Dissertations / Theses on the topic 'Bose gla'

The secosteroid hormone 1, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) exerts its effects through a receptor protein which is a member of a superfamily of steroid receptor proteins. The 1,25(OH)₂D₃ receptor protein (VDR) binds both hormone and DNA with high affinity , thereby transcriptionally regulating the expression of a number of genes. A fragment of the cDNA to the human VDR containing essentially the entire open reading frame was transcribed and translated in vitro. demonstrated to exhibit The resulting protein was then the physical and functional features, i.e. molecular weight, immunoreactivity, 1,25(OH)₂D₃ binding, and DNA-cellulose binding, of the native human receptor from the T47D cell line. This validates the authenticity of the cDNA in a cell free system and provides a biochemical means of generating this rare and labile macromolecule to use in heretofore difficult structure/function studies. The gene for rat bone gla protein (BGP) was isolated and 1250 bp including 1100 bp of 5' flanking DNA were placed upstream of the human growth hormone reporter gene. Following transient transfection into the osteoblast-like rat osteosarcoma cell line, ROS 17/2.8, the BGP promoter demonstrated a low level of basal activity that was increased approximately ten-fold by addition of 10⁻⁸ M 1,25(OH)2D3. A single 249 bp fragment (-523 to -274) was sufficient to confer hormone inducibility upon both heterologous and homologous promoters. Deletion studies, complemented by evaluation with synthetic oligomers, enabled localization of the 1,25(OH)₂D₃ response element (VDRE) to within 19 bp (-456 to -438), containing an element with an imperfect direct repeat (GGTGA(N₄)GGACA) and homology to other steroid responsive elements. This enhancer element was capable of strong synergism when present in multiple copies. Gel retardation assays demonstrated that partially purified VDR from chicken or rat bound specifically and with high affinity to a DNA fragment containing the putative VDRE and this binding was perturbed by monoclonal antibodies to the VDR. surprisingly, the 249 bp fragment, when linked in an antisense orientation with respect to the BGP promoter, blocked the basal and hormone dependent gene expression. However, a 245 bp fragment 5' to the 249 bp element (-1100 to -855) restored 20-fold inducibility when linked to the first fragment in the same orientation. The presence or absence of this distal element also modulated the 1,25(OH)₂D₃ response within the milieu of the native gene. Thus, the VDRE apparently cooperates with other elements to achieve the hormonal response observed in this gene.

Gentherapie und Zellersatz im zentralen Nervensystem (ZNS) werden durch die Blut-Hirn-Schranke behindert, die den Übertritt von Plasmabestandteilen und Zellen in das ZNS limitiert. Genetisch modifizierte Knochenmarkszellen können jedoch ungeachtet der intakten Blut-Hirn-Schranke in das ZNS einwandern und dort zu Mikroglia differenzieren. Neurone signalisieren eine Schädigung an benachbarte Glia und rekrutieren Zellen hämatopoetischen Ursprungs gezielt an den Ort der Läsion. Aus adulten Stammzellen des Knochenmarks können schliesslich Nervenzellen entstehen, die in die komplexe Architektur des ZNS integriert werden. Die Befunde liefern neue Ansätze für die Therapie von ZNS-Erkrankungen.
Gene therapy and cell replacement strategies in the central nervous system (CNS) are hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the CNS. Genetically modified bone marrow-derived cells are capable of entering the CNS in spite of the blood-brain barrier and they differentiate into microglia. Neurons signal damage to neighbouring glia and recruit cells of hematopoietic origin specifically to the sites of damage. Finally, adult bone marrow stem cells may generate new neurons which are incorporated into the complex cytoarchitecture of the CNS. The results provide a new approach for the therapy of CNS disorders.

Negli ultimi anni si è sviluppato un acceso dibattito in merito a come un’azienda è in grado di affrontare le scelte prese dalle generazioni precedenti di manager; il passato di un’impresa in principio non veniva considerato come un elemento determinante nel processo decisionale che porta al conseguimento di determinati risultati. Si è evidenziato come questo tipo di decisioni si ripercuotono nel tempo sull’immagine e sulla performance aziendali, generando effetti sia negativi che positivi. La responsabilità in merito all’eredità storica di un’azienda prende il nome di Historic Corporate Responsibility. Il contesto narrativo, l’engagement che l’azienda ha sui propri clienti e il passato della stessa sono elementi che concorrono a determinare gli effetti che un caso di Historic Corporate Responsibility può avere all’interno della governance. Questa tesi vuole analizzare gli effetti della responsabilità storica delle aziende sia sull’immagine che sulle performance, prendendo in esame casi celebri a titolo esemplificativo per poi concentrarsi sull’analisi di un’azienda e sull’individuazione di tematiche legate alla CHR.

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.

Abstract Heart failure (HF) and aortic valve stenosis (AS) are complex disorders affected by functional alterations and actively regulated remodelling of the heart and the aortic valve, respectively. In addition to structural proteins, such as collagens and elastin, the extracellular matrix (ECM) in the heart and the aortic valve comprises non-collagenous factors that are not strictly involved in the architecture but may modulate cardiac and valvular remodelling. In the present study the expression of non-collagenous fibrosis- and calcification-related ECM proteins was investigated in HF-associated cardiac remodelling from different origins as well as in fibrocalcific aortic valve disease leading to AS. The experimental models of pressure overload, myocardial infarction (MI) and chronic renal failure were used to study the cardiac expression of bone morphogenetic protein (BMP)-2, BMP-4, bone sialoprotein, matrix Gla protein (MGP), osteoactivin, osteopontin, periostin and/or pleiotrophin in vivo in cardiac remodelling. Human aortic valves, obtained from patients undergoing valve replacement, were studied to characterize the valvular expression of BMP-2, BMP-4, bone sialoprotein, MGP, osteoactivin, osteopontin, osteoprotegerin, periostin, pleiotrophin, and thrombospondins (TSPs) 1-4 in the different stages of fibrocalcific aortic valve disease. Left ventricular (LV) MGP expression was upregulated in vivo in non-uremic cardiac remodelling. In vitro results indicate that angiotensin II elevates MGP expression in cardiomyocytes and fibroblasts. Periostin gene expression was induced in cardiac but not in aortic valve remodelling and the cardiac induction in chronic renal insufficiency was associated with LV hypertrophy and blood pressure as well as the cardiac gene expression of other fibrosis-related genes. Bone sialoprotein and osteopontin were expressed in the aortic valves in parallel with calcification, and also in distinct models of cardiac remodelling. Osteoprotegerin protein expression in stenotic valves was weak regardless of a simultaneous increase in gene expression. BMPs were downregulated in AS and no change in LV gene expression was detected in uremic cardiac remodelling. All the studied TSPs were expressed in human aortic valves, and especially the expression of TSP-2 was shown to increase in fibrocalcific aortic valves simultaneously with decreased activation of the Akt/nuclear factor (NF)-κB-pathway. This study delineates distinct expression patterns of non-collagenous ECM proteins in pathological tissue remodelling in the heart and in the aortic valve, and thus emphasizes the role of ECM proteins as an important modulator of cardiac and aortic valve remodelling
Tiivistelmä Sydämen vajaatoiminnan ja aorttastenoosin taudinkuvaan kuuluvat toiminnallisten muutosten ohella aktiivisesti säädellyt soluväliaineen muutokset sydämen ja aorttaläpän rakenteessa. Soluväliaineen rakenteen muodostavien kollageenien ja elastiinin lisäksi soluväliaineessa on rakenteeseen kuulumattomia proteiineja. Tässä väitöskirjassa tutkittiin sidekudoksen kertymiseen ja kudosten kalkkiutumiseen osallistuvia soluväliaineen proteiineja sydämen vajaatoiminnassa sekä aorttastenoosiin johtavassa kalkkiuttavassa aorttaläppäviassa. Tutkimuksessa selvitettiin sydämen soluväliaineen proteiinien ilmentymistä painekuormituksen, sydäninfarktin ja pitkäaikaisen munuaisten vajaatoiminnan koemalleissa rotalla. Tutkittavia proteiineja olivat luun morfogeneettiset proteiinit 2 ja 4, luun sialoproteiini, matriksin Gla proteiini (MGP), osteoaktiviini, osteopontiini, periostiini ja pleiotropiini. Edellä mainittujen proteiinien lisäksi osteoprotegeriinin ja trombospondiinien 1-4 ilmentymistä tutkittiin kalkkiuttavan aorttaläppävian eri kehitysvaiheissa. Aorttaläpät oli kerätty tekoläppäleikkauspotilailta. Sydämessä MGP:n ilmentyminen lisääntyi kaikissa muissa paitsi munuaisten vajaatoiminnan koemallissa. Angiotensiini II nosti MGP:n ilmentymistä sydänlihassoluissa ja sidekudossoluissa. Periostiinin ilmentyminen lisääntyi sydämen uudelleenmuovautumisessa, muttei aorttaläppäviassa. Lisäksi munuaisten vajaatoiminnan aiheuttama periostiinin ilmentymisen muutos sydämessä liittyi sekä sydämen kasvuun, verenpaineen nousuun että muiden sidekudosta muokkaavien proteiinien ilmentymiseen. Luun sialoproteiinin ja osteopontiinin ilmentymiset erosivat toisistaan erilaisissa sydämen vajaatoiminnan malleissa, mutta aorttaläpissä niiden molempien ilmentyminen oli suhteessa läpän kalkkiutumiseen. Osteoprotegeriinin geenin ilmentyminen lisääntyi kalkkiutuneissa aorttaläpissä vaikkakin proteiinin määrä pysyi vähäisenä. Luun morfogeneettisten proteiinien ilmentyminen oli alentunut sairaissa läpissä, muttei sydämessä munuaisten vajaatoiminnan aikana. Aorttaläpissä ilmennettiin kaikkia trombospondiineita, joista trombospondiini-2:n ilmentyminen kasvoi sairaissa aorttaläpissä. Kalkkiutuneissa läpissä solunsisäinen Akt/NF-κB–signaalinvälitysjärjestelmä oli vaimentunut. Tutkimus osoittaa, että soluväliaineen proteiinien ilmentymistä säädellään eri tavoin sydämen vajaatoiminnassa ja aorttastenoosissa kudoksen uudelleenmuovautumisprosessin aikana

My PhD project was focused on understanding if endogenous bone-marrow cells (BMCs) could participate in the repair of mouse retina following N-Methyl-D-aspartate (NMDA) damage. We wanted to explore the possibility that endogenous BMCs could migrate from peripheral blood into the damaged retina and fuse with retinal neurons. We found that endogenous BMCs migrate into mouse retina upon NMDA damage and fuse with retinal neurons, mainly with Muller glia (MG) cells. MG cells undergo dedifferentiation and reprogramming after NMDA damage finally differentiating into ganglion and amacrine cells through a cell-fusion mediated mechanism. This neurogenic process was influenced by SDF-1/CXCR4 signaling pathway since when we manipulated it we were able to enhance or block the ability of MG cells to undergo reprogramming and to generate new neurons. All in all we described a novel mechanism by which murine MG cells can dedifferentiate through a cell fusion process with endogenous BMCs.
Mi proyecto de doctorado ha sido enfocado en entender si las células de la medula (BMCs) pueden participar en la reparación de la retina del ratón después de causar un daño con N-Methyl-D-aspartate (NMDA). Hemos querido explorar la posibilidad de que las BMCs pudiesen migrar desde la sangre periférica hasta la retina dañada y fusionarse con las neuronas retinianas. Hemos encontrado que las células endógenas de la médula son movilizadas hacia la retina del ratón después de causar el referido daño fusionándose con las neuronas de la retina, principalmente con las células gliales Müller (MG). Las células MG empiezan un proceso de desdiferenciación y reprogramación después de causar el daño de NMDA, y acaban diferenciándose en células ganglionares y amacrinas a través de un mecanismo de fusión celular. Este proceso neurogénico está influenciado también de la vía de señalación SDF-/CXCR4, cuando hemos manipulado esta vía de señalación celular hemos sido capaces de aumentar o bloquear la capacidad de las células MG de reprogramarse y de generar nuevas neuronas. En general hemos descrito un nuevo mecanismo mediante el cual las células MG pueden desdiferenciarse a través de un proceso de fusión celular con la células endógenas del la médula.

Knochen ist ein hierarchisch aufgebautes Material, gekennzeichnet durch eine erstaunliche Variabilität und Diversität. Knochenersatz- oder Biomaterialien sind wichtige Komponenten für künstliche Organe und werden auch als Gerüste für Tissue Engineering eingesetzt. Das Ziel dieser Dissertation ist die Vorhersage der Festigkeit von Knochen und Knochenersatzmaterialien auf Grund ihrer Zusammensetzung und Mikrostruktur mittels Mehrskalenmodellen. Die theoretischen Entwicklungen werden durch umfangreiche Experimente an kortikalen Knochen sowie an Biomaterialien aus Hydroxyapatit, Glas-Keramik und Titanium untermauert
Bone is a hierarchically organized material, characterized by an astonishing variability and diversity. Bone replacement or biomaterials are critical components in artificial organs, and they are also used as scaffolds in tissue engineering. The aim of this thesis is the prediction of the strength of bone and bone replacement materials, from their composition and microstructure, by means of multiscale models. The theoretical developments are supported by comprehensive experiments on cortical bone and on biomaterials made of hydroxyapatite, glass-ceramic, and titanium

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014
Cardiovascular disease is one of the main causes of death worldwide. Vascular calcification is a risk factor that strongly contributes to disease progression and to which the vitamin-K dependent family of proteins (VKDPs) appear to play a major role. Gla-rich protein, or GRP, was the last member of the VKDPs to be identified and has been associated with ectopic calcification in tissues such as skin, vasculature and cartilage in cases of dermatomyositis and pseudoxanthoma elasticum, chronic kidney disease and osteoarthritis, respectively, suggesting a possible role in the development and/or regulation of pathological calcification. Matrix Gla protein (MGP), another VKDP, is a recognized inhibitor of soft tissue calcification. Although its inhibitory mechanism is still not completely understood, distinct studies reported the binding of MGP to bone morphogenetic proteins (BMPs), known bone formation promoters in both skeletal and vascular tissue, antagonizing its function. However, these mechanisms of action are not enough to explain the numerous reported cases of calcification in humans leading us to hypothesize GRP as one of the missing regulators of calcification in soft tissues. Considering the reported data on MGP-BMP-2 interaction, and since an effect of BMP-2 on GRP expression has been previously demonstrated, we have focused in understanding the importance of GRP in calcification inhibition via interaction with MGP and BMP-2, either as a duplet or as a part of a larger protein complex. To further investigate these possibilities, we have engineered HEK293T cells to overexpress GRP and MGP and used their conditioned media in addition to recombinant BMP-2. Our immunoprecipitation assays demonstrate, for the first time, an interaction between GRP and BMP-2, supporting our hypothesis of GRP acting as a regulator of ectopic calcification via an interaction with BMP-2. Although our novel data indicate that GRP-BMP-2 interaction could be determining to vascular calcification, further functional studies will soon be performed to prove this hypothesis.
A doença cardiovascular é uma das principais causas de morte no mundo. A calcificação vascular é um factor de risco que contribui para a sua progressão e para o qual as proteínas dependentes da vitamina K (VKDPs) parecem ter um papel determinante. A proteína rica em Glas (GRP), a última das VKDPs a ser identificada tem sido associada a vários casos de calcificação ectópica na pele, vasculatura e cartilagem, em casos de dermatomiose e pseudoxanthoma elasticum, doença crónica do rim e osteoartrite, respectivamente, sugerindo um possível papel na regulação da calcificação patológica. A proteína Gla da matriz (MGP), outra VKDP, é reconhecida como um inibidor da calcificação dos tecidos moles. Embora o seu mecanismo de inibição não seja completamente conhecido, diversos estudos mostram a sua ligação a proteínas morfogenéticas do osso (BMPs), conhecidas por promoverem a formação de osso nos tecidos esquelético e vascular, antagonizando a sua função. Contudo, estas proteínas e os seus mecanismos de acção não são suficientes para explicar os inúmeros casos de calcificação ectópica em humanos levando-nos a propor a GRP como um dos reguladores adicionais envolvidos na inibição da calcificação. Considerando a interacção MGP-BMP-2 e o efeito da BMP-2 na expressão da GRP em condrócitos de ratinho, focámo-nos em investigar a importância da GRP na inibição da calcificação através do estudo da interacção com a MGP e BMP-2, quer como um dupleto ou como parte de um complexo proteico. Para este efeito, sobre-expressámos a GRP e MGP em células HEK293T e o seu meio condicionado, ao qual se adicionou BMP-2 recombinante, foi usado em ensaios de imunoprecipitação. Os nossos resultados demonstram, pela primeira vez, uma interação entre a GRP e BMP-2, apoiando a nossa hipótese de que a GRP actua como um regulador da calcificação ectópica e que esta poderá ocorrer através da interacção com a BMP-2. Neste momento, estamos a desenvolver estudos funcionais que julgamos ser determinantes para provar a nossa hipótese original que foi fortemente reforçada com os resultados deste trabalho.

Tese de Doutoramento, Biologia, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2005
As proteinas dependentes da vitamina K, proteína Gla do osso (BGP ou osteocalcina) e proteína Gla da matriz (MGP) são pequenas proteinas que ligam o cálcio, tendo sido recentemente descobertas em peixe.
The vitamin K dependent bone Gla protein (BGP or osteocalcin) and matrix Gla protein (MGP) are small calcium binding proteins only recently discovered in fish.

Osteocalcin (Oc) and Matrix Gia Protein (MGP) are vitamin K-dependente proteins known for their Ca2" binding capacity. Considering the problem of skeletal malformations in Mediterranean fish aquaculture, we investigated a possible correlation between vertebral deformities and changes of Oc and Mgp accumulation sites. ln this work we proposed to characterize bony and cartilaginous tissues from important fish for aquaculture, using molecular tools. The sites of gene expression and accumulation of osteocalcin and Mgp were investigated throughout Northern blot analysis and immunohistochemistry, following the cloning of those cDNAs not yet available, together with histological analysis. ln Scophthalmus maximus, oc mRNA was detected in vertebrae and branchial arches while mgp mRNA was highly expressed in branchial arches, followed by vertebrae, kidney and heart. ln S. maximus and Diplodus sargus, Mgp accumulated in chondrocytes from cartilages, in the vertebral mineralizing fronts and in notochord cells. Mgp was also found in scales of D. sargus. Ocaccumulated mainly in bone tissues, like the ceratobranchial bone and vertebral bone matrix. ln cultured Sparus aurata, Mgp accumulated mainly in vertebrae growth zones and in notochord cells while Oc was immunodetected in bone matrix of vertebrae and vertebral arches, both in non-deformed and in deformed vertebrae. We also identified Oc in the non-calcified notochord cells of deformed vertebrae, suggesting that abnormal skeletal development resulted in modifications on sites of osteocalcin expression and/or accumulation. Accordingly, histological analysis of normal and deformed vertebrae revealed calcification in blood vessel walls and pathological formation of chondroid bone, in the affected area of deformed vertebrae.
Osteocalcina (Oo) e Proteína Gla da Matriz (MGP2 são proteínas dependentes da vitamina K, com capacidade para ligarem Ca2+. Considerando o problema do desenvolvimento de malformações ósseas nos peixes de aquacultura no Mediterrâneo, investigou-se a existência de uma possível correlação entre a presença de deformações esqueléticas e alterações nos locais de acumulação da osteocalcina e Mgp. Neste trabalho, caracterizámos tecidos ósseos e cartilaginosos de peixes importantes em aquacultura por análise histológica e, determinámos locais de expressão e acumulação da osteocalcina e Mgp por hibridação tipo Northern e immunohistoquímica. Em Scophthalmus maximus, detectou-se expressão do ARNm da oc na vértebra e arcos branquiais. O ARNm da mgp teve maior expressão nos arcos branquiais, seguidos da vértebra, rim e coração. Em S. maximus e Diplodus sargus, observou-se acumulação de Mgp nos condrócitos de cartilagens, nas zonas de mineralização das vértebras e nas células da notocorda. A Mgp foi também detectada nas escamas de D. sargus. A acumulação de Oc foi detectada em tecidos ósseos, como o osso ceratobranquial e matriz óssea das vértebras. Em Sparus aurata, detectou-se Mgp nas zonas de crescimento das vértebras e nas células da notocorda, enquanto que a Oc foi detectada na matriz óssea de vértebras e arcos vertebrais, em vértebras deformadas e não deformadas. No entanto, identificou-se acumulação de Oc nas células não calcificadas da notocorda das vértebras deformadas, sugerindo que o desenvolvimento esquelético anormal levou a alterações da expressão e/ou acumulação desta proteína. Análises histológicas de vértebras normais e deformadas demonstraram calcificação nos vasos sanguíneos e formação patológica de osso condróide, na zona da vértebra afectada pela deformação.

Tese de dout. em Química, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2002
Matrix Gla protein (MGP) and Bone Gla Protein (BGP, osteocalcin) belong to the family of vitamin K dependent (VKD), Gla containing proteins. Matrix Gla protein (MGP) is a 10-15 kDa secreted protein with 4-5 residues of the Ca2+ binding y-carboxyglutamic acid residue (depending on the species). MGP was previously found to accumulate within the organic matrix of mammalian bone, from which it was originally purified. In mammals, birds and Xenopus, its mRNA was previously detected in extracts of bone, cartilage and soft tissues (mainly heart and kidney) while the protein was found to accumulate mainly in bone. However, at that time it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells since both co-exist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calciflcation. Interestingly, MGP was also found to accumulate in vértebra of shark, a cartilaginous fish. But to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. BGP is a small secreted protein with 46-50 residues including three potential Ca24 binding y-carboxyglutamic acid residues. This protein is the most abundant non-collagenous protein found in mammalian bone from which it was first isolated. BGP has been known for a number of years to be present in teleost fishes. nevertheless there is little information about the regulation of expression and tissue localization of this protein in lower vertebrate organisms and in particular in fish. This report describes, for the first time the identification of MGP in a teleost fish, with the protein purification and molecular cloning of MGP and BGP from the same teleost fish, Argyrosomus regius. The obtention of valuable biochemical tools such as specific antibodies and mRNA/DNA probes also permitted the study of tissue distribution/accumulation for MGP/BGP by Northern analysis, in situ hybridization and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in chondrocytes from branchial arches, with no expression detected in the different bone-like mineralized tissues analyzed while BGP mRNA was mainly located in bony tissues as expected. Accordingly,the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in chondrocyte-containing tissues. However and in contrast with results obtained for Xenopus and higher vertebrates, the protein does not significantly accumulate in vertebra of non-osteocytic teleost fish (such as A. regius) but only in its calcified cartilage. As previously seen in mammals and Xenopus, MGP was also found in soft tissues predominantly in heart and kidney. Our results indicate, in addition, that the presence of MGP mRNA in heart tissue is due, at least in físh, to the expression of the MGP gene in two specific cell types only, smooth muscle and endothelial cells, while no expression was found in the striated muscle fibers of the ventricle. In light of these results and in agreement with recent information on expression of the MGP gene in these same cell types in mammalian aorta, it is likely that the presence of MGP mRNA previously detected in Xenopus, birds and mammalian heart tissue may be due to expression of the gene only in smooth muscle and endothelial cells. Our results provide clear evidence that fish represem a valuable model system to study MGP/BGP gene expression and regulation, to bring further insight into its mode of action at the molecular levei throughout evolution.
As proteínas Matrix Gla Protein (MGP) e Bone Gla Protein (BGP, osteocalcina) pertencem ambas à família de proteínas secretadas dependentes da vitamina K que contêm Gla. A proteína Matrix Gla (MGP) com um peso molecular de 10-15 kDa que contém 4-5 resíduos do aminoácido y-carboxiglutamato que coordenam o ião Ca2+. Esta proteína foi originalmente purificada da matriz orgânica de osso de mamífero, onde foi detectada a sua acumulação. Em mamíferos, aves e Xenopus, o seu ARNm foi previamente detectado em extractos de osso, cartilagem e tecidos moles (nomeadamente coração e rim), enquanto a sua acumulação era essencialmente detectada em osso. No entanto, na época não era evidente se esta acumulação teria origem a partir de proteína sintetizada em cartilagem ou em células de osso, dado que ambas coexistem em estruturas de esqueleto de vertebrados superiores e Xenopus. Dados posteriores demonstram em mamíferos a acumulação de MGP em cartilagem calcificada da região costal, bem como em algumas zonas das válvulas do coração e em artérias calcificadas. Foi também detectada a acumulação de MGP em vértebra de tubarão, um peixe cartilagíneo. Até à data não existe informação acerca dos sítios de expressão ou acumulação de MGP em peixes teleosteos, os ancestrais dos vertebrados terrestres, que contêm no seu esqueleto estruturas mineralizadas com osso e cartilagem calcificada. A BGP é uma pequena proteína (46-50 resíduos) com três • ** 2"F T-* ' resíduos de y-carboxiglutamato responsáveis pela coordenação do ião Ca . Esta e a proteína não colagénica mais abundante encontrada em osso de mamífero, a partir do qual foi inicialmente isolada. Desde há alguns anos se sabe que a BGP se encontra presente em peixes teleosteos, no entanto é escassa a informação no que respeita à regulação da expressão e localização nos tecidos desta proteína em organismos vertebrados inferiores. Este trabalho descreve pela primeira vez a identificação da MGP num peixe teleósteo, assim como a purificação e a clonagem de ambas as proteínas BGP e MGP, esta pela primeira vez, isoladas do mesmo peixe Argyrosomus regius. A obtenção dos anticorpos específicos e das sondas de ARNm/ADN permitiram o estudo da distribuição/acumulação nos tecidos por análise de tipo "Northern", hibridação in situ e imunohistoquímica. Em tecidos mineralizados, o gene da MGP é predominantemente expresso em condrócitos de arcos branquiais, não lendo sido detectada expressão nos diferentes tipos de tecidos osseos analisados, enquanto que o ARNm da BGP foi essencialmente detectado em tecidoósseo, como esperado. Do mesmo modo, a acumulação da MGP foi detectada por análise imunohistoquímica, essencialmente na matriz extracelular de cartilagem calcificada. Em tecidos moles, o ARNm da MGP é detectado no coração, mas estudos realizados por hibridação in situ permitem concluir que as células responsáveis pela expressão do gene da MGP localizam-se no bulbus arteriosus e parede aórtica, ricos em células de músculo liso e células endoteliais. No entanto não foi detectada expressão nas fibras miocardicas do músculo estriado do ventrículo. Em contraste com os resultados obtidos para Xenopus e vertebrados superiores, não é detectada acumulação significativa da MGP em vértebra de peixe teleósteo não osteócitico, mas somente em cartilagem calcificada. Adicionalmente os resultados indicam também que a presença do ARNm da MGP no tecido do coração em peixe é devido à expressão do gene da MGP somente em dois tipos de células; células endoteliais e células de músculo liso, não tendo sido detectada expressão nas fibras miocárdicas do músculo estriado do ventrículo. À luz destes resultados e informação recente sobre a expressão do gene da MGP neste mesmo tipo de células em aorta de mamíferos, é provável que a presença de ARNm previamente detectados em Xenopus, aves e tecido do coração de mamíferos, seja restrito, a regiões ricas em músculo liso e células endoteliais. Os resultados obtidos evidenciam o papel do peixe como um sistema modelo no estudo da expressão e regulação dos genes da MGP/BGP e fornecer informação pertinente quanto ao modo de acção destas proteínas a nível molecular ao longo de evolução.

Tese de Doutoramento, Biologia Molecular, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2001
A proteína Bone Gla (BGP, osteocalcina) é uma pequena proteína dependenteda. vitamina K que apresenta resíduos de ácido glutâmico y-carboxilados. A presença destes aminoácidos modificados permite à proteína ligar-se a iões Ca2+ e interagir com os cristais de hidroxiapatite dos tecidos mineralizados.
The Bone Gla Protein (BGP, osleocalcin) is a small vilamin K-dependent protein which presenls Ihree y-carboxylated glutamic acid residues. lhe prcsence oí these modified amino acids enables the protein to bind to Ca2+ ions and to interact with hydroxyapatite ciystals of mineralized tissues.