Methods for preparing an array of potential small molecule inhibitors of Bone Morphogenetic Protein Receptor 1b (BMPR1b) are described. Target molecules were prepared from two general classes: (1) N9-aryl-N6-ureidoadenines, and (2) dicarbamyl iodoacetamides. Recent data from the Peterson lab indicated that both classes might bind to BMPR1b and thus inhibit this key receptor. Docking studies performed using Sureflex Dock suggested the N9-aryl-N6-ureidoadenines would bind to the active site of BMPR1b. In addition antiproliferative activities of dicarbamyl iodoacetamides previously synthesized in the Peterson lab pointed to this moiety as an attractive target for structure activity relationship (SAR) development. Compounds were prepared in good to excellent yields and 40 derivatives were screened for antiproliferative activity. Of the N9-aryl-N6-ureidoadenine derivatives, N9-phenyl-N6-N-phenylureaadenine was most potent and exhibited selective activity against HeLa cells (IC50 = 11± 1 uM). Dicarbamyl iodoacetamide derivatives had similar activities compared to the previously reported compound (JRS-150).

No description available.

Mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) are the most common genetic cause of heritable pulmonary arterial hypertension (PAH). However, given the reduced penetrance of BMPR2 mutations in affected families, a major outstanding question is the identity of additional factors or pathways that are responsible for the manifestation of clinical disease. Furthermore, limited human tissue is available for study and usually only from patients with end-stage disease, making it difficult to understand how PAH is established and progresses. Alternative human models of PAH are therefore required. This thesis describes the characterisation of the first human iPSC-derived smooth muscle cell (iPSC-SMC) model of PAH and elucidates the role of BMPR2 deficiency in establishing PAH-associated phenotypes in iPSC-derived SMCs. To achieve this, I used CRISPR-Cas9 gene editing to generate wild-type and BMPR2+/- iPSC lines with isogenic backgrounds which were subsequently differentiated into lineage-specific iPSC-SMCs that displayed a gene expression profile and responses to BMP signalling akin to those present in distal pulmonary artery smooth muscle cells (PASMCs). Using these cells, I found that the introduction of a single BMPR2 mutation in iPSC-SMCs was sufficient to recapitulate the pro-proliferative and anti-apoptotic phenotype of patient-derived BMPR2+/- PASMCs. However, acquisition of the mitochondrial hyperpolarisation phenotype was enhanced by inflammatory signalling and required an interaction between BMPR2 mutations and environmental stimuli provided by exposure to serum over time. Furthermore, I showed that BMPR2+/- iPSC-SMCs had an altered differentiation state and were less contractile compared to wild-type iPSC-SMCs, phenotypes which have not been observed previously in PAH-derived PASMCs. Finally, RNA sequencing analysis identified genes that were differentially expressed between wild-type and BMPR2+/- iPSC-SMCs and may hence provide further insights into PAH pathobiology. The iPSC-SMC model described in this study will be useful for identifying additional factors involved in disease penetrance and for validating therapeutic approaches that target BMPR2.

Pulmonary arterial hypertension (PAH) is a lethal disease, characterized by functional or structural abnormalities involving distal pulmonary arterioles that result in increased pulmonary vascular resistance (PVR) and ultimately right heart failure. Our objective is to establish a conditional transgenic system in mice, to test the hypothesis that lung EC apoptosis at the level of distal pulmonary arterioles is necessary and sufficient to cause a PAH phenotype. In a pilot study, the Fas-Induced Apoptosis (FIA) construct was expressed under the control of endothelial-specific Tie2 promoter in transgenic mice (i.e. EFIA mice). Administration of a small molecule dimerizing agent, AP20187, resulted in lung modest dose-dependent PAH, which was associated with proliferative vascular lesions localized to distal lung arterioles in a small proportion of mice. Due to the low level of transgene expression in preliminary EFIA lines, we re-designed the transgenic vector by incorporating a more robust endothelial promoter (superTie2). The new construct was transfected into HUVEC and BAEC and analyzed by monitoring immunofluorescence (DsRed). Data from the EFIA model suggests that EC apoptosis may be sufficient to induce a PAH phenotype with the characteristic lung vascular lesions. The EFIA model will allow us to better explore the mechanism that links distal lung EC apoptosis with reactive vascular cell proliferation in the pathogenesis of this devastating disease.

Melanosome biogenesis is a highly regulated endosomal maturation process wherein structural fibers harbouring immature melanosomes acquires its biosynthetic proteins through the secretory pathway and finally matures into a functional organelle. These processes were shown to be dependent on several cytosolic protein complexes such as AP (adaptor protein)-1, AP-3, BLOC (biogenesis of lysosome-related organelles complex)-1, -2 and -3; in addition to kinesin motor KIF13A and Rab GTPases 7, 32 or 38. Mutations in the subunits of these complexes or Rab38 result into defective melanosome maturation leading to occulocutaneous albinism, a clinical phenotype commonly observed in Hermansky-Pudlak syndrome (HPS). Moreover, molecular function of these complexes in regulating the biogenesis of melanosome is partially known. The delivery of cargo to maturing melanosomal membranes requires fusion machinery that includes Rab GTPases, tethering factors and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. However, the SNAREs involved in the transport of cargo to melanosomes is poorly understood. In this study entitled as “understanding the SNARE dynamics during melanosome biogenesis” we focus on functional role of endosomal Qa-SNARE protein, Syntaxin 13 (formally called STX12, herein referred to as STX13) in the organelle biogenesis and its transport in and out of melanosome. Moreover, these studies show that STX13-mediated cargo transport require a melanosomal membrane localized R-SNARE VAMP7 and these SNAREs are interdependent on each other in regulating their steady state distribution. In addition, this study illustrated the possible mechanism of SNARE recycling which occurs indirectly through AP-3 complex. Thus, these studies underscore the STX13‟s role in cargo transport to maturating melanosomes and its trafficking routes to and from the melanosomes. Chapter-I describes the literature review on melanosome biogenesis; Chapter-II lists the experimental procedures used in this study and Chapter-III to V focuses on results and discussion, segregated into three sections. Chapter-III: Screening and identification of endosomal SNAREs involved in the trafficking of melanosomal proteins. Our preliminary RNAi screen for SNAREs involved in melanosome biogenesis revealed STX13 as one of the Qa-SNARE affecting pigmentation and cargo transport. STX13, a recycling endosomal SNARE has been reported to interact with pallidin, a subunit of BLOC-1; however the functional role of this interaction in pigment formation is unknown. In addition, previous studies from our lab have shown that STX13 colocalize with endosomal Rab11 and partially with EEA1- or Rab5-positive organelles in melanocytes. Together, these observations insinuated us to characterize the functional role of STX13 in melanosome biogenesis. Upon STX13 inactivation, wild type mouse melanocytes showed hypopigmentation due to mistargeting of cargo such as TYRP1 and TYR to lysosomes. Knockdown of STX13 dramatically decrease the population of immature and mature melanosomes. Moreover, STX13 associate with the melanosome cargo on endosomal tubular structures. In addition, deletion of regulatory domain in STX13 increases the cargo transport to melanosomes due to its increased SNARE activity. This is possibly due to loss in intracellular regulation of SNARE occur through multiple factors such as SM (Sec1p/Munc18) proteins. Together this data suggests that STX13 mediates cargo transport to melanosomes from recycling endosomes. Chapter-IV: Functional characterization of the SNAREs involved in melanosomal maturation. Several in vitro studies have shown that a set of four SNAREs such as Qa, Qb, Qc (or Qbc) and R control the membrane fusion event duing the cargo transport. Additionally, this process is further regulated by SM proteins in in vivo. Electron microscopic studies in melanocytes have shown that melanosomal proteins were delivered to the melanosomal membrane through recycling endosomal tubular domains. Moreover, our RNAi screen show that STX13 possibly acts as Qa-SNARE in mediating the fusion events between melanosomal membranes and the endosomal tubular or vesicular intermediates. However, the role of other SNAREs for this membrane transport is unknown. It has been shown that the expression of VAMP family SNAREs such as VAMP3, VAMP7 and VAMP8 increased with melanogenesis upon differentiation of melanoma cells. VAMPs belong to the class of R-SNAREs, in which VAMP7 is known to interact with VARP (abbreviation) and AP-3 (mediates the trafficking of TYR) separately, and these molecules are known to regulate the cargo transport to melanosomes. However, the precise role of VAMP7 in pigment granule maturation is unknown. Therefore, we set out to characterize the functional role of VAMP7 in melanosome biogenesis. VAMP7 has been shown to localizes to multiple sub-cellular compartments and regulate the several transport steps in other cell types. Our study found that GFP-epitope tagged either human or rat VAMP7 localize to melanosomes at steady state in wild type mouse melanocytes. Knockdown of VAMP7 causes hypopigmentation of melanocytes and misroutes the cargo to lysosomes. Further, the inactivation of VAMP7 in melanocytes phenocopies the STX13 depletion, suggesting both the SNAREs are required for the melanosome biogenesis. In addition, knockdown of STX13 target the VAMP7 to lysosomes; while inactivation of VAMP7 affect the localization of STX13 to recycling tubular structures. Subsequently, the dominant active mutants of STX13 were not able to rescue the pigmentation or cargo transport defects in VAMP7 knockdown melanocytes. Together, the data suggests that STX13 functions from recycling endosomes and VAMP7 on melanosome membrane for the transport of cargo to melanosomes Chapter-V: Understanding the mechanism of STX13 recycling during melanosome biogenesis. At steady state, SNAREs are localized to the membranes of specific organelles where they mediate or regulate the membrane fusion. During this process, three or two Q-SNAREs on one membrane (in a trans-SNARE complex, possibly formed by Qa, Qb, Qc or Qbc) interact with a R-SNARE on another member to form a SNAREpin complex. Post-fusion, SNAREs are disassembled by SNAP and NSF proteins and then recycled back to the original compartment for next round of fusion. Here, we address the mechanism of post-fusion recycling of STX13 from melanosomes to endosomes. Previous studies have shown that STX13 mislocalize to melanosomes in AP-3-deficient melanocytes, suggesting a role for AP-3 in recycling the SNARE from melanosomes. Bioinformatic analysis of the N-terminal region of STX13 revealed the presence of two canonical adaptor binding motifs 3YGP6L and KETNE80L81L, resembling the tyrosine-based (YXXø) and dileucine-based motif [DE]XXXL[LI], recognized by several adaptor proteins. Point mutagenesis of these motifs in STX13 had no effect on their steady state distribution indicating that STX13 possibly uses non-canonical residues for its recycling. Further, deletion of the N-terminal region (either 1-129 or 14-129 aa) in STX13 redistributes the SNARE to melanosomes. Moreover, the activity and the trafficking of recycling defective STX13 mutants are dependent on another HPS complex, BLOC-2 and the SNARE, VAMP7. Absence of 1-129 region in STX13 or mutations in the subunits of AP-3 perturbs the steady state localization of STX13 suggesting an indirect role for AP-3 in recycling of STX13 to endosome via non canonical motifs present in its 1-129 aa region.

Pulmonary arterial hypertension (PAH) is a lethal disease, characterized by functional or structural abnormalities involving distal pulmonary arterioles that result in increased pulmonary vascular resistance (PVR) and ultimately right heart failure. Our objective is to establish a conditional transgenic system in mice, to test the hypothesis that lung EC apoptosis at the level of distal pulmonary arterioles is necessary and sufficient to cause a PAH phenotype. In a pilot study, the Fas-Induced Apoptosis (FIA) construct was expressed under the control of endothelial-specific Tie2 promoter in transgenic mice (i.e. EFIA mice). Administration of a small molecule dimerizing agent, AP20187, resulted in lung modest dose-dependent PAH, which was associated with proliferative vascular lesions localized to distal lung arterioles in a small proportion of mice. Due to the low level of transgene expression in preliminary EFIA lines, we re-designed the transgenic vector by incorporating a more robust endothelial promoter (superTie2). The new construct was transfected into HUVEC and BAEC and analyzed by monitoring immunofluorescence (DsRed). Data from the EFIA model suggests that EC apoptosis may be sufficient to induce a PAH phenotype with the characteristic lung vascular lesions. The EFIA model will allow us to better explore the mechanism that links distal lung EC apoptosis with reactive vascular cell proliferation in the pathogenesis of this devastating disease.

Transgenic plants containing genes from Bacillus thuringiensis have been used as an alternative to chemical insecticides for insect pest control. The vegetative insecticidal proteins (Vip) secreted during the vegetative growth phase of bacteria are considered a second generation of insecticidal proteins since they do not share any structural or sequence homology with previously used crystal proteins (Cry) as well as having a wide insecticidal spectrum. One of the target pests for this protein is the fall armyworm (FAW) (Spodoptera frugiperda), the most important corn pest in South America. Previously it has been controlled by insecticides and maize expressing Cry proteins, but has rapidly evolved resistance to many control practices and remains a top concern for sustainable biotechnology control efforts. Thus, resistance characterization involving mode of action and genetics of resistance can help with Insect Resistance Management strategies, and improve the durability of control. In this dissertation, using two selected FAW population resistant to Vip3Aa20 Bt protein (Vip-R1and Vip-R2) we generated comparative proteomic and transcriptomic data among resistant and susceptible colonies. In the chapter 2, we bring FAW biology/ecology and Brazilian agriculture landscape data to support the high adaptive potential of this pest to genetically modified maize expressing Bt Cry proteins in Brazil. Proteomics studies in the chapter 3 revealed that neither Vip-R1 nor Vip-R2 showed difference between resistant and susceptible colonies either for Vip3Aa20 activation through proteolysis assay nor protein binding to the receptor. Transcriptomic sequencing and RNA-seq analysis in the chapter 4 showed strong evidence of ABC transporter genes associated with resistance as well as genes related to G-protein signaling pathway as downregulated. These results will be discussed in context of providing best management practices for managing FAW resistance to Vip, and extending the durability of Vip technology. / Plantas Transgênicas expressando genes de Bacillus thuringiensis (Bt) tem sido usadas como alternativa ao controle químico para controle de insetos praga. A proteina Vip (Vegetative Insecticide Protein) cuja secreção é realizada durante fase de crescimento da bacteria é considerada como segunda geração de proteinas inseticidas em função desta não apresentar similaridade de sequencias com todas as outras proteinas cristal (Cry), apresentando ainda maior espectro de controle de pragas. Uma das pragas alvo desta proteina é a lagarta-do-cartucho do milho (Spodoptera frugiperda), considerada a mais importante na cultura do milho na América do Sul. Larvas desta espécie foram sempre controladas com inseticidas e mais recentemente, milho expressando proteínas Cry. No entanto, esta praga tem desenvolvido resistência para várias ferramentas de controle, trazendo preocupação para a sustentabilidade das taticas de controle geradas através da biotecnologia. Dessa forma, estudos de caracterização da resistencia envolvendo modo de ação e characteristicas genéticas envolvidas com resistência pode contribuir para melhorar estratégias de Manejo de Resistencia de Insetos (IRM) e aumentar a durabilidade destas tecnologias para o controle. Nesta dissertação, foi gerado dados proteômicos e de transcriptoma comparando uma população de S. frugiperda resistente a Vip3Aa20 com a susceptivel. No capítulo 2, abordamos as características de bio-ecologia da praga associado ao sistema de cultivo suportando o alto potencial adaptativo desta espécie para hibridos de milho expressando proteinas Bt no Brazil. No capitulo 3, estudos de proteômica mostrou que Vip-R1 e Vip-R2 quando comparado com SUS, não demostraram diferenças para ativação da proteina nem ausencia de ligação da proteína com receptor de membrana no intestino do inseto. Dados de transcriptoma descritos no capitulo 5 mostrou forte evidências de que a baixa expressão de genes relacionados ao sistema transportador ABC pode estar associado com resistência bem como genes da via de sinalização das proteínas G. Estes resultados serão discutidos em um contexto para suportar boas praticas de manejo de resistência para lagarta-do-cartucho e assim estender a durabilidade da tecnologia Viptera® no campo.

Vascular remodelling due to excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle (PASMCs) and endothelial cells (ECs) has been attributed to the pathogenesis of pulmonary arterial hypertension (PAH). It is an incurable cardiovascular disorder, which leads to right heart failure and death, if left untreated. Heterozygous germline mutations in the bone morphogenetic protein receptor type II (BMPR2) have been linked with the majority (~75%) of the familial form of the disease (HPAH). Mutations in the BMPR2 gene impinge upon the BMP signalling which perturbs the balance between BMP and TGF-β pathways leading to the clinical course of the disease. Current therapies were discovered prior to the knowledge that PAH has substantial genetic components. Hence, this study aims to identify novel therapeutic intervention and provide novel insights into how the dysfunctional BMPRII signalling contributes to the pathogenesis of PAH. This work demonstrates that cryptolepines and FDA approved drugs (doxorubicin, taxol, digitoxin and podophyllotoxin) inhibit the excessive proliferation and induce apoptosis in BMPR2 mutant PASMCs by modulating the BMP and TGF-β pathways. Moreover, established drug PTC124 has also been tested but has failed to promote translational readthrough. I have also shown that dysregulated apoptosis of PASMCs and HPAECs is mediated through the BMPRII-ALK1-BclxL axis. Finally, the siRNA screen targeting approximately 1000 genes has identified novel proteins including PPP1CA, IGF-1R, MPP1, MCM5 and SRC each capable of modulating the BMPRII signalling. Taken together, this study for the very first time has identified novel compounds with pro-BMP and anti-TGFβ activities which may provide therapeutic intervention prior to or after the onset of PAH. / Commonwealth Scholarship Commission in the UK / The full text will be available at the end of the embargo period, 31st July 2024.

40% of severe acute respiratory syndrome coronavirus two (SARS-CoV-2) severe cases develop acute kidney injury (AKI). Current treatment for renal complications limits financial and material resources available. To explore alternative treatments and accelerate research in case of future coronavirus outbreaks, a mouse model of coronavirus disease 2019-associated AKI (C19-AKI) would represent a critical biomedical research tool. The surrogate model of C19-AKI (SMC) developed consisted of angiotensin-converting enzyme two (ACE2) knockout (KO) mice receiving 400 ng/kg/min of angiotensin (Ang) II by osmotic minipump for eight days with a single injection of lipopolysaccharide (LPS; 10 mg/kg) on the seventh day of Ang II and euthanasia 24 hours after LPS. Similarly, to C19-AKI, the SMC exhibited albuminuria, elevated blood urea nitrogen, electrolyte imbalance, neutrophil infiltration, and upregulation of the G-coupled protein receptor (GPR)84 and pro-inflammatory and injury markers. GPR84 was found in bronchoalveolar lavage fluid neutrophils of coronavirus disease 2019 (COVID-19) patients, suggesting a potential implication of GPR84 in the disease. We hypothesised that GPR84 deletion or antagonism with GLPG-1205 could attenuate SMC’s indices of renal injury and inflammation. GLPG-1205 and GPR84 KO had no effects in the SMC model, as suggested by unchanged albuminuria, electrolytes, and markers expression. Interestingly, neutrophil infiltration was attenuated by GLPG-1205 only. The SMC is an interesting tool for therapeutic development for infections associated with renal injury, such as SARS-CoV-2. GPR84 role in the SMC needs to be further assessed.

No description available.