other members of the RAP complex, RAP-1 and -3 in processes resulting in anaemia. Antibodies to the RAP complex were shown to have the potential to mediate the destruction of RAP-2-tagged erythroid cells by phagocytosis or by complement activation and lysis. In addition, antibodies to RAP-1 and RAP-2 could induce the apoptotic death of RAP-2- tagged erythroblasts. The frequency and functionality of naturally occurring RAP-2 antibodies in the sera of anaemic and non-anaemic Cameroonian children were also investigated. All sera tested contained RAP-2-reactive antibodies by both immunofluorescence and flow cytometry. The anaemic group of children had higher levels of IgG than the non-anaemic ones, while the levels of IgM were similar. With respect to IgG subclasses, higher levels of IgG3 were seen in the non-anaemic individuals as compared to anaemic subjects. The non-anaemic individuals recognised a greater proportion of RAP-2-tagged RBCs and activated complement to a greater extent than the anaemic ones. Earlier studies observed that humans continuously exposed to malaria, recognised Pf332 extensively. Further studies revealed that Pf332 antibodies were able to inhibit parasite growth and cytoadherence in vitro. Making use of Pf332-C231, a sub-fragment of Pf332, we studied the effects/mode of action of C231-specific antibodies on P. falciparum parasite growth and development in vitro. The antibodies appeared to act mainly on late stage parasites by two main mechanisms: 1) through the induction of abnormal/pyknotic parasites, and, 2) RBC lysis (disintegration of RBCs), thus limiting parasite growth and development. The antibody isotype in this context was IgG. Following the removal of immune pressure, parasites resumed growth, albeit at a much slower rate. The results suggest that during natural infections, antibodies to C231 could play a role in parasite control. In summary, these data suggest that antibodies to both antigens could be instrumental in immune responses leading to disease control, but could also mediate pathology. / At the time of the doctoral defense, the following publication was unpublished and had a status as follows: Paper 3: Manuscript.

The inherited bone marrow failure syndrome Fanconi Anaemia (FA) is caused by mutations in any one of the multiple FANC genes, which encode proteins that collaborate in the FA/BRCA DNA damage response pathway. FA is characterised by extreme predisposition to acute myeloid leukaemia (AML). AML in FA is associated with typical chromosomal aberrations involving gains of the long arm of chromosome 3 (3q gains). These are linked to overexpression of the oncogene ecotropic viral integration site 1 (EVI1). Based on this clinical observation, the hypothesis that EVI1 confers leukaemic transformation, in particular in the context of FA, was tested. Mouse embryonic stem cells with either functional or disrupted FA/BRCA-pathway were used to model normal and FA-associated embryonic haematopoiesis, and the effect of EVI1 overexpression was assessed in this model. EVI1 functions were also investigated with respect to protein interactions, focusing on the interaction with the co-repressor C-terminal binding protein 1 (CtBP1), in the context of genotoxic stress. To study this, in vitro haematopoietic differentiation assays, flow cytometry, mass spectrometry, immunoprecipitations and immunofluorescence were employed. In vitro haematopoietic differentiation using mouse embryonic stem cells with defective Fanca was successfully developed and applied. The analysis revealed that EVI1 overexpression in haemangioblast-like cells prevented the generation of haematopoietic precursors through endothelial to haematopoietic transition. Studies into EVI1 protein interaction dynamics showed that DNA damage-induced phosphorylation of EVI1 modifies interaction with the co-repressor CtBP1. This interaction was demonstrated to be partially required for the EVI1-induced block of the development of haematopoietic precursors using the mESC-based model. An EVI1-mediated modulation of the FA phenotype characteristic G2-arrest and of the FA-associated embryonic haematopoiesis was not demonstrated. This study contributes to the understanding of the function of the EVI1 oncogene in normal and FA-associated haematopoiesis and the DNA damage response. FA-associated haematopoiesis and leukaemogenesis can be further studied using the embryonic haematopoiesis model developed here, and further studies can build on the data generated with respect to EVI1.

Blood transfusion forms a mainstay of the treatment of a variety of illnesses, and is lifesaving. Nonetheless, it is not without its risks and drawbacks. Blood transfusion is a cornerstone in the treatment of canine babesiosis. The development of blood alternatives has received attention in recent times. Blood alternatives offer much of what natural blood does but without many of the associated drawbacks. These include disease transmission, transfusion reactions, poor in vitro and in vivo shelf-life and special storage and administration requirements. One product, Oxyglobin®, is the first commercially available, veterinary-licensed, haemoglobin-based oxygen carrying solution (HBOCS). Although licenced for use in canine babesiosis, this colloidal “Oxygen Bridge” has never been evaluated against the gold standard of therapy, isovolumic packed red blood cell transfusion (pRBCT). This investigation was conducted to evaluate important aspects the equivalence of these two treatments in a field situation of naturally-infected dogs. Given the cost of HBOCS, they are unlikely to be commonly used by the practicing veterinarian in the treatment of canine babesiosis. Nonetheless, similarities in efficacy would bolster the case for and further research into blood substitutes of this and other classes, and may open the way to evaluation of HBOCS for falciparum malarial anaemia, a disease similar in many respects to canine babesiosis. / Dissertation (MMedVet)--University of Pretoria, 2007. / Companion Animal Clinical Studies / unrestricted

Despite haemolytic anaemia being the main consequence of Babesia rossi infection in dogs, the bone marrow response has been reported to be mild in the face of severe anaemia. A similar finding has been described in falciparum malaria and has been ascribed to either a decreased production of erythroid precursors or an inability of erythroid precursors to respond to hormonal stimulus. Recently, more information has become available on the use of various reticulocyte indices in an attempt to describe and explain the underlying pathogenesis of various anaemias as their release describes the recent functional state of the bone marrow. The objective of this study was to compare the admission absolute reticulocyte count (ARC) and reticulocyte indices in dogs naturally infected with B. rossi with dogs suffering from immune-mediated haemolytic anaemia (IMHA), unrelated to babesiosis, as well as healthy control dogs. The ARC and reticulocyte indices were also evaluated in five experimentally B. rossi infected dogs throughout the disease course. This was a retrospective observational study looking at the records generated on a haematology analyser, the ADVIA 2120 (Siemens, Munich, Germany). The haematocrit (HCT), ARC and other reticulocyte indices for 103 dogs, naturally infected with B. rossi was compared to 16 dogs with IMHA and 14 control dogs. The experimentally infected dogs consisted of five purpose-bred beagles that were infected with high and low dose B. rossi parasite inoculum. Differences between groups were assessed using the Mann-Whitney U test, whereas the Friedman’s ANOVA was used to assess the change over time during the disease course in the experimentally infected dogs. The Wilcoxon Signed Ranks test was then used to determine the differences within the experimentally infected groups. The median (IQR) HCT for the Babesia (0.16 L/L; 0.12 – 0.27; P < 0.001) and IMHA (0.15 L/L; 0.12 – 0.17; P < 0.001) groups were significantly lower than the control group (0.52 L/L; 0.45 – 0.57). For the Babesia and IMHA groups the HCT did not differ significantly. Compared to the control group (42.1 x109/L; 33.8 – 62.6), the median (IQR) ARC was significantly higher in the Babesia (82.1 x109/L; 48.6 – 174.9; P = 0.006) and IMHA (256.7 x109/L; 79.0 – 436.9; P = 0.004) groups. The ARC was significantly lower in the Babesia group compared to the IMHA group (P = 0.011), despite no significant difference for HCT between groups. On day four of the experimentally infected group, approximately 24 – 48 hours after a peripheral parasitaemia was observed, there was a sudden decrease in the ARC to less than its value on day one, which was inappropriate for the degree of anaemia observed. The reticulocytes of B. rossi naturally infected dogs were larger and more hypochromic with a greater difference in cellular haemoglobin (CH) between reticulocytes and mature erythrocytes compared to the control group, whereas the reticulocytes of the experimentally infected dogs were smaller and more hypochromic than day one of infection. All reticulocyte indices demonstrated significant differences on day four compared to day one of the experimental study. The changes noted in the reticulocyte indices are postulated to be a contribution of anaemia of inflammation (AID), shift reticulocytosis and iron-restricted erythropoiesis. This study concludes that the regenerative response in dogs naturally infected with B. rossi is inappropriate, despite the severity of anaemia observed, compared to dogs with IMHA. Furthermore, the findings of the experimentally group may indicate a possible direct suppressive action of the Babesia parasite on the bone marrow during the time of parasitaemia, resulting in insufficient erythropoiesis. Similar findings have been reported in falciparum malaria and other factors will require further investigation. / Dissertation (MSc)--University of Pretoria, 2020. / Companion Animal Clinical Studies / MSc / Unrestricted

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anaemia (FA) gene, following the discovery of two patients carrying dominant negative mutations. FA is a hereditary DNA repair disorder characterised by various congenital abnormalities, progressive bone marrow failure and cancer predisposition. The cellular and molecular pathology of FA is poorly understood, resulting in a severe lack of effective treatment options. In this thesis, I describe the first viable vertebrate model of RAD51 loss. Phenotypic characterisation of zebrafish rad51 loss-of-function mutants showed that they develop key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking agents and decreased size. Taking advantage of the unique properties of the zebrafish model, I show that some of these symptoms stem from both decreased proliferation, as well as increased apoptosis of embryonic haematopoietic stem and progenitor cells. Co-mutation of p$L was able to rescue the haematopoietic defects seen in the single mutants, but led to tumour development, underscoring the role of rad51 as a tumour suppressor. I further demonstrate that prolonged inflammatory stress can exacerbate the haematological impairment, leading to an additional decrease in kidney marrow cell numbers. In contrast, prolonged aldehyde-derived stress did not induce symptoms in the mutant fish. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signalling during embryogenesis leads to the haematological defects seen later in life in FA. It also strengthens the evidence for the involvement of haematopoietic stress, such as inflammation, in the development of bone marrow failure. Further research on this novel zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Chronic HIV-1 infection is characterized by extensive inflammation/immune activation and also by anaemia. Macrophages and neutrophils produce reactive oxygen species (ROS) which can cause damage to surrounding cells, including erythrocytes. Damaged erythrocytes may die by apoptosis (erythroptosis) or be tagged for clearance by monocytes/ macrophages. In this study we investigated HIV-1-associated anaemia and erythroptosis in asymptomatic, untreated HIV-1 infected individuals and how it relates to oxidative stress and immune activation. / AFRIKAANSE OPSOMMING: Chroniese MIV-1 infeksie word gekenmerk deur uitgebreide inflammasie/immuun aktivering en ook deur anemie. Makrofage en neutrofiele produseer reaktiewe suurstof spesies (ROS), wat kan skade aan omliggende selle, insluitend rooibloedselle veroorsaak. Beskadigde rooibloedselle kan sterf deur apoptose (erythroptosis) of gemerk vir klaring deur monosiete/makrofage. In hierdie studie het ons ondersoek MIV-1-verwante bloedarmoede en erythroptosis in asimptomatiese, onbehandelde MIV-1 besmette individue en hoe dit verband hou met oksidatiewe stres en immuun aktivering. / The Poliomyelitis Research Foundation (PRF

Objective: To identify the magnitude of anaemia and deficiencies of Fe (ID) and vitamin A (VAD) and their associated factors among rural women and children. Design: Cross-sectional, comprising a household, health and nutrition survey and determination of Hb, biochemical (serum concentrations of ferritin, retinol, C-reactive protein and alpha(1)-acid glycoprotein) and anthropometric parameters. Multivariate logistic regression examined associations of various factors with anaemia and micronutrient deficiencies. Setting: Kalale district, northern Benin. Subjects: Mother-child pairs (n 767): non-pregnant women of reproductive age (15-49 years) and children 6-59 months old. Results: In women, the overall prevalence of anaemia, ID, Fe-deficiency anaemia (IDA) and VAD was 47.7, 18.3, 11.3 and 17.7%, respectively. A similar pattern for anaemia (82.4 %), ID (23.6%) and IDA (21.2%) was observed among children, while VAD was greater at 33. 6%. Greater risk of anaemia, ID and VAD was found for low maternal education, maternal farming activity, maternal health status, low food diversity, lack of fruits and vegetables consumption, low protein foods consumption, high infection, anthropometric deficits, large family size, poor sanitary conditions and low socio-economic status. Strong differences were also observed by ethnicity, women's group participation and source of information. Finally, age had a significant effect in children, with those aged 6-23 months having the highest risk for anaemia and those aged 12-23 months at risk for ID and IDA. Conclusions: Anaemia, ID and VAD were high among rural women and their children in northern Benin, although ID accounted for a small proportion of anaemia. Multicentre studies in various parts of the country are needed to substantiate the present results, so that appropriate and beneficial strategies for micronutrient supplementation and interventions to improve food diversity and quality can be planned.

O uso de suplemento a base de ferro é fundamental para profilaxia e tratamento da anemia ferropriva, agravo freqüente em gestantes que vivem em paises em desenvolvimento, e que se não for tratada, pode ter conseqüências tanto para mulher quanto para o feto. Entretanto, muitas mulheres não aderem a terapêutica. O objetivo deste estudo foi identificar a utilização de suplemento à base de ferro no ciclo grávido-puerperal, em um grupo de mulheres.Trata-se de um estudo descritivo com abordagem quantiqualitativa realizado em uma Unidade Básica e Distrital de Saúde (UBDS) de Ribeirão Preto, e que teve como sujeitos um grupo de mulheres, usuárias de um serviço de saúde, que compareceram a UBDS para consulta de puericultura. A coleta foi realizada através de entrevista, coleta de sangue para dosagem de hemoglobina e consulta aos prontuários. A idade das mulheres variou entre 19 a 24 anos, sendo que destas 36,3% apresentaram ensino fundamental incompleto e 75% referiu não trabalhar. Quanto a realização de pré-natal, 75% realizou em Centro ou Posto de Saúde tendo como média de consultas durante a gestação oito. Durante a gestação e o puerpério o valor médio de hemoglobina encontrado nas entrevistadas foi de 12,1 g/dl e 12 g/dl, respectivamente. Quanto ao uso de suplemento a base de ferro na gestação e puerpério, 81,8% referiu ter usado durante a gestação e 66,9% no puerpério. Assim, conseguimos identificar que a maioria das gestantes fez uso do suplemento durante a gestação e puerpério e que apresentaram hemoglobina maior que 11 g/dl, ou seja, não apresentavam anemia. Os dados qualitativos revelam que as mulheres desconhecem a importância do uso do suplemento neste período, sendo mais evidente a preocupação com a saúde do feto. / The supplement use the iron base is basic for Prophylaxis and treatment of the ferropriva anemia, I aggravate frequent in gestantes that live in paises in development, and that if it will not be treated, it can in such a way have consequences for woman how much for the embryo. However, many women do not adhere the therapeutical one. The objective of this study was to identify the use of supplement to the base of iron in the pregnant-puerperal cycle, in a group of mulheres.Trata-if of a descriptive study with carried through quantiqualitativa boarding in a Basic and District Unit of Saúde (UBDS) of Ribeirão Preto, and that a group of women had as citizens, users of a health service, who had appeared the UBDS for puericultura consultation. The collection was carried through through interview, collection of blood for dosage of hemoglobina and consults to handbooks. The age of the women varied enters the 19 24 years, being that of these 36.3% had presented incomplete basic education and 75% related not to work. How much the accomplishment of prenatal, 75% carried through in Center or Rank of Health having as average of consultations during gestation eight. During the gestation and the puerpério the average value of hemoglobina found in the interviewed ones was of 12,1 g/dl and 12 g/dl, respectively. How much to the supplement use the base of iron in the gestation and puerpério, 81.8% related to have used during gestation and 66.9% in the puerpério. Thus, we obtain to identify that the majority of the gestantes made use the supplement during the gestation and puerpério and that they had presented 11 bigger hemoglobina that g/dl, or either, they did not present anemia. The qualitative data disclose that the women are unaware of the importance of the use of the supplement in this period, being more evident the concern with the health of the embryo.