Genome-wide copy number variation study in anorectal malformations

Anorectal malformations (ARMs, congenital obstruction of the anal opening) are among the most common birth defects requiring surgical treatment (2-5/10 000 live-births) and carry significant chronic morbidity. ARMs present either as isolated or as part of the phenotypic spectrum of some chromosomal abnormalities or monogenic syndromes. The etiology is unknown. To assess the genetic contribution to ARMs, we investigated single-nucleotide polymorphisms and copy number variations (CNVs) at genome-wide scale. A total of 363 Han Chinese sporadic ARM patients and 4006 Han Chinese controls were included. Overall, we detected a 1.3-fold significant excess of rare CNVs in patients. Stratification of patients by presence/absence of other congenital anomalies showed that while syndromic ARM patients carried significantly longer rare duplications than controls (P = 0.049), non-syndromic patients were enriched with both rare deletions and duplications when compared with controls (P = 0.00031). Twelve chromosomal aberrations and 114 rare CNVs were observed in patients but not in 868 controls nor 11 943 healthy individuals from the Database of Genomic Variants. Importantly, these aberrations were observed in isolated ARM patients. Gene-based analysis revealed 79 genes interfered by CNVs in patients only. In particular, we identified a de novo DKK4 duplication. DKK4 is a member of the WNT signaling pathway which is involved in the development of the anorectal region. In mice, Wnt disruption results in ARMs. Our data suggest a role for rare CNVs not only in syndromic but also in isolated ARM patients and provide a list of plausible candidate genes for the disorder.postprin

Modelling of chewing and aroma release during oral processing : model development, model validation and comprehensive examples for food design : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemical and Bioprocess Engineering, Massey University, Palmerston North, New Zealand

These Figures were removed for copyright reasons: 2-1 (=Lucas et al., 2002 Fig 1), 2-2 (=Hiiemae, 2004 Fig 1), 2-4 (=Mosca & Chen, 2017 Graphical abstract), 2-6 (=Zhang et al., 2019, Fig 1) and 2-11 (=Doyenette et al., 2014 Fig 1).Chewing is complex because of its sub-processes and interactions, and inter-individual differences between people. The development of mechanistic models can be a tool to explore these aspects and can lead to the development of foods with controlled digestion outcomes and improved sensory appeal. A mechanistic chewing model was developed based on selection and breakage processes and implemented using a discretised population balance to predict the changes in bolus particle size distribution during chewing. The model was successfully implemented on peanuts, which gave confidence for its implementation to cooked white rice, which is an aromatic food system and has strong correlations with in vitro digestion. The relationship between panellists physiological, chewing and aroma release parameters during mastication of white rice were investigated in vivo to provide insights for model development. The findings showed that the dynamic behaviour of aroma release of all five subjects followed a similar trend with the breakdown pathways where subjects with smaller particles size in their bolus had higher aroma release. The study paved the first step in understanding the role of chewing on aroma release of cooked white rice and provided a range of oral processing behaviours for model validation. A coupled chewing and aroma release model was developed and validated against experimental data. Adjusting the input parameters from the coupled model showed that the portion size, initial concentration of the studied aroma compound, initial liquid volume and the rice pasted fraction were the most sensitive product-related parameters. The oral cavity volume, pharynx volume, nasal cavity volume and the breathing frequency were the most sensitive physiological parameters. The physico-chemical parameter which had the most significant effect was the mass transfer coefficient in the saliva phase. Examples were also given to show the difference in aroma release when aroma compounds of varying partition coefficients were used. The work from this thesis constitutes the first step in the application of mechanistic chewing models as a tool for food design. The next step will be to expand these models to a wider range of food systems and to a larger number of individuals to improve the model reliability

Epigenetics of complex traits and diseases

Thousands of genetic and epigenetic variants have been identified for many common diseases including cancer through genome-wide association studies (GWAS) and epigenome-wide association studies (EWAS). To advance the complex interpretation of both GWAS and EWAS results, I developed new software tools (FORGE2 and eFORGE) for the analysis and interpretation of GWAS and EWAS data, respectively. Both tools determine the cell type-specific regulatory component of a set of target regions (either GWAS-identified genetic variants or EWAS-identified differentially methylated positions). This is achieved by detecting enrichment of overlap with histone mark peaks or DNase I hypersensitive sites across hundreds of tissues, primary cell types, and cell lines from the ENCODE, Roadmap Epigenomics, and BLUEPRINT projects. Application of both tools to publicly available datasets identified novel disease-relevant cell types for many common diseases, a stem cell-like signature in cancer EWAS, and also demonstrated the ability to detect cell-composition effects for EWAS performed on heterogeneous tissues. To complement these bioinformatics efforts and validate selected variants predicted by FORGE2, eFORGE and additional analyses, I performed conformation capture using 4C-seq to fine-map the 3D context of the genomic regions involved, uncovering novel interactions for autoimmunity-associated variants and IKZF3

Hepatocyte growth factor and epidermal growth factor in HIV associated preeclampsia.

Masters Degree. University of KwaZulu-Natal, Westville.Background: The survival or death of a cell is reliant upon growth factors. Hepatocyte and Epidermal Growth Factor (HGF and EGF) promote vital cellular processes such as cell survival, proliferation, differentiation, growth, invasion and repair via various pathways. Hence these growth factors facilitate normal pregnancy. In complications such as preeclampsia (PE), decreased trophoblast invasion results in defective spiral artery remodeling, which leads to decreased blood flow and a hypoxic micro-environment. In South Africa (SA), HIV infection and PE are the leading causes of maternal mortality and morbidity. In light of the high prevalence of HIV infection and PE in SA, this study aimed to determine the concentrations of HGF and EGF in HIV associated PE. Methods: Post ethics approval, serum samples were collected from normotensive HIV-negative (n = 20); normotensive HIV-positive (n = 20); preeclamptic HIV-negative (n = 20) and preeclamptic HIV-positive (n = 20) women. All HIV-positive women received Highly Active Anti-Retroviral Treatment (HAART). Quantification and analysis of HGF and EGF expression was attained by using the Bio-Plex multiplex immunoassay technique. Results: As expected there was a statistically significant difference between gestational age, systolic and diastolic blood pressures across the study groups (p<0.0001). No significant difference was noted in maternal age (p=0.16), parity (p=0.47) and maternal weight (p=0.36) across all study groups. Irrespective of pregnancy type, HGF was significantly increased in HIV-positive women vs HIV-negative women (p=0.0225). However, no statistical significance was found based on pregnancy type (p=0.8890). A significant decrease of HGF expression was noted between normotensive HIV-negative and normotensive HIV-positive women (p=0.0022). Irrespective of pregnancy type, EGF was found to be significantly elevated in HIV-positive compared to HIV-negative women (p=0.0055). In addition, preeclamptic women displayed a higher EGF level compared to normotensive women (p=0.003), regardless of HIV status. The Epidermal Growth Factor was significantly down-regulated in normotensive HIV-negative group vs normotensive HIV-positive (p<0.001), preeclamptic HIV-positive (p<0.001) and preeclamptic HIV-negative groups (p<0.001). Conclusion This novel study displays a significant up-regulation in the expression of HGF and EGF in HIV infection during pregnancy, reflecting an immune reconstitution following HAART. These findings may be caused due to the HIV accessory protein Tat that inhibits growth factor function thereby, negatively impacting cell migration. The up-regulation of EGF expression in PE, may be responsible for impaired trophoblast cell invasion. As anticipated in HIV associated PE, EGF expression increased in HIV infected pregnancies and PE. The expression Epidermal Growth Factor in HIV associated PE, may be used as a risk indicator, predicting PE development prior to the manifestations of clinical signs and symptoms

In silico analyzes of the involvement of GPR55, CB1R and TRPV1: response to THC, contribution to temporal lobe epilepsy, structural modeling and updated evolution

Introduction: The endocannabinoid (eCB) system is named after the discovery that endogenous cannabinoids bind to the same receptors as the phytochemical compounds found in Cannabis. While endogenous cannabinoids include anandamide (AEA) and 2-arachidonoylglycerol (2-AG), exogenous phytocannabinoids include Δ-9 tetrahydrocannabinol (THC) and cannabidiol (CBD). These compounds finely tune neurotransmission following synapse activation, via retrograde signaling that activates cannabinoid receptor 1 (CB1R) and/or transient receptor potential cation channel subfamily V member 1 (TRPV1). Recently, the eCB system has been linked to several neurological diseases, such as neuro-ocular abnormalities, pain insensitivity, migraine, epilepsy, addiction and neurodevelopmental disorders. In the current study, we aim to: (i) highlight a potential link between the eCB system and neurological disorders, (ii) assess if THC exposure alters the expression of eCB-related genes, and (iii) identify evolutionary-conserved residues in CB1R or TRPV1 in light of their function. Methods: To address this, we used several bioinformatic approaches, such as transcriptomic (Gene Expression Omnibus), protein–protein (STRING), phylogenic (BLASTP, MEGA) and structural (Phyre2, AutoDock, Vina, PyMol) analyzes. Results: Using RNA sequencing datasets, we did not observe any dysregulation of eCB-related transcripts in major depressive disorders, bipolar disorder or schizophrenia in the anterior cingulate cortex, nucleus accumbens or dorsolateral striatum. Following in vivo THC exposure in adolescent mice, GPR55 was significantly upregulated in neurons from the ventral tegmental area, while other transcripts involved in the eCB system were not affected by THC exposure. Our results also suggest that THC likely induces neuroinflammation following in vitro application on mice microglia. Significant downregulation of TPRV1 occurred in the hippocampi of mice in which a model of temporal lobe epilepsy was induced, confirming previous observations. In addition, several transcriptomic dysregulations were observed in neurons of both epileptic mice and humans, which included transcripts involved in neuronal death. When scanning known interactions for transcripts involved in the eCB system (n = 13), we observed branching between the eCB system and neurophysiology, including proteins involved in the dopaminergic system. Our protein phylogenic analyzes revealed that CB1R forms a clade with CB2R, which is distinct from related paralogues such as sphingosine-1-phosphate, receptors, lysophosphatidic acid receptors and melanocortin receptors. As expected, several conserved residues were identified, which are crucial for CB1R receptor function. The anandamide-binding pocket seems to have appeared later in evolution. Similar results were observed for TRPV1, with conserved residues involved in receptor activation. Conclusion: The current study found that GPR55 is upregulated in neurons following THC exposure, while TRPV1 is downregulated in temporal lobe epilepsy. Caution is advised when interpreting the present results, as we have employed secondary analyzes. Common ancestors for CB1R and TRPV1 diverged from jawless vertebrates during the late Ordovician, 450 million years ago. Conserved residues are identified, which mediate crucial receptor functions

Genetic screening algorithm for inflammatory back pain

Objective: To develop a single nucleotide polymorphism (SNP) based genetic-based algorithm among patients with low back pain to screen for axial spondyloarthritis (SpA). Methods: An 18-plex genetic assay was designed using a MassARRAY, consisting of SNPs associated with ankylosing spondylitis (AS), psoriasis, inflammatory bowel disease (IBD) and uveitis. 1172 AS cases and 848 controls have been analyzed over two cohorts. A machine learning algorithm was created using a J48/C4.5 decision tree model; the first decision was human leukocyte antigen B 27 (HLA-B*27) status. The initial algorithm was validated in an independent cohort. The discovery and validation cohorts were then combined and the final genetic-based screening algorithm was weighted. Results: The SNP based algorithm that included HLA-B*27 positivity had a precision, specificity and sensitivity of; 0.83, 0.83, and 0.80, respectively which is higher than the current HLA-B*27 based Assessment of Spondyloarthritis International Society (ASAS) classification criteria. The SNP based algorithm that included HLA-B*27 negativity had a precision, specificity and sensitivity of, 0.58, 0.32, and 0.69, respectively. Conclusions: This genetic screening algorithm is inexpensive, out performs the clinical arm of the current ASAS classification criteria and can potentially lead to earlier detection of axial SpA

Microfluidic front-end technologies for protein electrospray mass spectrometry

In the present work, a polymer microfabricated microsprayer is characterised and tested for the mass spectrometric analysis of biomolecules, in particular in the context of protein mass spectrometry and proteomics. As the core of this work deals with analytical sciences and device development, Chapter 1 puts this work in perspective with the emergence of proteomics and more generally systems biology. In particular, major epistemological concepts that allowed the emergence of these new disciplines among life science research are reviewed. It is shown that the new paradigm of integrative biology did not emerge ex nihilo, but was preceded by major epistemological shifts in medicine, physics, and engineering. As such, the rise of systems biology appears as a new episode in the balance between reductionist and holistic approaches in the history of biology and medicine, and its biomedical promises are discussed. Chapter 2 reviews the state of the art in the hyphenation of microfluidic devices with electrospray mass spectrometry, both from a technical and applicative standpoint. Chapter 3 and 4 present the characterisation of the new polymer microspray for the analysis of peptides, proteins and glycoconjugates when coupled with various electrospray ion sources and instruments. In particular, a detailed comparison with classical pulled nanospray capillaries is performed, that established the performances in terms of sensitivity, stability and applicable ranges of flow rates and solvents. Chapter 5 introduces a functionalised polymer microspray for online sample clean-up. Basically, a hydrophobic polyvinylidene fluoride membrane is interfaced at the inlet of the polymer microspray to serve as a capture solid-phase of hydrophobic compounds; once captured, they can be cleaned up, and further eluted by the spray solution containing organic solvents. The process is further investigated in the presence of compounds usually used in protein sample preparation, such as chaotropes, reducing agents or detergents. Further microchip developments include the introduction of a dual channel microsprayer in Chapter 6, that allows to spray two solutions at the same time; given the microchip design, the two solutions are mixed only in the Taylor cone, where the electrospray is generated, and their respective flow rates can be controlled independently. This unique feature allows to analyse pure aqueous samples with integration of minimum amounts of organic sheath flow to promote desolvation and ionisation. Moreover, when proteins are analysed, it allows to measure them in their folded state, as demonstrated by their charge state distribution in the mass spectrum. Further tuning of the organic sheath flow allows to denature the protein within the Taylor cone, which can be monitored by the evolution of the protein charge state distribution in the mass spectrum. Potential of this technology for protein thermodynamic stability measurement is discussed. Lastly, Chapter 7 presents in silico evaluation of two technologies developed in the laboratory, namely Off-Gel electrophoresis for isoelectric fractionation of proteins and peptides mixtures, and online counting of cysteine residues within peptides during their analysis by electrospray mass spectrometry, to provide useful information to speed-up proteome profiling: if one wants to seek within a whole digested proteomes for peptides that are unique, measurement of their mass alone is hardly sufficient to perform useful protein identification, even with very high resolution, high mass accuracy instruments. The purpose of these simulations is to estimate how much information peptide isoelectric point and number of cysteines within each peptide provide in terms of number of unique peptides (and hence unambiguous peptide identification without MS/MS). Applicability of this strategy for high-throughput proteome profiling and its limitations are further discussed

A Bioinformatics Study of Protein Conformational Flexibility and Misfolding: a Sequence, Structure and Dynamics Approach

This PhD Thesis titled "A Bioinformatics Study of Protein Conformational Flexibility and Misfolding: a Sequence, Structure and Dynamics Approach" comprises the results and conclusions obtained by us from the study of three different but somehow related research projects, covering aspects of the phenomenon of protein local conformational instability, its relationship with protein function, evolvability and aggregation, and the effect of genetic variations on protein conformational instability related to Conformational Diseases. These projects include the prediction of putative prion proteins in complete proteomes and the study of prion biology from a genomic perspective, the prediction of conformationally unstable protein regions and the existence of a structural framework for linking conformational instability to folding and function, and the establishment of a rationale for assessing the connection among mutations and disease phenotypes in Conformational Diseases.Esta tesis doctoral comprende los resultados y conclusiones obtenidos por nosotros a partir del estudio de tres proyectos de investigación diferentes pero de alguna manera relacionados, cubriendo los aspectos del fenómeno de la inestabilidad conformacional local de la proteína, su relación con la función de la proteína, la capacidad de evolución y agregación, y el efecto de las variaciones genéticas en la inestabilidad conformacional de la proteína relacionados con las enfermedades conformacionales. Estos proyectos incluyen la predicción de presuntas proteínas priónicas en proteomas complejos y el estudio de la biología de priones desde una perspectiva genómica, la predicción de las regiones de proteínas conformacionalmente inestables y la existencia de un marco estructural para la vinculación de la inestabilidad conformacional del plegado y la función, y el establecimiento de una razón fundamental para la evaluación de la relación entre las mutaciones y fenotipos de la enfermedad en enfermedades conformacionales

Assessment of a Nutritional Rehabilitation Model in Two Modern Broilers and Their Jungle Fowl Ancestor: A Model for Better Understanding Childhood Undernutrition

The World Health Organization, estimated that 22.9% of children under the age of 5 are stunted. The etiology of stunting is multifactorial and is associated with poor linear growth, villous atrophy, dysbiosis, and increased intestinal permeability. Inclusion of rye in poultry diets induces nutrient deficiencies and increases intestinal permeability, dysbiosis and decreases growth rates. The objective of this dissertation was to determine if chickens consuming a rye based diet exhibited a similar pathophysiology of stunted children to develop a relevant animal model. Therefore, early or late phase malnutrition was induced determine the effects of malnutrition on performance, bone mineralization, intestinal morphology and paracellular intestinal leakage across three diverse genetic backgrounds. 2015 Cobb chicken, 1995 Cobb chicken, and the Jungle Fowl were allocated into four different dietary treatments. Dietary treatments were (1) a control corn-based diet throughout the trial (corn–corn); (2) an early phase malnutrition diet where chicks received a rye-based diet for 10 days, and then switched to the control diet (rye–corn); (3) a malnutrition rye-diet that was fed throughout the trial (rye–rye); and (4) a late phase malnutrition diet where chicks received the control diet for 10 days, and then switched to the rye diet (corn–rye). Modern broilers in the rye-corn treatment group exhibited catch up growth and was able to fully recover all of the growth and bone parameters measured after the consumption of a rye based diet. However, the rye-corn group was unable to recover was the serum FITC-D indicating the gut was still leaky. 1995 broilers in the rye-corn group had significantly lower BW, BWG, and tibia strength and higher serum FITC-D than the corn-corn group indicating that these birds were not able to fully recover within the observed timeframe. Jungle fowl appeared to have a higher tolerance to the rye based diet, as there were minimal differences between dietary treatments for the parameters measured. This suggests that a rye-based diet is a viable approach to induce malnutrition in chickens and slower compensatory growth rate observed in the 1995 broilers was similar to that of stunted children in developing countries