Transcriptome analysis of cartilage in homoeostasis and osteoarthritis using deep sequencing technology

PhD ThesisOsteoarthritis (OA) is the most prevalent type of joint diseases. It is associated with the progressive degradation of articular cartilage and disease progression can lead to total destruction. In order to study the molecular changes in OA cartilage, I compared the transcriptome of OA and healthy cartilage using two technologies, expression microarrays and the recently introduced RNA sequencing (RNAseq) technology. RNAseq is based on the next-generation sequencing, enabling the investigation of the transcriptome in single nucleotide resolution. In this PhD project, I optimized the cartilage RNA extraction protocol first and then used the optimized protocol to extract RNAs from both OA and healthy cartilages samples. Before the RNAseq experiment, the quality of the RNA samples was checked with real-time PCR and gene expression microarray experiments. Using microarray and RNAseq data, I found novel OA associated genes and canonical pathways. With the RNAseq data, the knowledge of the OA transcriptome was further extended, including differentially expressed transcripts, novel transcripts in cartilage, alternative splicing events and differential allelic expressions. The performance of the RNAseq was also compared with the microarray technology, revealed both advantages and the limitations of the technology

Long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner where they function in various aspects of cell biology, often as key regulators of gene expression. In this study we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor which is essential for chondrocyte development by directing the expression of chondrocyte specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of MSCs. Depletion of one of these lncRNA, LOC102723505, which we termed ROCR (regulator of chondrogenesis RNA), by RNAi disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of ROCR, and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA

Altered RNA metabolism due to a homozygous RBM7 mutation in a patient with spinal motor neuropathy.

The exosome complex is the most important RNA processing machinery within the cell. Mutations in its subunits EXOSC8 and EXOSC3 cause pontocerebellar hypoplasia, spinal muscular atrophy (SMA) and central nervous system demyelination. We present a patient with SMA-like phenotype carrying a homozygous mutation in RBM7-a subunit of the nuclear exosome targeting (NEXT) complex-which is known to bind and carry specific subtypes of coding and non-coding RNAs to the exosome. The NEXT complex with other protein complexes is responsible for the substrate specificity of the exosome. We performed RNA-sequencing (RNA-seq) analysis on primary fibroblasts of patients with mutations in EXOSC8 and RBM7 and gene knock-down experiments using zebrafish as a model system. RNA-seq analysis identified significantly altered expression of 62 transcripts shared by the two patient cell lines. Knock-down of rbm7, exosc8 and exosc3 in zebrafish showed a common pattern of defects in motor neurons and cerebellum. Our data indicate that impaired RNA metabolism may underlie the clinical phenotype by fine tuning gene expression which is essential for correct neuronal differentiation

Identification of 34 Novel Proinflammatory Proteins in a Genome-Wide Macrophage Functional Screen

Signal transduction pathways activated by Toll-like Receptors and the IL-1 family of cytokines are fundamental to mounting an innate immune response and thus to clearing pathogens and promoting wound healing. Whilst mechanistic understanding of the regulation of innate signalling pathways has advanced considerably in recent years, there are still a number of critical controllers to be discovered. In order to characterise novel regulators of macrophage inflammation, we have carried out an extensive, cDNA-based forward genetic screen and identified 34 novel activators, based on their ability to induce the expression of cxcl2. Many are physiologically expressed in macrophages, although the majority of genes uncovered in our screen have not previously been linked to innate immunity. We show that expression of particular activators has profound but distinct impacts on LPS-induced inflammatory gene expression, including switch-type, amplifier and sensitiser behaviours. Furthermore, the novel genes identified here interact with the canonical inflammatory signalling network via specific mechanisms, as demonstrated by the use of dominant negative forms of IL1/TLR signalling mediators

Revealing the two-dimensional electronic structure and anisotropic superconductivity in a natural van der Waals superlattice (PbSe)1.14_{1.14}NbSe2_2

Van der Waals superlattices are important for tailoring the electronic structures and properties of layered materials. Here we report the superconducting properties and electronic structure of a natural van der Waals superlattice (PbSe)$_{1.14}$NbSe$_2$. Anisotropic superconductivity with a transition temperature $T_c$ = 5.6 $\pm$ 0.1 K, which is higher than monolayer NbSe$_2$, is revealed by transport measurements on high-quality samples. Angle-resolved photoemission spectroscopy (ARPES) measurements reveal the two-dimensional electronic structure and a charge transfer of 0.43 electrons per NbSe$_2$ unit cell from the blocking PbSe layer. In addition, polarization-dependent ARPES measurements reveal a significant circular dichroism with opposite contrast at K and K' valleys, suggesting a significant spin-orbital coupling and distinct orbital angular momentum. Our work suggests natural van der Waals superlattice as an effective pathway for achieving intriguing properties distinct from both the bulk and monolayer samples.Comment: 8 pages, 4 figure

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons

Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous—but not individual—depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α–Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability

Flat band magnetism and helical magnetic order in Ni-doped SrCo2_2As2_2

A series of Sr(Co$_{1-x}$Ni$_x$)$_2$As$_2$ single crystals was synthesized allowing a comprehensive phase diagram with respect to field, temperature, and chemical substitution to be established. Our neutron diffraction experiments revealed a helimagnetic order with magnetic moments ferromagnetically (FM) aligned in the $ab$ plane and a helimagnetic wavevector of $q=(0,0,0.56)$ for $x$ = 0.1. The combination of neutron diffraction and angle-resolved photoemission spectroscopy (ARPES) measurements show that the tuning of a flat band with $d_{x^2-y^2}$ orbital character drives the helimagnetism and indicates the possibility of a quantum order-by-disorder mechanism.Comment: 9 pages, 12 figures, Supplementary Material available upon request, accepted by Phys. Rev.

An iPSC Patient Specific Model of CFH (Y402H) Polymorphism Displays Characteristic Features of AMD and Indicates a Beneficial Role for UV Light Exposure

Age related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease, however these do not exist for the dry form. Complement factor H (CFH) polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, retinal pigment epithelium (RPE) damage and visual decline. We have derived and characterised induced pluripotent stem cell (iPSCs) lines from two patients without AMD and low risk genotype and two patients with advanced AMD and high risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H (FH), factor I (FI) and factor H like 1 (FHL-1). The iPSC RPE cells derived from high risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy and deposition of “drüsen” like deposits. The low and high risk RPE cells respond differently to intermittent exposure to UV light which leads to an improvement in cellular and functional phenotype only in the high risk AMD-RPE cells. Taken together our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing