Morphological analysis of cells by means of an elastic metric in the shape space

Shape analysis is of great importance in many fields, such as computer vision, medical imaging, and computational biology. This analysis can be performed considering shapes as closed planar curves in the shape space. This approach has been used for the first time to obtain the morphological classification of erythrocytes in digital images of sickle cell disease considering the shape space S1, which has the property of being isometric to an infinite-dimensional Grassmann manifold of two-dimensional subspaces (Younes et al., 2008), without taking advantage of all the features offered by the elastic metric related to the possibility of stretching and bending of the curves. In this paper, we study this deformation in the shape space, S2, which is based on the representation of closed planar curves by means of the square-root velocity function (SRVF) (Srivastava et al., 2011), using the elastic metric of this space to obtain more efficient geodesics and geodesic lengths between planar curves. Supervised classification with this approach achieved an accuracy of 94.3%, classification using templates achieved 94.2% and unsupervised clustering in three groups achieved 94.7%, considering three classes of erythrocytes: normal, sickle, and with other deformations. These results are better than those previously achieved in the morphological analysis of erythrocytes and the method can be used in different applications related to the treatment of sickle cell disease, even in cases where it is necessary to study the process of evolution of the deformation, something that can not be done in a natural way in the feature space

Plasmodium falciparum: programmed cell death in the erythrocytic stages

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. This thesis is presented as a series of publications and unpublished data. Johannesburg, 2015Plasmodium falciparum is responsible for the majority of global malaria deaths. During the pathogenic blood stages of infection, a rapid increase in parasitaemia threatens the survival of the host before transmission of slow-maturing sexual parasites to the mosquito vector to continue the life cycle. Programmed cell death (PCD) may provide the parasite with the means to control its burden on the host and thereby ensure its own survival. PCD in P. falciparum remains a poorly understood and controversial topic. A gathering body of evidence suggests P. falciparum is capable of PCD, but there are conflicting results regarding the phenotype. This study represents a comprehensive phenotypical characterisation of cell death in intraerythrocytic P. falciparum after various physiologically relevant stress stimuli, including high parasitaemia, heat stress simulating febrile paroxysms, and exposure to natural sunlight. The latter is a novel stimulus for PCD studies in P. falciparum. Biochemical markers of cell death, including DNA fragmentation, mitochondrial dysregulation and phosphatidylserine externalisation on parasitized erythrocytes, were used to provide a holistic description of cell death. Data showed that the combination of cell death markers varied with different stress stimuli and with the developmental stage of the parasite. An apoptosis-like phenotype, characterised by mitochondrial depolarisation, DNA fragmentation and phosphatidylserine externalisation, was suggested after stress from high parasitaemia. Heat stress affected ring stage parasites more severely than previous data suggested and induced an apoptosis-like phenotype. In contrast, late stage parasites showed markers of an autophagic-like cell death, including slight DNA fragmentation, phosphatidylserine externalisation and cytoplasmic vacuolisation. Sunlight exposure induced markers of PCD that included DNA fragmentation preceding mitochondrial hyperpolarisation, but the phenotype was not clear. The paradigm of PCD in P. falciparum is a dynamic and ever-evolving one that will continue to challenge our thinking and understanding of how the world’s deadliest parasitic killer can induce its own death to limit damage on the host. Evidence indicates that P. falciparum undergoes PCD and that the phenotype(s) may be unique. PCD is an important feature of P. falciparum biology and the elucidation of parasite PCD pathway(s) that differ from host mechanisms may yield novel drug targets

Automatic Segmentation and Classification of Red and White Blood cells in Thin Blood Smear Slides

In this work we develop a system for automatic detection and classification of cytological images which plays an increasing important role in medical diagnosis. A primary aim of this work is the accurate segmentation of cytological images of blood smears and subsequent feature extraction, along with studying related classification problems such as the identification and counting of peripheral blood smear particles, and classification of white blood cell into types five. Our proposed approach benefits from powerful image processing techniques to perform complete blood count (CBC) without human intervention. The general framework in this blood smear analysis research is as follows. Firstly, a digital blood smear image is de-noised using optimized Bayesian non-local means filter to design a dependable cell counting system that may be used under different image capture conditions. Then an edge preservation technique with Kuwahara filter is used to recover degraded and blurred white blood cell boundaries in blood smear images while reducing the residual negative effect of noise in images. After denoising and edge enhancement, the next step is binarization using combination of Otsu and Niblack to separate the cells and stained background. Cells separation and counting is achieved by granulometry, advanced active contours without edges, and morphological operators with watershed algorithm. Following this is the recognition of different types of white blood cells (WBCs), and also red blood cells (RBCs) segmentation. Using three main types of features: shape, intensity, and texture invariant features in combination with a variety of classifiers is next step. The following features are used in this work: intensity histogram features, invariant moments, the relative area, co-occurrence and run-length matrices, dual tree complex wavelet transform features, Haralick and Tamura features. Next, different statistical approaches involving correlation, distribution and redundancy are used to measure of the dependency between a set of features and to select feature variables on the white blood cell classification. A global sensitivity analysis with random sampling-high dimensional model representation (RS-HDMR) which can deal with independent and dependent input feature variables is used to assess dominate discriminatory power and the reliability of feature which leads to an efficient feature selection. These feature selection results are compared in experiments with branch and bound method and with sequential forward selection (SFS), respectively. This work examines support vector machine (SVM) and Convolutional Neural Networks (LeNet5) in connection with white blood cell classification. Finally, white blood cell classification system is validated in experiments conducted on cytological images of normal poor quality blood smears. These experimental results are also assessed with ground truth manually obtained from medical experts

On the molecular evolution of the Plasmodium falciparum

Research in the Plasmodium falciparum molecular evolution field has predominantly comprised three distinct areas: phylogenetics, host-parasite coevolution and evolutionary genomics. These areas have greatly enhanced our understanding of the early origins of the phylum Apicomplexa, the emergence of P. falciparum, and the co-evolution between parasite and human hereditary erythrocyte disorders. In addition, the genome sequencing projects have elucidated the complexity and extremely unusual nature of the parasite genome. Some aspects of parasite molecular evolution, however, are controversial, such as human pyruvate kinase (PK) deficiency and P. falciparum virulence coevolution. Other aspects, like Plasmodium whole genome evolution have remained unexplored. This thesis includes a collection of manuscripts that address aspects of the broad field of P. falciparum molecular evolution. The first deals with the limitations of bioinformatic methods as applied to P. falciparum, which have arisen due to the unusual nature of the parasite genome, such as the extreme nucleotide bias. Although conventional bioinformatics can partially accommodate and compensate for the genome idiosyncrasies, these limitations have hampered progress significantly. A novel alignment method, termed FIRE (Functional Inference using the Rates of Evolution) was therefore developed. FIRE uses the evolutionary constraints at codon sites to align sequences and infer domain function and overcomes the problem of poor sequence similarity, which is commonly encountered between P. falciparum and other taxa. A second aspect addressed in this thesis, is the host-parasite relationship in the context of PK deficiency. It was demonstrated that PK deficient erythrocytes are dramatically resistant to parasite infection, providing in vitro evidence for this phenomenon and confirming this aspect of host-parasite co-evolution. The unexplored field of parasite genome evolution was initiated in this thesis by investigating two major role-players in genome dynamics, mobile genetic elements (MGEs) and programmed cell death (PCD). MGEs were absent in P. falciparum, possibly due to a geno-protective mechanism, which increased the AT nucleotide bias. Interestingly, the parasite telomerase reverse transcriptase, which is a domesticated MGE, was identified. In addition, there is genomic evidence for the second determinant, a classical PCD pathway. Intriguingly, functional and structural evidence for a p53-like DNA-binding domain, which plays a key role in genome evolution, was obtained. Using MGEs and PCD as examples, a theoretical framework for investigating genome dynamics was developed. The framework proposes an ecological approach to genome evolution, in which a trade-off exists between two opposing processes: the generation of diversity by factors such as MGEs and the maintenance of integrity by factors like PCD. The framework is suggested for proposing and testing hypotheses to investigate the origins and evolution of the P. falciparum genome. Finally, a novel approach, termed Evolutionary Patterning (EP), was developed to limit the problem of parasite drug resistance and demonstrates the value of employing molecular evolution to address biomedical challenges. Some of this work, such as the FIRE method, the host-parasite co-evolution studies, the PCD findings and the EP approach have been incorporated in grant proposals and adopted in future projects. It is hoped that this research will be used to further our understanding of P. falciparum evolution and advance the efforts to control this deadly pathogen

Investigation of the use of histone deacetylase inhibitors for the treatment of inherited disorders of the glycolytic pathway

Histone acetylation by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs) regulate gene expression by activating or repressing transcription, respectively. HDAC inhibitors (HDACIs) are a diverse class of drugs used to treat haemoglobinopathies, urea cycle disorders and several types of malignancies. Recent evidence from genome-wide as well as gene-specific epigenetic studies suggest a model whereby active genes are more likely than silent genes to be hyperacetylated and increase their transcription levels in response to HDACIs, a process underpinned by the dynamic recruitment and antagonistic activities of HATs and HDACs. Based on this model and from a therapeutic perspective, I hypothesised that the ability of HDACIs to increase expression of active genes might be relevant for diseases caused by genes that encode proteins with enzymatic function. HDACI-mediated increase in gene transcription, even in the presence of missense, disease-causing mutations, might lead to increased enzymatic activity and amelioration of the cellular and clinical phenotype. I tested this hypothesis on a group of genes involved in the glycolytic and pentose phosphate pathway (GPPP) which, when mutated, cause chronic or episodic haemolytic anaemia. Using RT-qPCR (B cell lines) and gene expression profiling (primary, in vitro generated human erythroid precursors and CD4+ T cells) I found that of the 17 GPPP genes, only Glucose-6-Phosphate Dehydrogenase (G6PD) mRNA levels increased in response to HDACIs in a time-dependent manner. Epigenetic analysis in B cells by ChIP-qPCR showed that histone hyper-acetylation and increased recruitment of HATs and HDACs underpin the selective G6PD transcriptional activation in response to HDACIs. Pharmacological and genetic assays showed that increase in G6PD transcription was also dependent on Sp1, a generic transcription factor known to recruit both HDACs and HATs. Finally, I directly tested the hypothesis that HDACIs may increase enzymatic activity in G6PD deficient cells. Using B cell lines and primary erythroid cells from patients with G6PD deficiency, I found that HDACIs induce the same epigenetic changes in the mutant as in the wild type G6PD gene; more importantly, they lead to increased levels of the mutant mRNA and protein, associated with an up to 3-fold increase in enzymatic activity. These findings are potentially of great therapeutic significance for correction of G6PD deficiency in up to 300 million individuals worldwide with the polymorphic variants of G6PD deficiency (e.g., G6PDMed and G6PDA-).Open Acces

Investigating the effects of the disease-modifying anti-rheumatic drug methotrexate on the vascular endothelium

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease that associates with increased mortality from cardiovascular disease (CVD). Methotrexate (MTX), a folate analogue used as disease-modifying anti-rheumatic drug (DMARD), reduces CVD morbidity and mortality in RA patients, possibly by improving endothelial function. The aim of this study was to investigate the molecular effects of MTX in quiescent and activated vascular endothelial cells (EC). In EC pre-treated with tumour necrosis factor (TNF)-alpha, MTX independently increased the activity of mitogen-activated protein kinase (MAPK) p38 and Akt and caused cell cycle arrest due to folate depletion. In contrast, MTX did not affect cell signalling, proliferation or gene expression in EC exposed to different types of shear stress. To tease out the underlying mechanism, folate or one carbon metabolism (OCM) was investigated in EC cultivated under static or shear stress conditions. Endothelial OCM was fundamentally altered by shear stress, with potentially important implications for MTX uptake and function. Using endothelial colony forming cells (ECFC) isolated from RA patients before and after treatment initiation with MTX and hydroxychloroquine, it was shown that systemic inflammation primes ECFC towards a pro-inflammatory state that may be reversible with DMARD treatment. These data suggest that MTX acts on vascular EC in a folate-dependent manner. However, since endothelial OCM was largely downregulated by shear stress, this implies that MTX may have limited direct effects on EC in vivo and provides a potential explanation for the controversies around the vascular effects of folate and anti-folate therapy. Although findings in ECFC from patients with RA support anti-inflammatory effects of MTX containing therapy, the data in this thesis do not support a direct MTX-mediated anti-atherogenic effect on the endothelium but suggest that the drug may limit EC dysfunction in combination with other drugs indirectly by reducing systemic inflammation.Open Acces

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

Alterações de dosagem no genoma de doentes com atraso mental

Mestrado em BiotecnologiaDevelopment delay/Intellectual disability (DD/ID) is a serious and life-long condition which represents a challenge for families and public health services. It is characterized by suboptimal functioning of the central nervous system resulting in limitations both in intellectual functioning and in adaptive behavior, and it is observed in approximately 2-3% of the population worldwide. Establishing the disease etiology is important for clinical management, genetic counseling and coping strategies of the families. Array-based comparative genomic hybridization technique (aCGH), also called molecular karyotyping (MC), allows us to directly measure genomic copy number variations between the patient and a control DNA. The whole genome of an individual is represented in a high-resolution “virtual karyotype”, allowing the detection of submicroscopic alterations, undetectable by standard or highresolution karyotyping techniques. In Portugal, the guidelines for testing patients with DD/ID indicate the Gbanding karyotyping in first place and, whenever the result is normal, testing for the most common single gene disorders (Fragile X, for instance), for subtelomeric rearrangements and with specific FISH probes. However, the latter technologies are not suitable for whole genome scans in routine diagnosis, both because of the lower resolution levels (the case of conventional karyotyping), the extensive time consumption and high costs (the FISH case). The introduction of aCGH should contribute to the etiological classification of a large proportion of the DD/ID patients as well as to conclude about the utility of using these technologies for diagnosis of idiopathic DD/ID in the clinical context.O atraso mental (AM) é uma doença que apresenta desafios para a vida tanto nas famílias como na sociedade. É caracterizada por um funcionamento subóptimo do sistema nervoso central que manifesta limitações quer ao nível da capacidade intelectual quer do comportamento adaptativo e tem uma incidência de aproximadamente 2-3% de nados vivos em todo o mundo. A determinação da causa da doença é importante para o correcto encaminhamento clínico, aconselhamento genético e estratégias de coping a desenvolver pela família. A técnica de hibridação genómica comparativa (aCGH), também designada de cariótipo molecular (CM), permite medir variações no número de cópias entre o genoma do doente e de um controlo. A totalidade do genoma de um indivíduo é representado num cariótipo virtual de alta resolução, permitindo assim a detecção de alterações submicroscópicas indetectáveis por métodos de cariotipaem standard ou de alta resolução. Em Portugal, as recomendações para os testes genéticos a realizar num doente com AM indicam o cariótipo tradicional de bandas G como primeira abordagem e, sempre que o resultado seja negativo, a pesquisa relativa às patologias unigénicas mais comuns (Síndrome de X frágil, por exemplo), rearranjos subteloméricos e sondas de FISH específicas. No entanto, estas tecnologias não são aplicáveis para análise de todo o genoma no contexto de diagnóstico de rotina, tanto pela sua baixa resolução (caso do cariítpo convencional) como pela elevada laboriosidade e/ou custos monetários (caso do FISH). A introdução de aCGH pretende contribuir para a classificação etiológica de grande parte dos doentes com AM, assim como, concluir acerca da utilidade desta tecnologia para o diagnóstico de AM idiopático no contexto clínico

A study of iron nutrition and immunity in infancy

Motivation and study design: Iron deficiency is a common condition in infancy, particularly in lower socio-economic groups. In Cape Town it remains a problem in spite of public health measures taken against it: a recent survey found a prevalence of iron deficiency anaemia of 34% in healthy 1-year old term infants who had ready access to a municipal health clinic where iron fortified milk formula is sold at subsidized prices. The consequences of iron deficiency extend beyond anaemia- to involve all organ systems including the immune system. Since Helen Mackay's report in 1928 of a striking decrease in incidents of infection in infants treated with iron, clinicians have assumed that iron deficiency predisposes to infection. Despite a sound theoretical basis for this belief, the clinical evidence for the assumption is poor as studies to date have displayed methodological deficiencies. On the other hand, iron is also essential for the growth of micro-organisms. As such, supplemental iron may predispose to infection. Indeed, there is much laboratory and clinical evidence to show that excess iron can result in the recrudescence of quiescent infections and increase the virulence of newly acquired infections. Thus, the competition between host and parasite may sometimes hinge on the relative availability of iron and it has been speculated that excess iron in infant milk formula may increase susceptibility to infectious diarrhoeal disease. The problem addressed by this thesis was to determine the utility of increasing the level of iron fortification of infant milk formula. Three questions were posed: Does increasing the level of iron fortification of conventional infant milk formula improve the iron nutrition of normal infants fed on the formula? Does increased iron fortification of infant milk formula alter immunity as reflected by incidence of infection and laboratory tests of immune function? Are there any handful effects of increasing the quantity of iron in conventional infant milk formula? A double blind randomized trial was carried out in 1983 and 1984 to answer these questions. A group of 149 healthy, well-nourished infants from a lower socio-economic community of so called Cape Coloureds were followed from the age of 3 months to 1 year. Half of the infants, the Control group, were given a commercially available infant milk formula (Lactogen Full Protein) which has 8.3 mg Fe/ 100 g formula and 37 mg ascorbic acid/ 100 g. The other half of subjects, the Test group, were given the same milk formula but fortified with iron to a concentration of 40 mg Fe/ 100 g. The children were examined every 3 or 4 weeks and any infection or history of infection was noted. Laboratory tests were done at the start of the trial and again on completion. During the trial, laboratory tests were performed only if clinically indicated. The tests included full blood count and differential analysis, red cell zinc protoporphyrin, plasma ferritin, plasma and hair zinc and lymphocyte subtyping with monoclonal antibodies. Within each group, half of the infants were randomly selected for assay of neutrophil bactericidal activity. The other half were assayed for lymphocyte blastogenic response to stimulation with phytohaemagglutinin. Tests of delayed cutaneous hypersensitivity to Candida antigen and PPD were done and all children and their mothers had antibodies to tetanus and polio determined. Results: 74 infants in the Control group started the trial and 62 completed it. In the Test group, 75 infants began and 70 completed the study. Intake of milk and solid foods was not quantified, but the ages of weaning and of introduction of new foods were determined. The Control and Test groups did not differ significantly on any test item. The mean age of completion of weaning was 3.60 months for the Control group and 4.04 months for the Test group. The Control group was first given meat or fish at a mean age of 5.19 months; the Test. group had meat or fish introduced to their diets at a mean age of 4.36 months. These differences were not statistically significant. The children in the Control group were lighter and shorter than the Test group at the end of the year. Mean standard deviation scores for weight were 0.23 and 0.48 respectively (P = 20%), while for length the SD scores were -0.13 and 0.06 (P = 20%)