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The noradrenergic locus coeruleus (LC) is the principal source of brain norepinephrine, a neurotransmitter thought to play a major role in the pathology of major depressive disorder (MDD) and in the therapeutic action of many antidepressant drugs. The goal of this study was to identify potential mediators of brain noradrenergic dysfunction in MDD. Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-β superfamily, is a critical mediator of noradrenergic neuron differentiation during development and has neurotrophic and neuroprotective effects on mature catecholaminergic neurons. Real-time PCR of reversed transcribed RNA isolated from homogenates of LC tissue from 12 matched pairs of MDD subjects and psychiatrically normal control subjects revealed low levels of BMP7 gene expression in MDD. No differences in gene expression levels of other members of the BMP family were observed in the LC, and BMP7 gene expression was normal in the prefrontal cortex and amygdala in MDD subjects. Laser capture microdissection of noradrenergic neurons, astrocytes, and oligodendrocytes from the LC revealed that BMP7 gene expression was highest in LC astrocytes relative to the other cell types, and that the MDD-associated reduction in BMP7 gene expression was limited to astrocytes. Rats exposed to chronic social defeat exhibited a similar reduction in BMP7 gene expression in the LC. BMP7 has unique developmental and trophic actions on catecholamine neurons and these findings suggest that reduced astrocyte support for pontine LC neurons may contribute to pathology of brain noradrenergic neurons in MDD.

Multiple myeloma, or myeloma, is a bone marrow cancer which characterizes by uncontrolled proliferation of mutant plasma cells. It is a disease that claims many lives every year, mostly due to the absence of curative treatment. Finding a suitable treatment is therefor of great importance. One way to study different diseases is to use a gene editing method for knockdown or knockout of specific genes. The main aim of this project was to design guide RNAs, to be able to use CRISPR/Cas9 for knockout of the two genes BMPR1A and BMPR2 in different myeloma cell lines (KJON, INA-6 and IH-1). This, to be able to study the expression and function of these genes. Further aim of the project was to investigate potential SMAD activation by treatment with different bone morphogenetic proteins (BMPs). However, due to limited time this could not be carried through. Six guide RNAs were designed and ligated into pLentiCRISPRv2. Plasmid amplification was done by transformation of Escherichia coli. To check the quality of the plasmids, PCR, gel electrophoresis and Sanger sequencing was performed. The results from the gel electrophoresis showed that nine of the twelve samples for BMPR1A and seven of the thirteen samples for BMPR2, that were tested, were positive. The results from the Sanger sequencing confirmed that all guides that were tested (BMPR1A 3.2.3, BMPR1A 4.2.2, BMPR2 1.1.4 and BMPR2 2.1.2), were properly ligated into the plasmids. The main aim of the project was successfully accomplished, but additional work is needed for any further conclusions.

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No description available.

Connective tissue growth factor (CTGF/CCN2) and bone morphogenetic protein (BMP)-2 are both produced and secreted by osteoblasts. Both proteins have been shown to have independent effects in regulating osteoblast proliferation, maturation and mineralization. However, how these two proteins interact during osteoblast differentiation remains unknown. In Chapters 2 and 3, we utilized two cell culture model systems, osteoblasts derived from CTGF knockout (KO) mice and osteoblasts infected with an adenovirus, which over-expresses CTGF (Ad-CTGF), to investigate the effects of CTGF and BMP-2 on osteoblast development and function in vitro. To observe differences in osteoblast maturation and mineralization, we performed alkaline phosphatase (ALP) staining and activity and alizarin red staining, respectively. Contrary to a previously published report, osteoblast maturation and mineralization were similar in osteogenic cultures derived from KO and wild type (WT) calvaria in the absence of BMP-2 stimulation. Interestingly, in KO and WT osteoblast cultures stimulated with BMP-2, the KO osteoblast cultures exhibited increased alkaline phosphatase staining and activity and had larger, fused nodules stained with alizarin red than WT osteoblast cultures. This increase in osteoblast differentiation was accompanied by increased protein levels of phosphorylated Smad 1/5/8 and mRNA expression levels of bone morphogenetic protein receptor Ib. These data confirm enhanced osteoblast maturation and mineralization in BMP-2 induced KO osteoblast cultures. We also examined osteoblast differentiation in cultures that were infected with Ad-CTGF and in control cultures. Continuous over-expression of CTGF resulted in decreased ALP staining and activity, alizarin red staining, and mRNA expression of osteoblast markers in both unstimulated and BMP-2 stimulated cultures. Impaired osteoblast differentiation in cultures over-expressing CTGF was accompanied by decreased protein levels of phosphorylated Smad 1/5/8. In addition to the functional assays that we performed on WT and KO osteoblast cultures, we performed ChIP assays to investigate differences in binding occupancy of transcription factors on the Runx2 and Osteocalcin promoters in BMP-2 induced WT and KO osteoblast cultures. We demonstrate that in BMP-2 induced WT and KO osteoblast cultures, there was greater Smad 1 and JunB occupancy on the Runx2 promoter and Runx2 occupancy on the Osteocalcin promoter in BMP-2 induced KO osteoblast cultures compared to WT cultures. Collectively, the data demonstrate that CTGF acts to negatively regulate BMP-2 induced signaling and osteoblast differentiation. In Chapter 4, we synthesized an active His-tagged BMP-2 recombinant protein to track surface binding of BMP-2 in CTGF WT and KO osteoblasts. We amplified mature BMP-2 in genomic DNA, which was inserted correctly into a pET-28b(+) vector. We ran a SDS-PAGE gel and stained with Coomassie blue to show that we successfully induced BMP-2 in bacteria cells, extracted the protein using urea, and purified and eluted the protein using Nickel charged agarose beads and imidazole elution buffer. Furthermore, by Western blot analysis using anti-His antibody, we confirmed the presence of the His-tag on the BMP-2 protein. Lastly, ALP staining on osteoblast cultures stimulated with our synthesized BMP-2 exhibited increased staining compared to the unstimulated osteoblast cultures, which confirmed the activity of our His-tagged BMP-2 protein. Future studies utilizing this protein will demonstrate that CTGF acts as an extracellular antagonist by limiting the amount of BMP-2 available for receptor binding. / Cell Biology

In vitro differentiation of stem cells in biocompatible scaffolds in a bioreactor is a promising method for creating functional engineered tissue replacements suitable for implantation. Basic studies have shown that mechanical, chemical, and pharmaceutical stimuli enhance biological functionality of the replacement as often defined by parameters such as cell viability, gene expression, and protein accumulation. Most of the assays to evaluate these parameters require damage or destruction of the cell-scaffold construct. Therefore, these methods are not suitable for monitoring the development of a functional tissue replacement in a spatial and temporal manner prior to implantation. Bioluminescence imaging is a technique that has been utilized to monitor cell viability and gene expression in various in vivo applications. However, it has never been applied in an in vitro setting for the specific purpose of evaluating a cell-scaffold construct. This research describes the design of flow perfusion bioreactor system suitable for bioluminescence imaging. In the first experimental chapter, the system was tested using MC3T3-E1 cells transfected with a constitutive bioluminescent reporter. It was found that bioluminescence imaging was possible with this system. In the second experimental chapter, MC3T3-E1 cells transfected with BMP-2 linked bioluminescence reporter were cultured by flow perfusion for a period of 11 days. Bioluminescence was detectable from the cells starting at day 4, while peaking in intensity between days 7 and 9. Further, it was also found that bioluminescence occurred in distinct regions within the scaffold. These results indicate that these strategies may yield information not available with current assays. / Master of Science

Yes / Pulmonary Arterial Hypertension (PAH) is a devastating cardiovascular disorder characterised by the remodelling of pre-capillary pulmonary arteries. The vascular remodelling observed in PAH patients results from excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle (PASMCs) and endothelial cells (PAECs). We have previously demonstrated that mutations in the type II receptor for bone morphogenetic protein (BMPRII) underlie the majority of the familial and inherited forms of the disease. We have further demonstrated that BMPRII deficiency promotes excessive proliferation and attenuates apoptosis in PASMCs, but the underlying mechanisms remain unclear. The major objective of this study is to investigate how BMPRII deficiency impairs apoptosis in PAH. Using multidisciplinary approaches, we demonstrate that deficiency in the expression of BMPRII impairs apoptosis by modulating the alternative splicing of the apoptotic regulator, Bcl-x (B-cell lymphoma X) transcripts: a finding observed in circulating leukocytes and lungs of PAH subjects, hypoxia-induced PAH rat lungs as well as in PASMCs and PAECs. BMPRII deficiency elicits cell specific effects: promoting the expression of Bcl-xL transcripts in PASMCs whilst inhibiting it in ECs, thus exerting differential apoptotic effects in these cells. The pro-survival effect of BMPRII receptor is mediated through the activin receptor like kinase 1 (ALK1) but not the ALK3 receptor. Finally, we show that BMPRII interacts with the ALK1 receptor and pathogenic mutations in the BMPR2 gene abolish this interaction. Taken together, dysfunctional BMPRII responsiveness impairs apoptosis via the BMPRII-ALK1-Bcl-xL pathway in PAH. We suggest Bcl-xL as a potential biomarker and druggable target. / This work was supported by a fellowship (awarded to MTN) from the Department of Health via the NIHR Comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London, Heptagon Life Science Proof of Concept Fund (grant KCL24 to MTN), the Great Britain Sasakawa Foundation (grant B70 to MTN), the Royal Society (grant 43049 to MTN), the Medical Research Council (grant G900865 to RCT, MTN and NWM) and the University of Bradford (grants 003200, 66006/001NAS and DH005 to MTN). NS and MYB were supported by scholarships from the Commonwealth Scholarship Commission, UK and Scientific and Technological Research Council of Turkey (TUBITAK), respectively.

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Nous avons déterminé la capacité de régénération du tissu vasculaire in vivo des CSM traitées avec BMP2 à la dose de [100 ng.mL-1] dans un modèle rat. Nous avons ainsi rapporté qu’une prothèse revêtue de CSM traitée par BMP2 pendant 1 semaine et implantée 14 jours chez le rat permettait la reconstitution des trois tuniques de la paroi mimant la structure de l’aorte. La capacité proangiogénique des CSM était augmentée par BMP2 grâce à la mise en jeu de voies intracellulaires impliquant le facteur induit par l’hypoxie (HIF-1α) via JAK/STATs. Nous avons montré que les CSM migraient sous l’influence de BMP2 par stimulation de l’activité du complexe enzymatique NADPH oxydase via l’augmentation de l’expression des protéines PAK1, Vav2 et RAC1 GTPase/PI3K. Ce travail a confirmé l’intérêt de l’utilisation de CSM conjointement à rh-BMP2, une protéine impliquée dans l’embryogénèse vasculaire, pour la bioingénierie de la régénération vasculaire. / We determined the capacity to regenerate vascular tissue in vivo, of MSC treated with BMP2 at a dose of [100 ng.mL-1] in a rat model. We have reported that a prosthesis coated with CSM treated 1 week with BMP2 and implanted in rats 14 days allowed the reconstruction of the three tunics of the wall that mimic the structure of the aorta. The proangiogenic capacity of MSCs was increased by BMP2 through the intracellular pathways involving hypoxia inducible factor (HIF-1α) via JAK / STAT. We have shown that MSCs migrated under the influence of BMP2 by stimulating the activity of the enzyme complex NADPH oxidase via the increased expression of PAK1 protein, Vav2 and RAC1 GTPase/PI3K. This work confirmed the interest of the use of MSC in conjunction with rh-BMP2, a protein involved in vascular embryogenesis for bioengineering for vascular regeneration.