Nuclear import mechanism of EGFR in breast cancer cells

Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR) are internalised from the plasma membrane by endocytosis and may be transported to the nucleus. EGFR, a receptor for EGF and other RTKs, HER-2 and HER-4 has an important role in signalling; it contains transactivational activity and can function as a transcription co-factor to activate gene promoters. High nuclear accumulation of imported full length EGFR is associated with an increased tumour proliferation and a reduced survival in cancer patients. However, little is known about the mechanism by which membrane-bound proteins, such as EGFR, translocate from the cell surface into the cell nucleus; how nuclear membrane proteins cross through the NPC to reach the INM. The mechanism of translocation for soluble proteins is also presently unclear. EGFR nuclear import is mediated by importin α/β. And it is exported from the nucleus by the exportin CRM1. Sec61β which may reside in the inner nuclear membrane (INM) is required for the release of EGFR from the INM into the nucleus. Nuclear transport involves binding of nuclear localisation sequences (NLSs) within the cargo to a transport receptor (karyopherins or importin). Karyopherins interact with certain nuclear pore complex (NPC) proteins (nucleoporins). Membrane proteins can access the INM through the NPC membrane: by diffusion, using classical nuclear transport factors (the importin/Ran system); or by an ATP dependent mechanism. EGFR may use the former mechanism. This work concentrates to show by electron microscopy and by Immuno-Fluorescence that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the INM through the NPC, yet remaining a membrane-bound protein. We also confirm that importin regulates EGFR nuclear transport to the INM and in addition, Sec61β is required for EGFR release to the nucleoplasm. Altogether, this study of the mechanism of EGFR nuclear-cytoplasmic import in breast cancer cells, further confirms previous reports and provides an understanding of the nature and regulation of the nuclear EGFR pathway and the mechanism by which cell-surface EGFR is shuttled in the cell cytoplasm and channelled through the Golgi and Endoplasmic Reticulum (ER) compartments and into the nucleus through the NPC

Engineering biointerfaces to reveal collagen IV disease mechanisms

Basement Membranes (BMs) are specialised extracellular matrix (ECM) structures that underlie all endothelial and epithelial cells, and provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in the BMs major component collagen IV cause eye, kidney and cerebrovascular disease including intracerebral haemorrhaging (ICH). Haemorrhagic stroke accounts for 15% adult stroke and 50% paediatric stroke, and carries the worst prognosis and there are no therapeutic strategies. Mutations in the genes COL4A1/COL4A2 (collagen IV alpha chain 1 and 2) cause BM defects due to mutant protein incorporation in the BM or its absence by ER retention, and ER-stress due to intracellular accumulation of collagen IV. Despite this, the mechanism(s) of collagen IV mutations disease remain poorly characterised. To provide novel insights into mechanisms of collagen diseases, this study investigates the effect of defined engineered biointerfaces on cell behaviour/signalling, collagen secretion in COL4A2 mutant and wild-type cells. Atomic force microscopy and spectroscopy were employed together with confocal and biochemical analyses of cells cultured on engineered synthetic polymers, poly(ethyl acrylate) and poly(methyl acrylate), coated with ECM proteins, namely laminin, collagen IV and fibronectin. This enabled us to address the hypothesis that biomaterials may alter the behaviour of COL4A2+/G702D mutant cells by overcoming some of the defects caused by the mutation and rescuing the downstream effect of the ER stress. Of the ECM proteins that were used, only fibronectin was observed to undergo a drastic structural change depending on the substrate chemistry. On poly(ethyl acrylate), fibronectin was assembled into fibrillary networks upon adsorption, and these nanonetworks induced increased secretion of Col4a2 in COL4A2+/G702D cells than on poly(methyl acrylate) or control glass. The behaviour of the mutant cells appeared to be influenced by the underlying biointerface, increased levels of molecular chaperones and reduced ER area suggested an increased collagen IV folding capacity when the cells were cultured on the FN nanonetworks compared to the other surfaces. COL4A2+/G702D cells interacted with the adsorbed proteins and were able to mechanically translocate them. Enhanced formation of focal adhesions was also seen on FN-coated polymers, where ligand density and actin-myosin contractility accounted for the observed increase in cell adhesion strength. The stiffness of the mutant fibroblasts and of their ECMs was found to be 10 times lower than that of the wild-type cells; interestingly, mutant cells cultured on FN nanonetworks on poly(ethyl acrylate) were able to deposit a protein matrix with significantly higher Young modulus than on glass or poly(methyl acrylate). These findings suggest that biomaterials are able to influence the behaviour of these mutant cells through changes in the interfacial layer of adsorbed proteins presented to them. Collectively, these data provide an understanding of the effect of mutations on cell characteristic and a basis of concept that material may be employed to modulate effects of mutations of collagen/ECM molecules. Understanding the mechanisms through which these surfaces trigger a change in cell response will prove valuable for the development of new therapeutic approaches to address pathologies due to collagen IV mutations. In this respect, further investigation is needed to dissect the signalling pathways involved

Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin β1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells

Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin β1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells

DES ENTRAVES AU DEVELOPPEMENT SOCIO-ECONOMIQUE DE LA PROVINCE DU KASAI CENTRAL

Notre modeste recherche  qui a porté sur « Les entraves au développement socio-économique de la province du Kasaï central », est un problème majeur des politiques publiques qui exige la participation communautaire et des autorités compétentes dans la hiérarchie de la gestion d’une entité territoriale décentralisée.  La pertinence de ce thème nous a obligés à structurer le travail en trois points principaux notamment : le premier a porté sur l’approche conceptuelle, le deuxième a consisté à présenter la méthodologie en précisant la situation socio-économique de la province et le troisième était axé sur les entraves  au développement du Kasaï Central. Ceci étant, signalons que la problématique du développement socioéconomique de la province du Kasaï Central invite la participation de toutes les forces vives de la province, dirigeants tout comme dirigés, femmes, hommes, chômeurs, travailleurs, lettrés comme illettrés. Tout développement est un processus et exige l’implication de tout le monde sans exception. Dans le cas sous examen, la problématique relative au développement socioéconomique du Kasaï Central n’est pas singulièrement liée aux écueils sus énumérés,  mais aussi elle est liée aux mentalités culturelles des administrés qui n’ont pas encore compris le rôle qu’ils doivent jouer dans le processus du développement. Dans le souci de voir le développement socioéconomique comme un fait réel dans la province du Kasaï central, il est impérieux de lutter contre les causes..Notre modeste recherche  qui a porté sur « Les entraves au développement socio-économique de la province du Kasaï central », est un problème majeur des politiques publiques qui exige la participation communautaire et des autorités compétentes dans la hiérarchie de la gestion d’une entité territoriale décentralisée.  La pertinence de ce thème nous a obligés à structurer le travail en trois points principaux notamment : le premier a porté sur l’approche conceptuelle, le deuxième a consisté à présenter la méthodologie en précisant la situation socio-économique de la province et le troisième était axé sur les entraves  au développement du Kasaï Central. Ceci étant, signalons que la problématique du développement socioéconomique de la province du Kasaï Central invite la participation de toutes les forces vives de la province, dirigeants tout comme dirigés, femmes, hommes, chômeurs, travailleurs, lettrés comme illettrés. Tout développement est un processus et exige l’implication de tout le monde sans exception. Dans le cas sous examen, la problématique relative au développement socioéconomique du Kasaï Central n’est pas singulièrement liée aux écueils sus énumérés,  mais aussi elle est liée aux mentalités culturelles des administrés qui n’ont pas encore compris le rôle qu’ils doivent jouer dans le processus du développement. Dans le souci de voir le développement socioéconomique comme un fait réel dans la province du Kasaï central, il est impérieux de lutter contre les causes.

Oral fexinidazole for stage 1 or early stage 2 African Trypanosoma brucei gambiense trypanosomiasis: a prospective, multicentre, open-label, cohort study

BACKGROUND: Staging and treatment of human African trypanosomiasis caused by Trypanosoma brucei gambiense (g-HAT) required lumbar puncture to assess cerebrospinal fluid (CSF) and intravenous drugs that cross the blood-brain barrier for late-stage infection. These procedures are inconvenient in rural health systems of disease-endemic countries. A pivotal study established fexinidazole as the first oral monotherapy to be effective against non-severe stage 2 g-HAT. We aimed to assess the safety and efficacy of fexinidazole in early g-HAT. METHODS: In this prospective, multicentre, open-label, single-arm cohort study, patients with stage 1 or early stage 2 g-HAT were recruited from eight treatment centres in the Democratic Republic of the Congo. Primary inclusion criteria included being older than 15 years, being able to ingest at least one complete meal per day (or at least one sachet of Plumpy'Nut®), a Karnofsky score higher than 50, evidence of trypanosomes in the blood or lymph but no evidence of trypanosomes in the CSF, willingness to be admitted to hospital to receive treatment, having a permanent address, and being able to comply with the follow-up visit schedule. Exclusion criteria included severe malnutrition, inability to take medication orally, pregnant or breastfeeding women, any clinically important medical condition that could jeopardise patient safety or participation in the study, severely deteriorated general status, any contraindication to imidazole drugs, HAT treatment in the past 2 years, previous enrolment in the study or previous intake of fexinidazole, abnormalities on electrocardiogram that did not return to normal in pretreatment repeated assessments or were considered clinically important, QT interval corrected using Fridericia's formula of at least 450 ms, and patients not tested for malaria or not having received appropriate treatment for malaria or for soil-transmitted helminthiasis. Patients were classified into stage 1 or early stage 2 g-HAT groups following evidence of trypanosomes in the blood, lymph, and absence in CSF, and using white-blood-cell count in CSF. Patients received 1800 mg fexinidazole once per day on days 1-4 then 1200 mg fexinidazole on days 5-10. Patients were observed for approximately 19 months in total. Study participants were followed up on day 5 and day 8 during treatment, at end of treatment on day 11, at end of hospitalisation on days 11-18, at week 9 for a subset of patients, and after 6 months, 12 months, and 18 months. The primary endpoint was treatment success at 12 months. Safety was assessed through routine monitoring. Analyses were done in the intention-to-treat population. The acceptable success rate was defined as treatment efficacy in more than 80% of patients. This study is completed and registered with ClinicalTrials.gov (NCT02169557). FINDINGS: Patients were enrolled between April 30, 2014, and April 25, 2017. 238 patients were recruited: 195 (82%) patients with stage 1 g-HAT and 43 (18%) with early stage 2 g-HAT. 189 (97%) of 195 patients with stage 1 g-HAT and 41 (95%) of 43 patients with early stage 2 g-HAT were finally included and completed the 10 day treatment period. Three patients with stage 1 g-HAT died after the 10 day treatment period and before the 12 month primary follow-up visit, considered as treatment failure and were withdrawn from the study. Treatment was effective at 12 months for 227 (99%) of 230 patients (95% CI 96·2-99·7): 186 (98%) of 189 patients (95·4-99·7) with stage 1 and 41 (100%) of 41 patients (91·4-100·0) with early stage 2, indicating that the primary study endpoint was met. No new safety issues were observed. The most frequent adverse events were headache and vomiting. In total, 214 (93%) of 230 patients had treatment-emergent adverse events, mainly common-terminology criteria for adverse events grades 1 to 3. None led to treatment discontinuation. INTERPRETATION: Fexinidazole is a valuable first-line treatment option in the early stages of g-HAT. FUNDING: Through the Drugs for Neglected Diseases initiative: the Bill & Melinda Gates Foundation, the Republic and Canton of Geneva (Switzerland), the Dutch Ministry of Foreign Affairs (also known as DGIS; Netherlands), the Norwegian Agency for Development Cooperation (also known as Norad; Norway), the Federal Ministry of Education and Research (also known as BMBF) through KfW (Germany), the Brian Mercer Charitable Trust (UK), and other private foundations and individuals from the HAT campaign

Discovery of synergistic material-topography combinations to achieve immunomodulatory osteoinductive biomaterials using a novel in vitro screening method: The ChemoTopoChip

Human mesenchymal stem cells (hMSCs) are widely represented in regenerative medicine clinical strategies due to their compatibility with autologous implantation. Effective bone regeneration involves crosstalk between macrophages and hMSCs, with macrophages playing a key role in the recruitment and differentiation of hMSCs. However, engineered biomaterials able to simultaneously direct hMSC fate and modulate macrophage phenotype have not yet been identified. A novel combinatorial chemistry-topography screening platform, the ChemoTopoChip, is used here to identify materials suitable for bone regeneration by screening 1008 combinations in each experiment for human immortalized mesenchymal stem cell (hiMSCs) and human macrophage response. The osteoinduction achieved in hiMSCs cultured on the “hit” materials in basal media is comparable to that seen when cells are cultured in osteogenic media, illustrating that these materials offer a materials-induced alternative to osteo-inductive supplements in bone-regeneration. Some of these same chemistry-microtopography combinations also exhibit immunomodulatory stimuli, polarizing macrophages towards a pro-healing phenotype. Maximum control of cell response is achieved when both chemistry and topography are recruited to instruct the required cell phenotype, combining synergistically. The large combinatorial library allows us for the first time to probe the relative cell-instructive roles of microtopography and material chemistry which we find to provide similar ranges of cell modulation for both cues. Machine learning is used to generate structure-activity relationships that identify key chemical and topographical features enhancing the response of both cell types, providing a basis for a better understanding of cell response to micro topographically patterned polymers

Study of doped barium manganites : electrical and magnetic properties

The electrical and magnetic properties of barium manganite solid solutions Ba4-xAxMn3O10, Ba6-xAxMn5O16, (A = La, Sr and Nd), Ba4-xSrxMn3-yFeyO10 and Ba6Mn5-yFeyO16 have been investigated.  Initially, Ba4Mn3O10 and Ba3.9La0.1Mn3O10 were synthesised.  XPS1 measurements suggested that the latter phase was substoichiometric in oxygen.  A.C. impedance showed that the La-doped sample displayed insulator-like behaviour, whereas the pure one exhibited a metallic to insulator transition.  ND2 results showed both samples are antiferromagnetic (with TN = 170 K for Ba4Mn3O10).  In addition, a decrease of the ordered Mn magnetic moment was observed in the La-doped sample. SEM3 and a.c. impedance data for Ba4-xSrxMn3O10 revealed that conductivity drops as grain size decreases.  It has also been shown that doping with a smaller ion causes a decrease in the Mn magnetic moment and can increase the resistivity of the sample.  Doping with a higher valence ion prompts a change in the conduction mechanism leading to semiconductor behaviour above room temperature.  A loss of oxygen induces a creation of Mn3+ - O – Mn4+ interactions which may affect the magnetic properties. Comparison between the properties of Ba3.9La0.1Mn3O10 and Ba3.9Nd0.1Mn3O10­  was one of the major contributions of this thesis.  The former is antiferromagnetic, whilst the latter became ferromagnetic below 100 K. Both compounds exhibit semiconductor behaviour above room temperature.  However, Ba3.9La0.1Mn3O10 has lower resistivity than Ba3.9Nd0.1Mn3O10.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The government's environmental initiatives and their relationship to sustainable lifestyle among the Swedish people : A study of aid investments impact on sustainable living in the donor country

Swedish government aid is divided into three main themes: democracy and human rights, the environment and climate, and gender equality and women's role in development. These three themes in aid are considered by the government to be vital in order to fight poverty and create fair and sustainable development. This study will examine two of the four focus areas designated under the theme of "environment and climate". The study observes the relationship between projects in energy and water and how it promotes a sustainable lifestyle. A survey on environmental habits will be conducted. The purpose of the study is to see if international aid projects funded by the Swedish government are able to promote a sustainable lifestyle in the Swedish population. The study has revealed the following: there is a lack of knowledge among the respondents of how the government conducts its environmental efforts; there is an interest in the environment and environmental issues among the respondents; the respondents consider environmental issues important. The investigation has also revealed that water projects have a greater tendency to promote sustainable lifestyles among the respondents because it had the greatest degree of influence on individuals’ actions. However, the investigation also showed that the action readiness for environmental behavior is generally low but that development aid for water and electricity can promote a sustainable lifestyle in the donor country in conjunction with other factors. Regeringen har tre prioriterade biståndsteman för att bekämpa fattigdomen och för en rättvis och hållbar utveckling, dessa är: demokrati och mänskliga rättigheter, miljö och klimat samt jämställdhet och kvinnors roll i utveckling. Jag undersöker två av fyra inriktningar under temat miljö och klimat. Inom denna studie kommer jag att undersöka hur relationen mellan satsningar på energi- och vattenprojekt och vår livsstil ter sig. En egen enkätundersökning har utförts om miljövanorna hos svenskar. Tanken är att se om internationella biståndsprojekt som Sverige bedriver främjar en hållbar livsstil genom att influera svenskarnas handlingsberedskap. Det som undersökningen har påvisat är följande: att det bland respondenterna saknas kunskap om hur regeringen bedriver sitt miljöarbete på ett internationellt plan, bland annat genom biståndsarbeten; att det hos de flesta respondenterna fanns ett miljöintresse och att miljöfrågan och miljöarbete ansågs vara viktiga; att vattenprojekt har en större benägenhet att främja en hållbar livsstil hos respondenterna eftersom det hade störst påverkan på deras handlingar; att det förekommer en svag handlingsbenägenhet för ett miljöbeteende hos respondenterna och att biståndsprojekt gällande vatten och el kan i samverkan med andra faktorer vara en faktor som främjar en hållbar livsstil inom givarlandet.