Investigating nephronophthisis using a novel murine and cell model

PhD ThesisNephronophthisis (NPHP) is a major cause of pediatric renal failure. Currently there is little understanding of the aetiology of the disease. In order to identify the molecular events leading to NPHP, we have created a novel mutant mouse strain containing a truncating mutation in the Cep290 gene. Patients with mutations in CEP290 present with a ciliopathy phenotype that includes retinal dystrophy, cerebellum defects and NPHP. Characterisation of Cep290LacZ/LacZ mice confirms that they display all the features of the human condition. Microarray analysis of newborn kidney tissue was used to explore initiating events leading to NPHP. Ciliopathies have recently been associated with either disrupted Wingless integrated (Wnt) or sonic hedgehog (Shh) signaling. We show that mutant kidneys display abnormal Shh signaling in the absence of Wnt signaling abnormalities. Primary cell cultures of collecting duct (CDT) cells (isolated from Cep290LacZ/LacZ mice and wild-type litter mates crossed with the “immorto” mouse) were established and characterised. CDT cells expressed the mineral corticoid receptor (MR) and the epithelial sodium channel (ENaC) alpha subunit. The CDT cell lines formed epithelial layers and formed tubules when maintained in 3D culture media. Cep290LacZ/LacZ CDT cells displayed ciliogenesis abnormalities as well as abnormal spheroids with loss of lumen when grown in 3D culture. Pharmacological activation of Shh signaling (purmorphamine) partially rescues the spheroid and ciliogenesis defects in Cep290LacZ/LacZ CDT cells. This implicates abnormal Shh signaling in the onset of NPHP and suggests that targeted treatment of Shh antagonists have therapeutic potential

Promoting Resilience amongst Young People Transitioning from Care to Independent Living: Experiences of Residential Social Care Workers

The capacity for a young adult to develop and overcome obstacles in life underpins the concept of resilience building (Gilligan, 1997). A key role of the social care worker is to ensure that young adults growing up in the care system are afforded the best possible outcomes. This includes social care workers helping young people build their resilience in preparation for the transition into independent living and aftercare. The research reported here examines the experiences and perspectives of two residential social care workers regarding the promotion of resilience amongst young people transitioning from residential care to independent living. From analysis of the data collected, three themes were identified: the importance of a secure base, maintaining consistent and long-term relationships and challenges faced by social care workers in building the resilience of care leavers. Findings concur with previous research, which identified the importance of young people having a secure base in their lives as well as long-term relationships as a source of support to aid them in their transition from care to independence. This research in exploring some of the challenges of helping build the resilience of care leavers highlights the possible value of allowing young people to engage in positive risk taking opportunities in order to build important life skills for independent livin

Investigating Embryonic Expression Patterns and Evolution of AHI1 and CEP290 Genes, Implicated in Joubert Syndrome

Joubert syndrome and related diseases (JSRD) are developmental cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy and nephronophthisis (a cystic kidney disease). We have utilised the MRCWellcome Trust Human Developmental Biology Resource (HDBR), to perform in-situ hybridisation studies on embryonic tissues, revealing an early onset neuronal, retinal and renal expression pattern for AHI1. An almost identical pattern of expression is seen with CEP290 in human embryonic and fetal tissue. A novel finding is that both AHI1 and CEP290 demonstrate strong expression within the developing choroid plexus, a ciliated structure important for central nervous system development. To test if AHI1 and CEP290 may have co-evolved, we carried out a genomic survey of a large group of organisms across eukaryotic evolution. We found that, in animals, ahi1 and cep290 are almost always found together; however in other organisms either one may be found independent of the other. Finally, we tested in murine epithelial cells if Ahi1 was required for recruitment of Cep290 to the centrosome. We found no obvious differences in Cep290 localisation in the presence or absence of Ahi1, suggesting that, while Ahi1 and Cep290 may function together in the whole organism, they are not interdependent for localisation within a single cell. Taken together these data support a role for AHI1 and CEP290 in multiple organs throughout development and we suggest that this accounts for the wide phenotypic spectrum of AHI1 and CEP290 mutations in man

Clinical and Functional Characterization of URAT1 Variants

Idiopathic renal hypouricaemia is an inherited form of hypouricaemia, associated with abnormal renal handling of uric acid. There is excessive urinary wasting of uric acid resulting in hypouricaemia. Patients may be asymptomatic, but the persistent urinary abnormalities may manifest as renal stone disease, and hypouricaemia may manifest as exercise induced acute kidney injury. Here we have identified Macedonian and British patients with hypouricaemia, who presented with a variety of renal symptoms and signs including renal stone disease, hematuria, pyelonephritis and nephrocalcinosis. We have identified heterozygous missense mutations in SLC22A12 encoding the urate transporter protein URAT1 and correlate these genetic findings with functional characterization. Urate handling was determined using uptake experiments in HEK293 cells. This data highlights the importance of the URAT1 renal urate transporter in determining serum urate concentrations and the clinical phenotypes, including nephrolithiasis, that should prompt the clinician to suspect an inherited form of renal hypouricaemia

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Brain multiplexes reveal morphological connectional biomarkers fingerprinting late brain dementia states

Accurate diagnosis of mild cognitive impairment (MCI) before conversion to Alzheimer’s disease (AD) is invaluable for patient treatment. Many works showed that MCI and AD affect functional and structural connections between brain regions as well as the shape of cortical regions. However, ‘shape connections’ between brain regions are rarely investigated -e.g., how morphological attributes such as cortical thickness and sulcal depth of a specific brain region change in relation to morphological attributes in other regions. To fill this gap, we unprecedentedly design morphological brain multiplexes for late MCI/AD classification. Specifically, we use structural T1-w MRI to define morphological brain networks, each quantifying similarity in morphology between different cortical regions for a specific cortical attribute. Then, we define a brain multiplex where each intra-layer represents the morphological connectivity network of a specific cortical attribute, and each inter-layer encodes the similarity between two consecutive intra-layers. A significant performance gain is achieved when using the multiplex architecture in comparison to other conventional network analysis architectures. We also leverage this architecture to discover morphological connectional biomarkers fingerprinting the difference between late MCI and AD stages, which included the right entorhinal cortex and right caudal middle frontal gyrus

Multimodal and Multiscale Deep Neural Networks for the Early Diagnosis of Alzheimer’s Disease using structural MR and FDG-PET images

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease where biomarkers for disease based on pathophysiology may be able to provide objective measures for disease diagnosis and staging. Neuroimaging scans acquired from MRI and metabolism images obtained by FDG-PET provide in-vivo measurements of structure and function (glucose metabolism) in a living brain. It is hypothesized that combining multiple different image modalities providing complementary information could help improve early diagnosis of AD. In this paper, we propose a novel deep-learning-based framework to discriminate individuals with AD utilizing a multimodal and multiscale deep neural network. Our method delivers 82.4% accuracy in identifying the individuals with mild cognitive impairment (MCI) who will convert to AD at 3 years prior to conversion (86.4% combined accuracy for conversion within 1–3 years), a 94.23% sensitivity in classifying individuals with clinical diagnosis of probable AD, and a 86.3% specificity in classifying non-demented controls improving upon results in published literature