Modern Tools for Genetic Engineering

Site-specific endonucleases create double-strand breaks within the genome and can be targeted to literally any genetic mutation. Together with a repair template, a correction of the defective locus becomes possible. This book offers insight into the modern tools of genome editing, their hurdles and their huge potential. A new era of in vivo genetic engineering has begun

MicroRNA expression profiling in peripheral blood mononuclear cells and serum of type 2 diabetic, pre-diabetic and normo-glycaemic individuals

Thesis (MSc)--Stellenbosch University, 2016.ENGLISH ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs that play a fundamental role in cellular function by regulating messenger RNA gene expression. Alterations in miRNA expression are implicated in metabolic dysregulation, with several studies reporting the involvement of miRNAs in the pathophysiology of Type 2 diabetes (T2D). Recently, circulating miRNAs have attracted considerable interest as biomarkers to identify individuals at risk for T2D, thus we hypothesised that circulating miRNA could be used as markers for T2D progression. The aim of this study was to determine whether miRNA expression profiles differ between diabetic, pre-diabetic and normo-glycaemic individuals. Individuals were recruited from local communities and classified as diabetic, pre-diabetic or normo-glycaemic according to World Health Organization criteria, whereafter miRNAs were extracted from peripheral blood mononuclear cells (PBMCs) and serum of age-, gender-, ethnicity- and BMI-matched diabetic (n=4), pre-diabetic (n=4) and normo-glycaemic (n=4) individuals. MiRNAs extracted from PBMCs were sequenced using the Illumina HiSeq 2500 platform, and validated by quantitative real time PCR (qRT-PCR) in PBMCs and serum of these individuals. Moreover, bioinformatics was conducted using various target prediction programs (TargetScan, DIANA and PITA) and the DAVID functional gene annotation tool to assign biological significance to the differentially expressed miRNAs identified by sequencing. Sequencing showed that 267 (pre-diabetics vs. normo-glycaemics), 277 (diabetics vs. normo-glycaemics) and 267 (pre-diabetics vs. diabetics) miRNAs were differentially expressed between groups. Of these, five differentially expressed miRNAs (miR-27b, miR-379, miR-21, miR-98 and miR-143) were selected for validation by qRT-PCR in PBMCs. Only miR-143 and miR-27b were significantly differentially expressed using qRT-PCR, although the results for miR-143 were different compared to the sequencing data. MiR-143 was upregulated in pre-diabetics compared to normo-glycaemic individuals (1.40-fold, p≤0.01), whereas sequencing showed upregulation of miR-143 in diabetics compared to pre-diabetics (1.75-fold, p≤0.05). The differential expression of miR-27b was consistent between qRT-PCR (1.55-fold; p=0.07) and sequencing (1.15-fold; p<0.01), where both methods showed upregulation in pre-diabetics compared to normo-glycaemic individuals. The expression of miR-27b was similarly upregulated in serum of pre-diabetics compared to normo-glycaemic individuals (2.0-fold; p≤0.05). Furthermore, five novel miRNAs identified by sequencing were successfully validated in PBMCs of diabetic, pre-diabetic and normo-glycaemic individual. Sequencing and qRT-PCR showed that miR-27b was upregulated in PBMCs and serum of pre-diabetics compared to normo-glycaemic individuals. Bioinformatics identified peroxisome proliferator-activated receptor gamma (Pparg) as a target for miR-27b. PPARG is an insulin sensitizing agent, thus we speculate that increased miR-27b expression in pre-diabetes suppresses Pparg, thereby inhibiting insulin signaling and subsequently decreasing glucose uptake. The increased insulin and glucose levels observed in the pre-diabetic individuals support this idea, although further work is required to confirm this hypothesis. In conclusion, we showed that miRNA profiles differ during T2D progression, and are able to discriminate between diabetic, pre-diabetic and normo-glycaemic individuals. To our knowledge, this is the first study to report differential expression of miR-27b during T2D, suggesting its potential as a biomarker that could be incorporated into predictive models for the identification of high risk individuals. However, miRNA profiling in a larger sampleAFRIKAANSE OPSOMMING: MikroRNAs (miRNAs ) is klein nie-koderende RNAs wat 'n fundamentele rol in sellulêre funksie speel deur regulering van boodskapperRNA geenuitdrukking. Verskeie studies ïmpliseer veranderings in miRNA ekspresie met metaboliese disregulering en in die patofisiologie van Tipe 2-diabetes (T2D). Onlangs het sirkulerende miRNAs groot belangstelling uitgelok as biomerkers om individue te identifiseer wat „n verhoogde risiko vir T2D het. Ons hipotese stel dus voor dat sirkulerende miRNA gebruik kan word as merkers vir T2D siekteprogressie. Die doel van hierdie studie was om vas te stel of miRNA geenuitdrukkings profiele verskil tussen diabete, prediabete en normoglisemiese individue. Individue wat uit plaaslike gemeenskappe gewerf is, is volgens die Wêreld Gesondheid Organisasie riglyne geklassifiseer as diabete, pre diabete of normoglisemiese individue. Hierna is miRNAs uit die perifere bloed mononukleêreselle (PBMS) en serum van ouderdom, geslag, etniesiteit en liggaamsmassa-indeks vergelykbare diabete (n=4), prediabete (n=4) en normoglisemiese individue (n=4), geïsoleer. Die geenvolgordebepaling van die geïsoleerde miRNAs is bepaal deur „n Illumina HiSeq 2500 platform, en bevestig deur kwantitatiewe “real time PCR” (qRT-PCR). Verder, is bioinformatika uitgevoer met behulp van verskeie teikenvoorspellings programme (TargetScan, Diana en PITA) asook David se funksionele geenannotasie instrument om biologiese betekenis aan die differensieel uitgedrukte miRNAs, te koppel. Geenvolgordebepaling het getoon dat 267 (prediabete vs. normoglisemies), 277 (diabete vs. normoglisemies) and 267 (prediabete vs. diabete) miRNAs differensieel uitgedruk word. Hiervan is vyf differensieel uitgedrukte miRNAs (miR-27b, miR-379, miR-21, miR-98 en miR-143) gekies vir bevestiging deur qRT-PCR in PBMS. MiR-143 en miR-27b differensiasie was deur qRT-PCR bevestig, hoewel die qRT-PCR resultate vir miR-143 verskil het met die geenvolgordebepaling data. Met qRT-PCR is miR-143 opgereguleer in die prediabete teenoor normoglisemiese individue (1,40-voudig, p≤0.01), terwyl met geenvolgordebepaling miR-143 in diabete teenoor prediabete (1,75-voudig, p≤0.05) opgereguleer was. Daar was ooreenstemming in die differensiële uitdrukking van miR-27b tussen die qRT-PCR (1,55-voudig; p=0,07) en geenvolgordebepaling (1,15-voudig; p<0,01), waar albei metodes opregulering gewys het in die prediabete teenoor normoglisemiese individue. In die serum monsters was die uitdrukking van miR-27b soortgelyk opgereguleer in prediabete (2,0-voudig; p≤0.05). Verder is vyf unieke miRNAs geïdentifiseer deur geenvolgordebepaling wat suksesvol bevestig is in PBMS van diabete en prediabete. Bioinformatika het Pparg geïdentifiseer as 'n teiken vir miR-27b. PPARG is 'n insuliensensiterings agent, dus spekuleer ons dat hoër miR-27b ekspresie, in prediabete Pparg onderdruk, wat die insuliensein demp en tot verlaagde glukose opname lei. Die verhoogde insulien en glukose vlakke wat in prediabete voorkom ondersteun hierdie idee, alhoewel verdere werk nodig is om hierdie hipotese te bevestig. Ten slotte, het ons getoon dat miRNA profiele tydens die T2D siekteprogressie verskil, en in staat is om tussen diabete, prediabete en normoglisemiese individue te diskrimineer. Tot ons kennis, dit is die eerste studie wat differensiele uitgedrukking van miR-27b in T2D rapporteer, en die potensiële toepassing as 'n nie-indringende biomerker uitwys. Dit kan moontlik in voorspellende modelle geïnkorporeer kan word vir die identifisering van hoë risiko individue. Maar verdere studies met groter monster getalle en prospektiewe longitudinale studies is nodig om die kliniese toepaslikheid te evalueer

A systems-level approach to the evolution of ageing

Ageing as a biological process is ubiquitous in life. In humans, ageing and its related conditions are revealed with improvements in health care conditions. Evidence for ageing is also apparent in most other organisms including unicellular species. Many of the pathways and mechanisms involved in ageing are evolutionarily conserved across the tree of life which provides an exceptional opportunity to study simpler organisms and extend the results to more complex forms of life. There is a rapidly growing body of data from organisms of varying levels of complexity, but there is a shortage of attempts in coherently making use of these data. A systems-level approach is necessary to bridge the gap between different biological levels, integrate the available information, and enable the synthesis of unifying hypotheses. Also, given the evolutionary nature of the question at hand (i.e. ageing), a successful hypothesis needs to be able to account for evolutionary considerations. In this thesis, I take a theoretical approach and try to explain a number of aspects of ageing from a systems-level perspective in an evolutionary context. Among the topics that will be covered are the following: (i) intra-islet pancreatic beta-cell dynamics, (ii) antioxidant defence system in pancreatic beta-cells, (iii) metabolic evolution of the glucose homeostatic system, and (iv) asymmetric damage segregation in unicellular organisms. In (i), I investigate the dynamics of beta-cell number within pancreatic islets and link the results to pathophysiology of diabetes and its various stages. In (ii) and (iii), I provide a unifying hypothesis for the paradoxical and unequivocal observation that metabolically active beta-cells have a weak antioxidant defence system and interestingly, that they are particularly weak in females. In (iv), I show how asymmetric segregation of damage at the time of mitosis is a fundamental step toward ageing and then evaluate whether and by how much asymmetry is optimal in a given organism under certain environmental conditions. I use a variety of techniques including deterministic and stochastic modelling in this thesis. The shared essence of these projects is an attempt to put data of various sources together in a unifying, systems-level evolutionary framework in order to better understand some aspects of the ageing process and its consequences.EThOS - Electronic Theses Online ServiceUK Dorothy Hodgkin Postgraduate AwardGBUnited Kingdo

Understanding intestinal and pancreatic hormone secretion in health and type 2 diabetes: (pre-)clinical studies and technical innovations

The gastrointestinal (GI) tract and pancreatic islets are key components of the endocrine system, responsible for the release of an array of peptide hormones, which orchestrate metabolic homeostasis through regulation of energy intake, nutrient digestion, absorption and metabolism. Of numerous hormones released from the gut, the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), govern the secretion of both insulin and glucagon from pancreatic islets. Together, these hormones play a critical role in maintaining glucose homeostasis. Disrupted secretion and/or action of the incretins and pancreatic hormones underpins the development of type 2 diabetes (T2D) - a global epidemic characterised by elevated blood glucose concentrations and associated with devastating micro- and macro-vascular complications. Accordingly, an improved understanding of the physiology and pathophysiology of GI and pancreatic hormones in health and T2D is of major relevance to the development of effective strategies to both prevent and better manage T2D. This thesis comprises a series of clinical and preclinical evaluations that provide novel insights into the determinants of GI and islet hormone secretion (Chapters 3-6). In addition, it details the cross-disciplinary collaborative development of two ‘organ-on-a-chip’ platforms for dissecting the secretory function of both intestinal tissues and pancreatic islets (Chapters 7-8). Chapter 1 provides an overview of the secretion and action of GI hormones arising from the complex interaction between luminal nutrients/bioactive compounds and the gut mucosa, and details conventional and innovative research tools/platforms that are indispensable for the investigation of GI hormone secretion. Chapter 2 summarises the molecular mechanisms underlying insulin secretion from pancreatic islets, with a focus on the role of Ca2+ signalling, and systematically reviews the development of diverse research platforms that are fundamental to progressing islet research. Given the substantial sex-related differences in glucose metabolism and risk of T2D, the study described in Chapter 3 explores the sex disparity in incretin hormone secretion, and compares the incretin and glycaemic responses to standardised intraduodenal glucose infusions within the physiological range of gastric emptying between healthy young men and women. While insulin resistance and consequently a relative deficiency in insulin secretion are recognised as key metabolic derangements in T2D, there is accumulating evidence indicating that excessive glucagon secretion also underpins the development of dysglycaemia during both the fasting and postprandial phases. In the liver, insulin and glucagon signalling pose counter-regulatory effects on hepatic glucose production. Alterations in hepatic function have the potential to disrupt hepatic insulin and glucagon signalling, leading to pathological changes in insulin and glucagon secretion. The study reported in Chapter 4 evaluates the relationships of blood glucose, plasma insulin, C-peptide and glucagon, both during fasting and after 75g oral glucose, with serum liver enzymes in healthy and T2D subjects, and in T2D subjects before and after a mixed meal. Given the major role of the rate of gastric emptying (GE) in determining nutrient digestion and absorption, GE may influence the glucagon and glycaemic responses in T2D. Therefore, Chapter 5 further examines the relationships of plasma glucagon and blood glucose with the rate of gastric emptying (GE) of a standardised mashed potato meal in individuals with well-controlled T2D. Strategies that are effective for modulating GI and pancreatic hormone secretion have the potential to improve glycaemic control in T2D. The recent recognition that the GI tract can detect a range of physiological and pharmacological bitter substances via a family of type 2 monomeric G-protein-coupled receptors, namely bitter taste receptors (BTRs), to release GI hormones has led to growing interest in the administration of bitter tastants to stimulate GI hormone secretion for the management of metabolic disorders, including T2D. However, the effects of bitter substances beyond the GI tract have received little attention. Chapter 6 reports the effect of a bitter substance, denatonium benzoate (DB), on insulin secretion in a series of in vitro and ex vivo experiments using a rodent pancreatic β-cell line, INS-1 832/13 cells, and isolated mouse pancreatic islets. In the latter, the effects of DB on the secretion of other islet hormones, including glucagon, GLP-1 and somatostatin, were also characterised. While the currently available cell/tissue models and in vivo tools have substantially advanced the knowledge on the physiology and pathophysiology of incretins and islet hormones, there is rising demand for sophisticated biomimetic platforms to address the increasingly complicated biological challenges and improve the translational success from benchtop to bedside. To this end, the development of a gut-on-a-chip (GOC) system is described in Chapter 7 which facilitates continuous monitoring of dynamic GLP-1 secretion from primary mouse intestinal tissue. Similarly, the development and customisation of a microfluidic sensing platform is described in Chapter 8, allowing quantification of the dynamic changes of Ca2+ and insulin concurrently, enabling investigation of the secretory function of isolated islets.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202