| 215491. |
- Mira-Pascual, Laia
(författare)
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Role of tartrate-resistant acid phosphatase in bone remodeling
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tartrate-resistant acid phosphatase is a metalloenzyme that exists as two isoforms: the monomeric TRAP 5a and the proteolytically cleaved TRAP 5b, responsible for phosphatase activity, which is highly expressed in osteoclasts (OCs). TRAP 5b has been used as a serum marker of bone resorption, as it correlates with the absolute number of OCs and with resorption markers such as CTX-I. Despite being used as biomarker for bone metabolic diseases, little is known about the role of TRAP isoforms in OCs and thus bone remodeling (the process of bone degradation by osteoclasts, and bone formation by osteoblasts). Therefore, this thesis aimed to investigate the role of TRAP isoforms in bone remodeling. To enable the investigation of TRAP 5a and 5b we (1) developed a sandwich TRAP 5a/5b ELISA for the quantification of human TRAP isoforms. This ELISA was then used for (2) evaluating the expression and secretion pattern of TRAP 5a and 5b in healthy individuals and during OC differentiation. Additionally, we used the ELISA to (3) investigated if TRAP protein levels correlate to osteoarthritis (OA) or rheumatoid arthritis (RA). Here we correlated the phosphorylation status of the known TRAP in vivo substrate, osteopontin, to the TRAP isoforms. (4) Using a competitive inhibitor for TRAP 5b, we studied the role of TRAP in OCs differentiation. Lastly, we (5) developed a high throughput system to identify a subclone in a murine cell line that is a more homogeneous and stable OC precursors that could be used as a screening tool for OC biology studies. A double TRAP 5a/5b sandwich ELISA was developed and designed as a two-step process. Using the ELISA, we showed that in vitro cultures of OCs secrete not only TRAP 5b but also TRAP 5a and that both isoforms were present intracellular establishing that 5b can also be formed intracellular in OCs. Correlation between TRAP 5a and 5b indicated a dependence between TRAP 5a and formation of 5b. There was a positive correlation in both serum from healthy men, and media from in vitro OC cultures of not only 5b but also TRAP 5a with CTX-I further suggesting that TRAP 5a also originates partly from OCs. Measurement of TRAP 5a and 5b in synovial fluid from OA and RA patients revealed a correlation between low TRAP 5b/ TRAP 5a ratio and phosphorylated osteopontin. This suggested that synovial fluid from RA patients contained an insufficient amount of TRAP 5b increasing levels of phosphorylated OPN leading to a higher OC activation and bone destruction. Inhibition of TRAP 5b using the competitive inhibitor, 5-phenylnicotinic acid, decreased the number of OCs formed and the expression of several OC markers. However, some OCs were able to fuse and resorb bone. In this thesis we show that measurement of TRAP isoforms protein is an important tool in research and possibly also in diagnostic to understand the biological implications of TRAP 5a and 5b in OCs, which may lead to therapeutic targeting of certain isoforms for inflammatory and metabolic bone diseases. We further show that TRAP is involved in the bone remodeling process in OCs and defects in TRAP may cause alterations in OCs function and differentiation.
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| 215492. |
- Mira Veiga, Joana, et al.
(författare)
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Identifying Sources of Marine Litter
- 2016
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Marine litter is a global problem causing harm to marine wildlife, coastal communities and maritime activities. It also embodies an emerging concern for human health and safety. The reduction of marine litter pollution poses a complex challenge for humankind, requiring adjustments in human behaviour as well as in the different phases of the life- cycle of products and across multiple economic sectors. The Marine Strategy Framework Directive (MSFD) requires European Member States to monitor marine litter and implement programmes of measures to reduce its occurrence. A crucial step in monitoring and effectively addressing marine litter is the identification of the origin and the pathways that lead to litter entering the marine environment. A given site or region can be subject to litter pollution from a number of sources, which can be local, regional or even distant, as litter can be transported to a specific area by ocean currents and wind drift. For this reason, pinpointing the origin of the different items that make up marine litter is a difficult task and will always have an inherent degree of associated uncertainty. Plastic food packaging recorded in the marine environment, for example, can consist of a diverse selection of items, which can be generated from a number of sources, which in turn can be sea-based or land-based and originate from near or distant regions. A wide variety of methods have been used over the years to determine the sources of marine litter, from simple counts of items believed to originate from a given source to more complex statistical methods. This report provides a brief overview of the main methods used and outlines one of the most promising approaches for determining sources – a Matrix Score Technique based on likelihoods, which considers the possibility that specific items originate from more than one source. Furthermore, it presents a series of other parameters that can be used to analyse data-sets, with regard to the use, origin and risk of items recorded in the marine or coastal environments. These can further support decision-making when considering preventive measures. Finally, recommendations to help the process of identification of sources are given, from the early stage of data collection and site characterization to bringing in the knowledge of local stakeholders to better determine where litter is coming from and what needs to be done to prevent it.
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| 215493. |
- Mirabdollah, A., et al.
(författare)
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Optimization of a protoplast transformation method for Bacillus Subtilis, Bacillus megaterium, and Bacillus Cereus by a plasmid pHIS1525.SplipA
- 2009
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- During the past years of gene cloning studies, Escherichia coli has always been a foremost host cell for exogenous genes expressions owing to its high level of protein production and excretion. However, problems relating to low level of extracellular production of some proteins specially the accumulation of cloned proteases within the cells have moved the attentions from E.coli to bacilli bacteria such as B. megaterium, B.subtilis, and B.cereus due to their secretion ability of many different enzymes. Bacillus megaterium is widely used for high-level expression of heterologous proteins with little or no degradation. Bacillus subtilis is a naturally competent host cell for uptake of exogenous DNA, resulting in attractive industrial applications. Bacillus cereus has sporulation capability which makes it suitable for several industrial uses. A conventional approach for transferring DNA into protoplasts or intact cells of bacillus bacteria is chemical transformation, using chemicals through chilling and then shock-heating of the suspension of cells to induce reversible permeabilization of the cell membrane to make it possible for the external DNA to enter into the cells. In most cloning experiments, the transformation with plasmid DNA is performed using Polyethylene glycol (PEG)-induced competence cells. In this study, a PEG-induced protoplast transformation protocol was developed for three different bacillus strains of Bacillus megaterium ATCC®14945, Bacillus Subtilis ATCC®6051, and Bacillus Cereus ATCC®14579. In all cases a plasmid pHIS1525.SPlipA, well working vector in B.megaterium, was applied. Protoplasts were formed in RHAF medium after treating the cells with lysozyme. Two factors, the incubation time and the lysozyme concentration have been found to play the most important role in effective protoplast formation. These two factors were further optimized in this study to elaborate a chemical transformation procedure which can possibly work for other bacillus strains as well. The optical density (A420) and the number of colony-forming units (CFUs) were determined to find the optimal conditions for each strain. The results indicate that PEG-induced protoplast transformation is a sufficient technique when using a plasmid pHIS1525.SPlipA in Bacillus genus.
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| 215494. |
- Mirabella, Roseanne, et al.
(författare)
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Civil Society Education : International Perspectives
- 2015
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Ingår i: Journal of Nonprofit Education and Leadership. - Urbana, IL : Sagamore publishing. - 1046-6819 .- 2157-0604. ; 5:4, s. 213-218
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Over the last few decades, the world has experienced an unprecedented growth in the size and scope of civil society organizations (Boli & Thomas, 1999; Kaldor, Moore, & Selchow, 2012).1 On par with these developments is the ever increasing significance of what these organizations assumingly can and should do to mitigate and solve some of the more pressing social and environmental issues we currently face locally and globally. Yet despite the growing numbers and allotted importance of civil society organizations, relatively little is known globally about how we prepare, train, and educate present and future leaders and professionals in these organizations, nor have more normative issues been sufficiently addressed, such as how we should be preparing individuals for leading, managing, or administering these organizations.
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| 215495. |
- Mirabella, Roseanne, et al.
(författare)
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Civil Society Education : National Perspectives
- 2019
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Ingår i: Journal of Nonprofit Education and Leadership. - 1046-6819 .- 2157-0604. ; 9:1
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 215496. |
- Mirabella, Roseanne, et al.
(författare)
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Civil Society Education : International Perspectives
- 2015
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Ingår i: Journal of Nonprofit Education and Leadership. - : Sagamore Publishing. - 1046-6819 .- 2157-0604. ; 5:4
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 215497. |
- Mirabella, Roseanne, et al.
(författare)
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Civil Society Education : Western Perspectives
- 2022
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Ingår i: Journal of Nonprofit Education and Leadership. - 1046-6819 .- 2157-0604. ; 12:1
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- This is the third themed issue in the Journal of Nonprofit Education and Leadership (JNEL) in which researchers around the world examine regional and national training and education programs for individuals with civil society leadership roles. When we as editors set our sails in the early years of the last decade, the third issue was always our planned destination. However, as with most voyages, things do not always go according to plan. As we now proudly present the third issue, we are already hard at work with two more themed issues—one with a focus on a set of countries in Southeast Asia and another with a focus on regional mappings and studies. Over time have we come to realize that this voyage has no one harbor, to arrive at once and for all, and that the true delight is the cruise itself.
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| 215498. |
- Mirabelli, Pierfrancesco
(författare)
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Inhibitors of corneal inflammation and angiogenesis : Prospectives and challenges
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pathologic angiogenesis is involved in cancer and several blinding conditions such as wet age-related macular degeneration, proliferative retinopathies and corneal neovascularization.In these dieseases, the angiogenic triggers are hypoxia and inflammation, and both involve the main angiogenic mediator, which is Vascular Endothelial Growth Factor (VEGF). Among available treatments, anti-VEGF often shows limited or temporary efficacy, while steroids are potentially responsible for many side-effects. This thesis presents a series of linked studies aimed at elucidating the early pathologic changes leading to inflammation and corneal neovascularization, and how various treatments affect this process. In this thesis, anti-inflammatory and anti-angiogenic treatments are applied in corneal neovascularization models, to identify VEGF-independent pathways and other novel factors as future therapy targets, as well as to investigate the endogenous modulation of angiogenesis.A model of experimental neovascularization in the rat cornea was used as main model, where the neovascular response is triggered by a surgical suture placed into the cornea. Investigational treatments (anti-Vegf, dexamethasone, IMD0354, Gap27, or control substances) were then given topically, with the exception of IMD0354, which was given systemically. The effects in the cornea were studied in vivo with slit lamp photography to assess and quantify macroscopic vessel growth and using in vivo confocal microscopy (IVCM) to study cell infiltration and limbal vessel dilation and detect microscopic vessel sprouts; these examinations were performed longitudinally. Genomic analysis with RNA microarray, selected gene expression with q-RT-PCR, and selected protein expression in tissue (immunohistochemistry, immunofluorescence, Western blot) were performed at different time-points. Moreover, other experiments on cell cultures (HUVEC and HCEC), organ cultures (human corneas), ex vivo models (aortic rings) and in vivo studies (zebrafish vasculogenesis) were performed.Dexamethasone suppressed limbal vasodilation and corneal neovascularization more than anti-Vegf, despite no difference in inflammatory cell infiltration into the cornea. Five-hundred eleven fewer genes were differentially expressed in dexamethasone-treated corneas relative to naïve corneas, compared to anti-Vegf. Among them, several major pro-angiogenic and pro-inflammatory factors and chemokines were suppressed only by dexamethasone and represent novel candidate factors to target in order to improve anti-VEGF treatment. On the other hand, selective inhibition of a single inflammatory pathway (NF-κB), despite showing similar early effects as dexamethasone in suppressing tissue inflammation, was not effective enough to suppress new vessel growth. The same factors suppressed by dexamethasone are also inhibited in endogenous modulation of angiogenesis. Surprisingly, dexamethasone activated several complement factors, which could possibly be beneficial in the anti-angiogenic response.In a different therapeutic approach, promoting cell migration to accelerate epithelial wound closure similarly was not sufficient to avoid inflammation and angiogenesis in the cornea.In conclusion, new and more effective treatments are needed for corneal inflammation and neovascularization with fewer side-effects. In this thesis, several novel factors and mechanisms related to inflammation are identified, factors that are not addressed by anti-Vegf therapy, and therefore represent interesting objects for further study, as they have the potential to be targets for adjuvant therapy. Specific anti-inflammatory treatment as well as therapeutic activation of endogenous regulatory pathways, and potentially complement modulation, might represent new strategies to improve anti-angiogenic therapy, but when used alone they do not seem to avoid corneal neovascularization.
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| 215499. |
- Mirabet, Vincent, et al.
(författare)
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The self-organization of plant microtubules in three dimensions enables stable cortical localization and sensitivity to external cues
- 2017
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Ingår i: bioRxiv. - : Cold Spring Harbor Laboratory.
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Many cell functions rely on the ability of microtubules to self-organize as complex networks. In plants, cortical microtubules are essential to determine cell shape as they guide the deposition of cellulose microfibrils, and thus control mechanical anisotropy in the cell wall. Here we analyze how, in turn, cell shape may influence microtubule behavior. Using a computational model of microtubules enclosed in a three-dimensional space, We show that the microtubule network has spontaneous configurations that could explain many experimental observations without resorting to specific regulation. In particular, we find that the preferred localization of microtubules at the cortex emerges from directional persistence of the microtubules, combined with their growth mode. We identified microtubule parameters that seem relatively insensitive to cell shape, such as length or number. In contrast, microtubule array anisotropy depends strongly on local curvature of the cell surface and global orientation follows robustly the longest axis of the cell. Lastly, we found that the network is capable of reorienting toward weak external directional cues. Altogether our simulations show that the microtubule network is a good transducer of weak external polarity, while at the same time, it easily reaches stable global configurations.Author summary Plants exhibit an astonishing diversity in architecture and shape. A key to such diversity is the ability of their cells to coordinate and grow to reach a broad spectrum of sizes and shapes. Cell growth in plants is guided by the microtubule cytoskeleton. Here, we seek to understand how microtubules self-organize close to the cell surface. We build upon previous two-dimensional models and we consider microtubules as lines growing in three dimensions, accounting for interactions between microtubules or between microtubules and the cell surface. We show that microtubule arrays are able to adapt to various cell shapes and to reorient in response to factors such as signals or environment. Altogether, our results help to understand how the microtubule cytoskeleton contributes to the diversity of plant shapes and to how these shapes adapt to environment.
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| 215500. |
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