Mutation analysis of the promoter region of CYBRD1, HFE, LTF, HAMP and SLC40A1 in a Tuberculosis cohort

Thesis (MSc)--Stellenbosch University, 2014.ENGLISH ABSTRACT: Tuberculosis (TB) is an epidemic disease characterised by wet, persistent coughing, weight loss, fever, fatigue, and blood in the sputum. It has been reported that one in every three individuals is currently infected with Mycobacterium tuberculosis, the causative agent of TB, and that 10% of them will develop the active disease. The high prevalence and low penetrance of this disease has resulted in increased research performed to ascertain what factors play a role in susceptibility to M. tuberculosis infection. Some factors known to play a role in a minority of cases may include: HIV infection, diabetes, alcohol abuse and old age, but racial differences in susceptibility have also been observed. However, the influence of genetic factors is gaining popularity in current research. M. tuberculosis requires iron to proliferate, which it must appropriate from its host. For this reason the genes involved in the metabolism of iron in the human host are of particular interest when considering susceptibility to M. tuberculosis infection. In order to determine whether the expression of these genes may influence disease susceptibility, the promoter region of the CYBRD1, HAMP, HFE, LTF and SLC40A1 genes have been targeted for investigation. The aim of this study was to determine whether DNA variation in the promoter region of these genes involved in iron metabolism is associated with M. tuberculosis susceptibility. The study cohort consisted of 49 TB patients and 51 healthy, unrelated, population-matched controls, all of whom were Black, Xhosa-speaking individuals. Initially, 15 patient samples were randomly selected for exploratory screening, utilising semi-automated bi-directional sequencing analysis. In this manner, 40 variants were identified of which 30 were previously described. The novel variants included CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; and LTF: -1377 T/G, -457 T/C, -437 C/G. A number of loci demonstrated a statistically significant difference in allele and genotype frequencies, and in iron parameter levels when comparing the patient and control groups and for each variant separately. In silico analyses revealed the creation and/or abolishment of several transcription factor binding sites (TFBSs) due to the presence or absence of certain identified variants. The change in the composition of TFBSs in the promoter region may lead to differential expression of the gene. This study served as a pilot investigation to identify promoter region variants in the candidate genes involved in iron metabolism, in TB patients. The results presented here indicate that the identified variants (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) could possibly contribute to the increased absorption of iron in the TB patient group, which could subsequently increase the occurrence of pathogenic infection. The findings of this study could further aid in the elucidation of the exact mechanism(s) by which iron, its metabolism, and the genes involved affect disease susceptibility, more specifically, M. tuberculosis susceptibility.AFRIKAANSE OPSOMMING: Tuberkulose (TB) is „n epidemiese siekte gekarakteriseer deur nat, aanhoudende hoes, gewigsverlies, moegheid, en bloed in die speeksel. Dit is gerapporteer dat een in elke drie individue tans geïnfekteer is met Mycobacterium tuberculosis, die veroorsakende agent van TB, en dat 10% van dié individue die aktiewe vorm van die siekte sal ontwikkel. Die hoë voorkoms en lae effek van hierdie siekte het daartoe gelei dat meer navorsing mettertyd gedoen is om die faktore wat „n rol mag speel in M. tuberculosis infeksie, te bepaal. Sommige faktore bekend vir hul rol in „n minderheid van gevalle sluit in: MIV infeksie, diabetes, alkoholmisbruik en bejaardheid, maar etniese verskille in vatbaarheid vir die siekte is ook al waargeneem. Die waarskynlikheid van genetiese invloed op die ontwikkeling van TB word ook meer deur navorsers ondersoek. M. tuberculosis benodig yster om te vermeerder, wat dit moet bekom vanaf die gasheer. Vir hierdie rede is die gene betrokke by yster metabolisme in die menslike gasheer veral van belang vir die oorweging van vatbaarheid vir M. tuberculosis. Om te bepaal of die uitdrukking van hierdie gene moontlik „n invloed het op vatbaarheid vir die siekte, was die promoter areas van die CYBRD1, HAMP, HFE, LTF en SLC40A1 gene geteiken. Die doel van hierdie studie was om te bepaal of DNS variasie in die promoter area van hierdie gene betrokke in yster metabolisme moontlik verband kan hou met vatbaarheid vir M. tuberculosis. Die studie kohort het uit 49 TB pasiënte en 51 gesonde, onverwante, populasie-gepaarde kontroles, waarvan almal Swart, Xhosa-sprekende individue was, bestaan. Aanvanklik was 15 pasiënt monsters lukraak gekies vir ondersoekende sifting, deur die gebruik van semi-outomatiese twee-rigting volgordebepalings. Op hierdie manier is 40 variante geïdentifiseer waarvan 30 voorheen beskryf is. Die nuwe variante sluit in CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; en LTF: -1377 T/G, -457 T/C, -437 C/G. „n Aantal loci het statisties betekenisvolle verskille getoon in alleel en genotipe frekwensies, en in yster parameter vlakke met die vergelyking van die pasiënt groep met die kontrole groep. In silico analise het verder die skepping en/of afskaffing van verskeie transkripsiefaktor bindingsetels (TFBSs), as gevolg van die teenwoordigheid of afwesigheid van sekere variante, getoon. Die verandering in die samestelling van TFBSs in die promoter area kan lei tot differensiële uitdrukking van die geen. Dié studie het gedien as „n voorlopige ondersoek om te bepaal of promoter area variante, geïdentifiseer in kandidaat gene betrokke by yster metabolisme, „n invloed het in die ontwikkeling van TB. Die resultate wat hier gewys word dui aan dat die geïdentifiseerde variante (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) moontlik die verhoogde opname van yster kan veroorsaak, wat later die toename van die patogeniese infeksie kan veroorsaak. Die bevindinge van hierdie studie kan moontlik bydra tot die toeligting van die presiese meganisme(s) waardeur yster, yster metabolisme, en die betrokke gene vatbaarheid vir siekte, meer spesifiek M. tuberculosis vatbaarheid, beïnvloed

Analysis of the multicomponent ALEX array data to examine patterns of sensitization in Cape Town, South Africa

IntroductionThis study analysed allergen sensitization patterns in Cape Town, a biodiversity-rich region with a Mediterranean climate, using ALEX® and ALEX²® multiplex component-resolved diagnostics tools. It aimed to address gaps in allergen sensitisation pattern data and complement aerobiological monitoring.Methods and resultsA retrospective review of 708 adults and children attending two tertiary allergy clinics (2019–2024) found that house dust mites were the most common allergens, affecting 50%–60% of participants, with Der p 23 particularly prevalent (53%). Grass pollen sensitization was also high (46%), with 85% sensitised to the C4 grass Bermuda. Tree pollen sensitisation occurred in 29% with 14% sensitised to a diverse range of trees but neither London plane nor Cypress currently recommended in limited testing panels. Common food allergens included fruits (30%), seafood (27%), and nuts (25%), often linked to pollen cross-reactivity.ConclusionOur study confirms a known pattern of aeroallergen sensitisation for a coastal temperate region, with increasing pollen sensitisation, particular C4 grasses. Clinicians should be aware of the diversity of tree pollen sensitisation, cross-reactivity patterns between food and pollen sensitisations and rates of minor allergen sensitisations for Blomia and animal danders when considering allergen-immunotherapies

Scaling up the effects of inbreeding depression from individuals to metapopulations

Abstract Inbreeding is common in nature, and many laboratory studies have documented that inbreeding depression can reduce the fitness of individuals. Demonstrating the consequences of inbreeding depression on the growth and persistence of populations is more challenging because populations are often regulated by density- or frequency-dependent selection and influenced by demographic and environmental stochasticity. A few empirical studies have shown that inbreeding depression can increase extinction risk of local populations. The importance of inbreeding depression at the metapopulation level has been conjectured based on population-level studies but has not been evaluated. We quantified the impact of inbreeding depression affecting the fitness of individuals on metapopulation persistence in heterogeneous habitat networks of different sizes and habitat configuration in a context of natural butterfly metapopulations. We developed a spatial individual-based simulation model of metapopulations with explicit genetics. We used Approximate Bayesian Computation to fit the model to extensive demographic, genetic, and life-history data available for the well-studied Glanville fritillary butterfly (Melitaea cinxia) metapopulations in the Åland islands in SW Finland. We compared 18 semi-independent habitat networks differing in size and fragmentation. The results show that inbreeding is more frequent in small habitat networks, and consequently, inbreeding depression elevates extinction risks in small metapopulations. Metapopulation persistence and neutral genetic diversity maintained in the metapopulations increase with the total habitat amount in and mean patch size of habitat networks. Dispersal and mating behavior interact with landscape structure to determine how likely it is to encounter kin while looking for mates. Inbreeding depression can decrease the viability of small metapopulations even when they are strongly influenced by stochastic extinction-colonization dynamics and density-dependent selection. The findings from this study support that genetic factors, in addition to demographic factors, can contribute to extinctions of small local populations and also of metapopulations. This article is protected by copyright. All rights reserved.Peer reviewe

Causes of hatching failure in endangered birds

About 10 per cent of birds’ eggs fail to hatch, but the incidence of failure can be much higher in endangered species.Most studies fail to distinguish between infertility (due to a lack of sperm) and embryo mortality as the cause of hatching failure, yet doing so is crucial in order to understand the underlying problem. Using newly validated techniques to visualize sperm and embryonic tissue, we assessed the fertility status of unhatched eggs of five endangered species, including both wild and captive birds. All eggs were classified as ‘infertile’ when collected, but most were actually fertile with numerous sperm on the ovum. Eggs of captive birds had fewer sperm and were more likely to be infertile than those of wild birds. Our findings raise important questions regarding the management of captive breeding programmes

INVESTIGATING THE REGULATION OF GROWTH MECHANISMS IN TWO DISTINCT BRANCHES OF PHOTOSYNTHETIC LIFE

Photosynthetic organisms often have limited mobility and rely on a variety of environmental, physiological, and chemical signals to regulate aspects of growth and development. In this thesis, I investigated how two such organisms, one a flowering plant and the other a heterokont alga, incorporate external signaling cues to make decisions regarding reproduction. My dissertation research is focused on 1) investigating molecular mechanisms of crosstalk between photoperiod and shade in regulating asexual reproduction in the wild strawberry Fragaria vesca, and 2) elucidating the mechanism of a bacterium-derived agent in the stimulation of cell division in the marine diatom Phaeodactylum tricornutum. First, strawberry, including woodland strawberry Fragaria vesca, is capable of a form of asexual reproduction by producing horizontal stems with daughter plants at the nodes. These horizontal stems, referred to as stolon, are derived from axillary meristems at the base of the leaves. Depending on the signals the axillary meristem receives, it will give rise to either a branch crown (a flowering shoot) or a stolon. Stolon allows for asexual reproduction to maintain the superior hybrid genotype and hence is of great significance agriculturally. Daughter plants derived from stolon are sold and propagated in strawberry farming. In this work, I have shown that a key regulatory protein FveRGA1 in GA signaling pathway functions as a repressor of stolon development. I further expanded this work by showing that the light quality (shade) signaling pathway interacts with the GA signaling to regulate stolon development. I identified and demonstrated FvePIF3 as a key transcription factor that positively regulates stolon initiation under far-red light (shade). Understanding the mechanisms underlying axillary meristem cell fate determination could advance biotechnology to increase strawberry production. Second, I have discovered and characterized a bacterium-based growth stimulation of the diatom Phaeodactylum tricornutum. Specifically, I noticed that a culture of P. tricornutum that had been accidentally contaminated with bacteria exhibited faster growth. I subsequently identified the responsible bacterium as Bacillus sp, which stimulated rapid Phaeodactylum cell division when added to the Phaeodactylum culture. I experimentally determined that the growth stimulating agent was heat labile and proteinase K-resistant. Further, I showed that the mother cell lysate of Bacillus sp. under sporulation was just as effective in promoting Phaeodactylum. In collaboration with Dr. Jon Clardy lab, we identified the growth-stimulating compounds as two distinct peptide-signaling molecules. The work revealed that the peptides may be previously under-reported signaling molecules for cross-kingdom communications. In addition to the fundamental discovery of novel signaling mechanisms between bacterium and algae, this work may facilitate large-scale diatom culture in biomass production for biofuel and biopharma

DISTRIBUTION OF EMBRYOS BETWEEN SIDES IN SWINE

Using Parker and Bullard’s data (Proc. Amer. Acad. Arts Sci. 49: 399–426, 1913) on 1,000 litters of swine, a predictable pattern was determined for the distribution of implants between left and right uterine horns. In contrast to mice, left and right horns had almost equal numbers of implants. Variability of left–right distributions was less than binomial due to intrauterine migration (P &lt; 0.05 in litter sizes 4–11). Deviations from the common pattern are described. Embryonic mortality appeared to enhance symmetry between sides only slightly. </jats:p