A novel and environmentally friendly method for preserving and depilating sheepskin : comprehensive physical, biochemical and molecular analyses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand

Figures are re-used with the publishers' permission.The first step of leather processing, depilation – in other words, removing hair from skins – accounts for one-third of the leather-making industrial waste due to the production of sulfide and alkaline water waste from the process. This study describes a method that preserves and depilates unwashed sheepskins using milk or milk by-products, including whey and permeate. It doesn’t require the use of harsh chemicals or temperature control, and results in skin that is ready to be tanned. In order to evaluate the products of this process, the depilated skin surface was evaluated not only by eye, but with scanning and transmission electron microscopy (SEM and TEM) which showed there was no apparent damage to the grain or fraying of the collagen bundles. The depilated skin was also processed to leather which was subjected to tear, tensile and shrinkage measurements which were shown to be almost identical to leathers made using the traditional process. Quantitative biochemical analyses, including collagen quantitation, collagen crosslink analysis and glycosaminoglycan (GAG) analysis of sheepskins depilated with this process showed no significant differences in both collagen, and collagen crosslink concentrations in contrast to the 10-fold increase seen in the GAG concentration. A quantitative proteomic analysis showed there was a higher retention of proteins found in the basement membrane of the skin, supporting the observation that permeate depilated skins were smoother than their sulfide depilated counterparts and produced leather with a superior surface. It also showed the loss of specific proteins around the hair follicle, hinting at a possible mechanism for depilation. To further investigate this a microbiological survey of the process was conducted. Traditional culturing methods were used to isolate and identify microorganisms present in the depilation solution after the wool had been removed. Two bacterial species (Lactococcus lactis and Lactobacillus plantarum) and two fungal species (Geotrichum candidum and Yarrowia lipolytica), were routinely identified, all of which are known to secrete a number of hydrolytic enzymes and antimicrobial compounds. This was followed by a metagenomic study of changes in the microbial community over the time course of the depilation. Although there were only 13 dominant bacterial genera identified during this study, the biggest change was a concomitant increase in the relative abundance of Lactococcus lactis that matched the decrease in Acinetobacter sp. by the end of the depilation treatment, controlling the proliferation of other putrefying organisms. In conclusion, this preliminary study has paved the way for the development of a depilation process that is kind to the environment, but more research is needed to investigate its potential use with other animal skins

High-resolution genome-wide scan of genes, gene-networks and cellular systems impacting the yeast ionome

Peer reviewedPublisher PD

Socio-demographic determinants of anaemia and nutritional status in the Democratic Republic of Congo, Uganda and Malawi

Anaemia is a worldwide public health concern. Anaemia is multifactorial and its related factors are classified according to their position in the pathophysiological process. Socioeconomic and demographic factors such as poor education, cultural norms such as food taboos can predispose children and women to anaemia through immediate causes such as physiological, biological, diet and infections. However, socioeconomic and demographic factors associated with anaemia are not widely reported and it is difficult to find published literature on this subject, which could be due to the lack of data. The objective of this research is to provide an understanding of socioeconomic and demographic factors related with anaemia among children and women and the links between anaemia during childhood and child nutritional status which can be used as a basis for policy formulation, planning and implementation.Almost three quarters of children and half of women in DRC (2007), Uganda (2006) and Malawi (2004) are anaemic. Multilevel ordinal regression models were fitted for anaemia among children and multilevel logistic regression models for anaemia among women. The models showed variations in anaemia prevalence within the countries at the community level. However, country level interactions indicate that there are no significant differences in the risk of anaemia in children and women between these countries. Endogenous switching regression models were fitted to the data to explore the link between anaemia and child’s health outcomes. Anaemia is endogenous to children’s nutritional status (weight-for-age z-scores) which should be accounted for. The prevalence of anaemia is high in DRC (71%), Uganda (74%) and Malawi (73%) and anaemia is a severe public health problem in the three countries. Although it will take considerable time for the three countries to control anaemia, it is not an impossible task. By improving nutrition and iron status, and treating helminth and malaria infections, the prevalence of anaemia can decrease as observed in Malawi in 2010. More effort is needed to identify the pathways through which anaemia within each country may be addressed

The Role of SPAG1 in the Cytoplasmic Assembly of Axonemal Dynein Arms in Human Airway Epithelia

Motile cilia are microtubule-based hair-like organelles that extend from the apical surface of cells and are essential for the propulsion of fluid or cells across the respiratory, ependymal, oviduct, and nodal epithelium. Dysfunctional motile cilia, due to genetic variants in proteins associated with the structure, regulation, and biogenesis of motile cilia, result in primary ciliary dyskinesia (PCD), a genetically heterogeneous, rare disorder characterized by chronic oto-sino-pulmonary disease, infertility, and laterality defects. The most common ciliary defect seen in PCD is absence of the multiprotein motor complexes known as axonemal dynein arms. While the composition and regulation of dynein arms have been extensively studied, how these large, complex structures are assembled remains poorly understood. Mutations in the gene sperm-associated antigen 1 (SPAG1) cause PCD with defects in both the inner and outer dynein arms, resulting in completely immotile cilia. Due to the observed dynein defects and the cytoplasmic location of SPAG1, it has been proposed to be a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium. To further elucidate SPAG1’s role in dynein assembly, we examined its expression, interactions, and ciliary defects caused by its absence in human airway epithelia. A previously uncharacterized 60-kDa isoform of SPAG1 was discovered. The full-length and 94-kDa isoforms of SPAG1 expression corresponded with ciliogenesis, while the 60-kDa isoform was constitutively expressed. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains, and canonical components of the R2TP complex, and we identified protein interaction domains between SPAG1 and another DNAAF, DNAAF2. Examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants demonstrated that the 60-kDa SPAG1 can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In SPAG1 mutants, dynein heavy chain (DHC) protein levels and their interaction with dynein intermediate chains (DICs) were reduced. In summary, our data demonstrate that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding of the DHCs and/or binding of the DHCs to the DIC complex.Doctor of Philosoph

The epidemiology of Plasmodium vivax among adults in the Democratic Republic of the Congo

Reports of P. vivax infections among Duffy-negative hosts have accumulated throughout sub-Saharan Africa. Despite this growing body of evidence, no nationally representative epidemiological surveys of P. vivax in sub-Saharan Africa have been performed. To overcome this gap in knowledge, we screened over 17,000 adults in the Democratic Republic of the Congo (DRC) for P. vivax using samples from the 2013-2014 Demographic Health Survey. Overall, we found a 2.97% (95% CI: 2.28%, 3.65%) prevalence of P. vivax infections across the DRC. Infections were associated with few risk-factors and demonstrated a relatively flat distribution of prevalence across space with focal regions of relatively higher prevalence in the north and northeast. Mitochondrial genomes suggested that DRC P. vivax were distinct from circulating non-human ape strains and an ancestral European P. vivax strain, and instead may be part of a separate contemporary clade. Our findings suggest P. vivax is diffusely spread across the DRC at a low prevalence, which may be associated with long-term carriage of low parasitemia, frequent relapses, or a general pool of infections with limited forward propagation

The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues–Which Methods Are Useful When?

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase “blocks” during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims

Phylogenetics and Biogeography of Emilia Cass. (Asteraceae, Senecioneae)

A thesis submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. June 2017, Johannesburg, South Africa.A molecular phylogenetic and biogeographic study of the palaeotropical genus Emilia Cass., tribe Senecioneae in the Asteraceae, was undertaken as very little was known about these aspects of the genus, which are informative for taxonomic and conservation practices and contribute to our understanding of its evolution. The investigation aimed to: (1) assess the species recognised by Jeffrey (1997) in the large-headed, morphologically variable and widespread Emilia coccinea complex using a phenetic approach, and evaluate how applicable the morphological and phenetic species concepts are to this complex; (2) elucidate the phylogenetic relationships of Emilia species, genera Bafutia, Emiliella and closely allied genera in the Senecioneae, using molecular DNA sequence data; (3) examine the pattern and timing of diversification in Emilia and correlate this pattern with morphological trends in the genus; and (4) identify centres of diversity and endemism for Emilia in southern Africa1, and compare and assess the following spatial biodiversity indices: species richness (SR), corrected weighted endemism (CWE), phylogenetic diversity (PD), and phylogenetic endemism (PE), and their application to the conservation of Emilia in a chosen region (viz. Zimbabwe). The phenetic study of the E. coccinea complex was based on 134 herbarium specimens spanning the altitudinal and geographical ranges of this complex and using multivariate analyses (cluster analysis and ordinations). Parsimony and Bayesian phylogenetic analyses, based on molecular plastid trnL-trnF and nuclear ITS sequence data, were conducted on a representative sample of Emilia species together with other closely related Senecioneae genera to provide the basis for a taxonomic revision of the genus. Phylogenetic relationships of Emilia species, including Jeffrey’s sectional classification of Emilia, the distinctness of the morphologically similar species in the E. coccinea complex, and the generic status of similar genera Bafutia and Emiliella were then evaluated from reconstructed phylogenies. The biogeographic diversification history of Emilia was traced using the present distribution of species and a reconstructed nuclear ITS phylogeny. Dated molecular phylogenetic hypotheses constructed using BEAST were used to estimate the time of divergence in Emilia, and linked with optimized evolutionary patterns of morphological features to trace evolutionary trends in the genus. Centres of diversity and endemism were mapped and identified in southern Africa 1 Southern Africa is defined here as the countries south of the Democratic Republic of Congo and Tanzania (Angola, Botswana, Lesotho, Malawi, Mozambique, Namibia, South Africa, Swaziland, Zambia, and Zimbabwe). using distribution data obtained from PRECIS data sets, field work and herbarium specimens. Four spatial biodiversity indices (SR, CWE, PD, and PE), two of which incorporate evolutionary history of the genus (PD, PE), were examined for overlap in southern African countries and also evaluated for potential use in conservation planning in Zimbabwe by assessing the distribution/ranges of the ten Zimbabwean Emilia species and their occurrence in currently protected areas that include national parks and botanical reserves. Additionally, the conservation status of the species found in Zimbabwe was assessed using the ‘IUCN Red Lists Categories and Criteria’. Five of the eight species (viz., Emilia emilioides, E. jeffreyana, E. praetermissa, E. subscaposa, and E. vanmeelii) in the E. coccinea complex are phenetically and genetically distinct; E. lisowskiana is not distinct as three E. coccinea sensu stricto specimens clustered with it in the multivariate analysis and it is unresolved in the molecular analyses. Two species (E. caespitosa and E. coccinea) are indistinguishable from each other in both the phenetic and molecular analyses as they overlap significantly in many morphological characters, habitats, and co-occur in most areas suggesting that they are either indistinct and should be synonymized, or possibly that they have hybridized in areas where they co-occur. A key with useful combinations of morphological characters separating the eight species in the E. coccinea complex is provided. The molecular phylogenetic analyses revealed that Emilia is not monophyletic, and that Bafutia and Emiliella are nested within it, indicating that these genera do not warrant separate generic status and should be combined with Emilia. Jeffrey’s sectional classification is not supported by the reconstructed phylogenies and there are no distinguishable morphological patterns evident amongst the clades to warrant the proposal of any meaningful sectional delimitation. Emilia baumii and E. graminea are grouped outside Emilia in both the nuclear and plastic-based molecular analyses and exclusion of these species from the genus is recommended, although additional molecular markers are needed to support this exclusion. Well-supported topological incongruences are revealed between nuclear ITS and plastid trnLtrnF phylogenies suggesting that hybridization and/or introgression have played a role in the history of Emilia, as with many other senecionoid genera. Emilia, a mainly tropical genus, is hypothesised to have originated in southern Africa during the Mid-Miocene (ca. 14.19 Mya) coinciding with a period of global climate cooling following the mid-Miocene Climatic Optimum (ca. 15 Mya). Early diversification occurred northwards into diverse habitats in Africa with further diversification in most Emilia clades occurring during the Late Miocene and occupying various habitats such as savannas, grasslands, and forest edges. At least five independent dispersals out of southern Africa to Madagascar, ascribed to long distance wind dispersal, occurred during the Pliocene. The successful diversification of Emilia in Africa could have been enhanced by its prevalent annual life form postulated to be either ancestral or evolved early (ca. 13.32 Mya) in its history. Narrow leaves, radiate capitula, and non-yellow florets have all arisen independently several times in Emilia. Emilia species are unevenly distributed in southern Africa with the highest number of species occurring in Zambia (12 species), followed by Zimbabwe (10 species) and Malawi (seven species). Centres of greatest diversity for Emilia species are found in northern and southern Malawi (including the Nyika and Zomba plateaus respectively) and Zimbabwe (Eastern Highlands and areas surrounding Harare). Two recognized centres of endemism, which are also part of the Austro-temperate Region, viz. the Chimanimani-Nyanga Centre in the Eastern Highlands and the Nyika Plateau Centre, are amongst the centres with the highest diversity of species of Emilia. Only six Emilia species are endemic or near endemic to southern Africa, a low number compared to other senecionoid genera in the Savanna and Austro-temperate Floras. With the exception of endemism, three of the spatial biodiversity indices (SR, PD, and PE) investigated in this study were congruent thus providing additional conservation information. These three biodiversity indices overlap in some of the following areas: northern Malawi, Harare region and eastern highlands of Zimbabwe and therefore should be prioritized for biodiversity conservation: thus fulfilling one of the mandates of the Convention on Biological Diversity. Phylogenetic diversity and PE provides further conservation information in eastern and north-western Zambia that could have been missed by using SR alone. These phylogenetic indices (PD and PE) should therefore be prioritized in the conservation of Emilia species, and other taxa in similar floras, thus mitigating the problems of climate change as areas that have an evolutionary history and contain geographically restricted traits are conserved. In an assessment of the conservation status of species of Emilia in Zimbabwe, three species (E. limosa, E. protracta, and E. tenellula) are rare and/or threatened and are habitat specialists e.g. in swampy areas thus warranting protection and one (E. baumii) is Data Deficient and should be investigated further. The majority of Emilia species are categorised as Least Concern. The current protected areas in Zimbabwe cover most areas where 70% of Emilia species occur, including those areas with high PD and PE, the exception being the Harare region where populations are unprotected. Conservation efforts should therefore be extended to these unprotected areas. In addition to traditional conservation approaches, it is recommended that conservation prioritization of Emilia species in southern Africa and possibly the whole of Africa, as well as other genera with similar distribution patterns in Savanna and/or Austro-temperate Floras, should integrate SR, PD, and PE since phylogenetic indices (PD and PE) provide information on evolutionary history and spatially restricted diversity which are necessary for understanding and maximizing conservation of evolutionary diversity.XL201

Interactive and computational methods for molecular modelling applied to the bacterial ribosome

Rechnerische Methoden zur effektiven Modellierung von großen Biomolekülen wurden entwickelt und dann auf die Struktur des bakteriellen Ribosoms angewendet. Basierend auf der im Englischen molekulardynamisch (molecular dynamics) genannten Technik wurden Algorithmen entworfen, um definierte Gruppen von Atomen zu starren Clustern zusammenzufassen; dadurch läßt sich die Anzahl der Parameter stark vermindern und damit der Rechenprozeß beschleunigen (Clustered Molecular Dynamics (CMD)). Die im molekulardynamischen Ansatz zur Beschreibung der strukturellen Details von Partikeln verwendete Energiepotentialfunktion wurde um zwei Terme erweitert. Diese berücksichtigen gröbere biochemische Strukturzusammenhänge, die sich aus Cross-linking Techniken und aus der Cryo-Elektronen-Mikroskopie ergeben. Grob- und detailstrukturelle Eigenschaften der Potentialfunktion wurden spezifiziert und die CMD Technik wurde in den interaktiven Modellbildungsprozeß einbezogen um ein Strukturmodell des bakteriellen Ribosoms zu generieren. Dieses weist einen hohen Plausibilitätsgrad auf, da es den zugrundegelegtenA computational multi-scale modelling approach for the refinement of large biomolecules was designed and then applied to the structure of the bacterial ribosome. Algorithms were developed, allowing defined groups of atoms to be clustered into rigid objects, which greatly reduces the number of parameters in the molecular dynamics approach and thus speeds up the computational process considerably (clustered molecular dynamics). The energy potential function, which is used in molecular dynamics to describe structural details of a particle, was extended to include terms that describe high-level biochemical constraints resulting from cross-linking techniques and cryo-electron microscopy. High- and low-level features of the potential function were specified, and the clustered molecular dynamics technique was integrated into the interactive model building process, t

Genome-wide analysis of mitochondrial DNA copy number reveals loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses was used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 × 10(–15)) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 × 10(–8)) and mtDNA replication (p = 1.2 × 10(–7)). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell-related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 × 10(–4)). SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00439-021-02394-w