Pathogenic variants in SLF2 and SMC5 cause segmented chromosomes and mosaic variegated hyperploidy

Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability

The study of senescent pathology in Caenorhabditis elegans

The aim of this work is to identify the biological mechanisms of ageing, which remain poorly understood. One model organism widely used by biogerontologists is C. elegans. Here lifespan is, one assumes, a function of life limiting senescent pathologies. However, the cause of senescent pathologies and the identity of those pathologies that limit life are unclear. Therefore the main goals of my PhD were to understand where senescent pathologies come from and to identify the pathologies that limit worm lifespan. Using Nomarski microscopy, pathology within various tissues of the germline and soma was examined during ageing. The C. elegans intestine is the major somatic organ and is a likely location for lethal, senescent pathology. It undergoes major atrophy during ageing, which is demonstrated here to be driven by intestinal biomass conversion into yolk. To determine the pathology state at death, we performed necropsy analysis on the corpses of elderly worms. This revealed that the majority of early deaths occurred with an enlarged pharynx, reflecting severe bacterial infection. Combining survival and pathology data into a new pathology-centred approach has allowed new insights to be obtained into the determinants of late life disease and lifespan. It has long been widely believed that ageing was caused by molecular damage. However the recently proposed hyperfunction theory, which is related to the antagonistic pleiotropy theory, suggests that a major contributory cause of ageing is actually quasi-programmes. These are biological programmes essential for early-life fitness that continue to operate in a non-adaptive fashion in older organisms, such as intestinal biomass conversion to yolk. Thus hyperfunction may contribute to the development of age-related pathologies, some of which will cause death

The Automatic Tracking Of <i>Caenorhabditis elegans</i> And Its Use In Determining Genetic Function

Even with its simple nervous system, the nematode worm Caenorhabditis elegans can display a range of complex behaviours. Movement can be viewed as the main output of the C. elegans nervous system, and aberrations in the worm’s locomotion can be used as an indicator for genetic function in mutant strains of C. elegans. Automated tracking of C. elegans locomotion has been used to determine phenotypic fingerprints for ~300 mutant C. elegans strains. Two methods of creating phenotypic fingerprints were used. The first based on pre-determined micro-behaviours previously described in worms, but never before analysed using automated tracking. The second used the tracking data itself to determine micro-motifs, repeated sets of behaviours observed at least twice in at least two mutant or wild-type strains. Both methods of clustering successfully grouped together strains with mutations in genes known to interact together, verifying that the technique is able to detect meaningful connections between mutant strains. The following step was to determine whether the technique can be used to establish connections between genes on unknown function. A pair of strains with mutations in DEG/ENaC subunit encoding genes clustered strongly together using the micro-motif method, due to similar defects in their behaviours upon turning. The function of these genes, asic-2 and acd-5, was unknown. Upon further investigation it was found that the two genes are expressed in different classes of neurons, the IL2s in the case of asic-2 and the ASIs in the case of acd-5. Following investigation into behaviours known to be modulated by these two neuron classes it was found that the mutant strains displayed mutant phenotypes in similar behaviours, but that their mutant phenotypes are opposing. Mutations in asic-2 cause increased lifespan and healthspan and a reduction in dauer entry in response to exogenous, purified ascarosides. Mutations in acd-5 cause decreased lifespan and healthspan and a reduction in dauer entry in response to crude dauer pheromone. This suggested that the two genes were unlikely to be working in the same pathway, but do function in similar pathways. Calcium imaging is a technique used in C. elegans to measure responses in excitable cells, in this case in neurons. Many calcium indicators are available for use in this technique, one in particular is GCaMP. GCaMP has undergone many rounds of targeted mutations with the aim to increase the molecule’s dynamic range and dissociation constant. At the time of commencing this project, new variants of GCaMP, known as GCaMP6s, became available, and had yet to be tested in C. elegans neurons. The effectiveness of a total of 6 new variants was tested in the gentle touch neurons of C. elegans. It was found that the alterations made to GCaMP5G in order to make the GCaMP6 variants did not result in improved dynamic range or dissociation constant in the PLM of C. elegans

Pathogenic mutations in components of the SMC5/6 complex cause segmented chromosomes and mosaic variegated hyperploidy

The structural maintenance of chromosomes (SMC) complexes are a highly conserved family of ring shaped ATPases which have many important functions in maintaining genome stability. Whilst cohesin (SMC1/3) and condensin (SMC2/4) have well characterised roles in cellular division, the precise functions of SMC5/6 are less well-defined, although the complex is believed to function primarily in DNA repair and replication. SMC5/6 comprises of several subunits, including SMC6, SMC5 and a number of non-SMC elements (NSEs), six of which have been identified in yeast (Nse1-6). Whilst only four NSE subunits have been identified in human cells (NSE1-4), SLF1 and SLF2, functional paralogues of Nse5 and Nse6, have been shown to promote the recruitment of SMC5/6 to damaged chromatin. The importance of SMC5/6 for human development is highlighted by that fact that mutations in SMC5/6 complex components are associated with two separate childhood syndromes. Individuals with NSE2 variants present with primordial dwarfism, extreme insulin resistance, and primary gonadal failure, whilst patients with mutations in NSE3 exhibit structural chromosome abnormalities and pulmonary disease. In this thesis I will explore the function of SMC5/6, and its impact on human disease, further, by characterising patients with mutations in two SMC5/6 associated genes: SLF2 and SMC5. Using patient derived lymphoblastoid and fibroblast cell lines, alongside U-2-OS SLF2 CRISPR mutants, I will show that SLF2 and SMC5 promote proper replication and repair and suggest novel functions for these factors in mitosis and promoting efficient replication through G4-quadruplex DNA secondary structures

From Ecological Epitome to Medical Model: An investigation into Applications for the use of Daphnia in Heart Science.

The primary aim of this research was to determine whether Daphnia might become a model for cardiovascular concentration-response trials. This would provide a high throughput means of testing cardiac therapeutics without resort to small mammal trials. We found Daphnia are inappropriate in this context due to high population variance and sensitivity to small, subtle, environmental changes. A new aim was developed to determine whether beat-to-beat variation could be correlated with an individual’s response to toxic insult. Further, to develop more accurate and efficient means of gathering heart rhythm data by recording heart movement from whole live Daphnia. This opens the way to individualising cardio therapeutics; by correlating the stability of individual hearts with response to cardiac insult, regression analysis provides a means of finding a prediction tool. Daphnia are a convenient example here, but successful scoring systems might also be applied to the human heart via analysis of ECG readouts. Collecting signals from whole live Daphnia did not fulfil the goal of gathering heart data as this instead recorded limb movement. However, this provides a means of improving toxicology testing in aquatic ecology. This thesis offers three contributions to knowledge: 1. Daphnia are an inappropriate model for cardiovascular therapeutic dose-response trials due to extreme environmental sensitivities. 2. Baseline heart rhythm can be correlated with paired response to cardiac insult, with significance at the 0.01 alpha level, using an adjusted version of the Lyapnov equation; Finite Time Growth (Wessel, 2010). However, this is only if population variation is adequate. It is better applied to a natural in situ population than a homegenic lab population. 3. A novel technique for measuring Daphnia electromechanical movement records feeding limbs rather than the heart. This offers a novel and more efficient technique for aquatic ecotoxicology, where visual observation or films of the same are currently used

The Largest Unethical Medical Experiment in Human History

This monograph describes the largest unethical medical experiment in human history: the implementation and operation of non-ionizing non-visible EMF radiation (hereafter called wireless radiation) infrastructure for communications, surveillance, weaponry, and other applications. It is unethical because it violates the key ethical medical experiment requirement for “informed consent” by the overwhelming majority of the participants. The monograph provides background on unethical medical research/experimentation, and frames the implementation of wireless radiation within that context. The monograph then identifies a wide spectrum of adverse effects of wireless radiation as reported in the premier biomedical literature for over seven decades. Even though many of these reported adverse effects are extremely severe, the true extent of their severity has been grossly underestimated. Most of the reported laboratory experiments that produced these effects are not reflective of the real-life environment in which wireless radiation operates. Many experiments do not include pulsing and modulation of the carrier signal, and most do not account for synergistic effects of other toxic stimuli acting in concert with the wireless radiation. These two additions greatly exacerbate the severity of the adverse effects from wireless radiation, and their neglect in current (and past) experimentation results in substantial under-estimation of the breadth and severity of adverse effects to be expected in a real-life situation. This lack of credible safety testing, combined with depriving the public of the opportunity to provide informed consent, contextualizes the wireless radiation infrastructure operation as an unethical medical experiment

IDENTIFICATION AND CHARACTERIZATION OF THE GENETIC AETIOLOGY OF A RARE DISEASE KNOWN AS ORAL-FACIAL-DIGITAL SYNDROME TYPE II OR MOHR SYNDROME

Ph.DDOCTOR OF PHILOSOPH

Molecular Mechanisms of piRNA Biogenesis and Function in Drosophila: A Dissertation

In the Drosophila germ line, PIWI-interacting RNAs (piRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. We examined the genetic requirements for the biogenesis and function of piRNAs in both female and male germ line. We found that piRNAs function through the PIWI, rather than the AGO, family Argonaute proteins, and the production of piRNAs requires neither microRNA (miRNA) nor small interfering RNA (siRNA) pathway machinery. These findings allowed the discovery of the third conserved small RNA silencing pathway, which is distinct from both the miRNA and RNAi pathways in its mechanisms of biogenesis and function. We also found piRNAs in flies are modified. We determined that the chemical structure of the 3´-terminal modification is a 2´-O-methyl group, and also demonstrated that the same modification occurs on the 3´ termini of siRNAs in flies. Furthermore, we identified the RNA methyltransferase Drosophila Hen1, which catalyzes 2´-O-methylation on both siRNAs and piRNAs. Our data suggest that 2´-O-methylation by Hen1 is the final step of biogenesis of both the siRNA pathway and piRNA pathway. Studies from the Hannon Lab and the Siomi Lab suggest a ping-pong amplification loop for piRNA biogenesis and function in the Drosophila germline. In this model, an antisense piRNA, bound to Aubergine or Piwi, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. We isolated the loss-of-function mutations in ago3, allowing a direct genetic test of this model. We found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. Moreover, we also discovered a second Ago3-independent piRNA pathway in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line