Vesicle trafficking provides the solution to the 'sorting problem' - how the eukaryotic cell maintains the distinct identities, and thus functional properties, of its membrane-bound organelles. During vesicle trafficking, proteins are selectively sorted into membrane bound transport intermediates by vesicle adaptors, which include those of the highly conserved adaptor protein (AP) complex family. Each AP complex has a distinct subcellular localisation and functions in the sorting of a specific subset of transmembrane cargo proteins. Adaptor protein complex 4 (AP-4) is one of the more recently identified AP complexes, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder, suggesting an important role in neuronal development and homeostasis. However, the pathomechanisms that underly the neuronal pathology in AP-4 deficiency are currently unknown. AP-4 is proposed to function in protein sorting at the trans-Golgi network (TGN), so AP-4 deficiency can be thought of as a disease of missorting. The aim of this study was to apply unbiased global proteomic approaches to define the composition of AP-4 vesicles and to identify physiological cargo proteins of the AP-4 pathway. Using 'Dynamic Organellar Maps' and comparative analysis of vesicle-enriched fractions from wild-type and AP-4-depleted cells, three ubiquitously expressed transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, were found to be mislocalised in AP-4-deficient cells. Two novel cytosolic AP-4 accessory proteins, RUSC1 and RUSC2, were also identified. Further proteomic analyses confirmed the interactions between these proteins. AP-4 deficiency was found to cause missorting of ATG9A in diverse cell types, including patient derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A and SERINC-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the 'ATG9 reservoir' required for autophagosome biogenesis. This study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

<p>Sposobnost insekata da se prilagode različitim ekolo&scaron;kim uslovima je veoma dobro<br />dokumentovana; oni predstavljaju najrasprostranjeniju grupu životinja na planeti, sa vrstama koje naseljavaju različita kopnena i vodena stani&scaron;ta, od tropskih predela do polova. Razumevanje mehanizama koji omogućavaju insektima da prežive ekstremne temperature i zadrže vitalne funkcije tokom dugog perioda dormancije je kao model sistem od interesa za mnoge naučne oblasti. Na osnovu načina na koji preživljavaju temperature ispod 0&ordm;C insekte možemo podeliti u tri grupe:<em> i)</em> insekti koji toleri&scaron;u formiranje leda u ekstraćelijskom prostoru,<em>ii)</em> insekti koji ne toleri&scaron;u zamrzavanje i moraju da ga izbegnu,&nbsp; a to&nbsp; čine superhlađenjem svojih telesnih tečnosti i<em>&nbsp; iii) </em>insekti koji preživljavaju zahvaljujući gubitku vode kroz permeabilnu kutikulu, &scaron;to je nazvano krioprotektivna dehidratacija. Zajednička odlika organizama sa različitim mehanizmima adaptacije na niske temperature je setbiohemijskih jedinjenja&nbsp; čija se fiziolo&scaron;ka funkcija razlikuje u zavisnosti da li organizam pripada grupi koja toleri&scaron;e ili ne toleri&scaron;e zamrzavanje. To su nukleatori kristalizacije leda, krio/anhidroprotektanti i antifriz proteini.<br />Cilj ovih istraživanja je bio ispitivanje molekularne osnove otpornosti na niske temperature dve vrste polarnih kolembola&nbsp;<em> Onychiurus arcticus </em>i <em>Cryptopygus</em> <em>antarcticus </em>kombinujući fiziolo&scaron;ki, biohemijski i molekularno biolo&scaron;ki pristup. Ispitivane vrste izbegavaju zamrzavanje svojih telesnih tečnosti primenjujući različite strategije preživljavanja. Za antarktičku vrstu&nbsp;<em> C. antarcticus </em>karakteristična je brza promena tačke superhlađenja, kao i njena bimodalna distribucija tokom leta, kada neke jedinke mrznu na&nbsp; vi&scaron;im temperaturama (manje otporne na hladnoću), a druga na nižim (otpornije na hladnoću). Ova bimodalna distribucija tačke superhlađenja je dobro dokumentovana, ali slabo razja&scaron;njena na molekularnom nivou. Druga, arktička vrsta<em>&nbsp; O. arcticus&nbsp;</em> koristi strategiju preživljavanja zimskih temperature koje idu i do -25&ordm;C nazvanu krioprotektivna dehidratacija. Na ovaj način, količina slobodne vode u telu se značajno redukuje, a akumulira se trehaloza koja deluje kao&nbsp; krio/anhidroprotektant. Iako je krioprotektivna dehidratacija opisana i kod drugih vrsta<br />insekata, molekularni mehanizmi koji se nalaze u osnovi ovog fenomena su veoma slabo razja&scaron;njeni.<br />Za karakterizaciju genoma generisano je 16379 EST sekvenci za&nbsp;<em> O. arcticus </em>i 1180 za&nbsp; <em>C. antarcticus</em>. To su ujedno i prvi javno dostupni podaci u bazama podataka o genomima ove dve vrste koji predstavljaju značajnu osnovu za komparativne genomske analize. Činjenica da kod obe analizirane vrste, oko 60% EST sekvenci nije pokazalo statistički značajnu sličnost sa proteinima iz baza podataka ukazuje na specifičan patern genske ekspresije kao adaptivni odgovor ispitivanih vrsta na niske temperature.<br />Sa ciljem da se identifikuju geni uključeni u preživljavanje niskih temperatura konstruisani su mikroereji, za&nbsp;<em> O. arcticus</em> &scaron;tampanjem 6912 cDNK u duplikatu, a za&nbsp; <em>C. antarcticus</em> &scaron;tampanjem 672 cDNK u duplikatu.. Analizom sekvenci identifikovanih putem homologije sa dostupnm bazama podataka kod C. antarcticusuočen je jasan trend povećane ekspresije gena koji kodiraju strukturne proteine u grupi koja je otporna na hladnoću. Ove strukturne proteine uglavnom&nbsp; čine kutikularni proteini, &scaron;to je u skladu sa rezultatima nedavnih istraživanja kod kolembola, da je presvlačenje proces tokom kog se snižava tačka superhlađenja, odnosno da varijacije u tački superhlađenja mogu nastati kao posledica endogenih fiziolo&scaron;kih procesa tokom presvlačenja. Kod&nbsp;<em> O. arcticus </em>analizom EST sekvenci i mikroereja identifikovani su<br />potencijalni geni i biohemijski putevi povezani sa krioprotektivnom dehidratacijom, a istakli bi gene uključene u metabolizam ugljenih hidrata, gene za akvaporine, proteine toplotnog stresa, LEA proteine i enzime antioksidativne za&scaron;tite.</p> / <p>The ability of insects to adapt to diverse ecological conditions iswell documented; they &nbsp;are the most diverse fauna on earth, with different species occupying arange of terrestrial and aquatic habitats from the tropics to the poles. Understanding the mechanisms by which insects survive such extreme temperatures and retain viability for longperiods in the dormant state is of great interest to many scientific fields. Insects have evolved three main strategies to survive sub-zero temperatures:&nbsp; i) freeze tolerance,&nbsp; ii) freeze avoidance and&nbsp; iii) cryoprotective dehydration. The main biochemical compounds involved in surviving sub-zero temperatures are same for different strategies but their physiological&nbsp; role is different. They include: ice nucleating agents (INAs), cryo/ anhydroprotectants, and antifreeze proteins (AFPs).&nbsp;</p><p>The aim of this study was to determine molecular adaptations to extreme cold&nbsp; environments, combining physiology, biochemistry and molecular biology&nbsp; pproaches, in thePolar Collembola:&nbsp; Cryptopygus antarcticus and&nbsp; Onychiurus arcticus. Both species are freeze avoiding but employ different strategies for surviving low temperatures. The Antarctic springtail&nbsp; C. antarcticusis capable of rapid cold hardening with a bi-modal distribution of super cooling points (SCP) with high (less cold-hardened) and low (more&nbsp; cold-hardened) groups of animals present even during the growing season in summer. This bimodal distribution has been well documented, but is poorly understood. The Arctic springtail&nbsp; O. arcticusemploys the strategy known as cryoprotective dehydration to survive winter temperatures as low as &nbsp; -25&ordm;C. With this technique, the amount ofavailable water in the body&nbsp; is reduced to almost zero and also there is an accumulation of trehalose, which acts as a cryo/anhydroprotectant. Although cryoprotective dehydration has been described in &nbsp;other&nbsp; insects, the molecular mechanisms behind this phenomenon are poorly understood.</p><p>A total of 16,379 EST clones were generated for O. arcticus and 1180 for C. antarcticus. This represents the first publicly available sequence data for this two species providing useful data for comparative genomic analysis. The fact that around 60% of the clones for both species showed no sequence similarity to annotated genes &nbsp;in the datasets, suggests a specific pattern of gene expression in these species as adaptation to low temperatures.</p><p>Two microarrays were constructed to identify genes involved in&nbsp; surviving low temperatures, one for C. antarcticus by printing 672 clones in duplicate and the other&nbsp; for O. arcticus by&nbsp; printing 6912 clones in duplicate. An analysis of those where putative function could be inferred via database homology, in C. antarcticus there was aclear pattern of up-regulation of structural proteins being associated with the cold tolerant group.&nbsp; These structural proteins mainly comprised cuticle proteins and provide support for the recenttheory that summer SCP variation within Collembola species could be a consequence of moulting, with moulting population having lowered SCPs. In O. arcticus EST and microarrayanalysis revealed clones&nbsp;and biochemical pathways associated with cryoprotective dehydration with a particular&nbsp; reference to genes involved in carbohydrate metabolism, aquaporin&nbsp; genes, heat shock&nbsp; proteins, LEA proteins and antioxidant enzymes.</p>

Durant les dernières décennies, la vitamine D a été décrite comme un micronutriment d'intérêt, bénéfique pour la santé humaine, mais les mécanismes régissant son absorption intestinale ont été peu investigués. Il a longtemps été admis que son absorption était régie par un phénomène de diffusion passive. Nous avons tout d'abord montré qu'elle n'était pas uniquement passive mais impliquait des transporteurs membranaires du cholestérol tels que SR-BI, CD36 et NPC1-L1 et que des molécules pharmaceutiques utilisées pour inhiber l'absorption du cholestérol (Ezétimibe - Ezetrol®) pouvaient également diminuer celle de la vitamine D. Dans un second temps, nous avons montré que des composés présents au sein de notre alimentation comme les phytostérols pouvaient limiter l'absorption de la vitamine D. Au contraire, certains acides gras libres comme l'acide oléique pouvaient augmenter sa biodisponibilité. Ces données suggèrent que les autres composés lipidiques que nous consommons quotidiennement peuvent améliorer ou a contrario limiter l'absorption de la vitamine D. Pour finir, nous nous sommes intéressés aux mécanismes moléculaires en temps réel entre divers vecteurs lipidiques et les boucles extracellulaires de SR-BI et CD36 grâce à la technique de la résonnance plasmonique de surface. Nous avons montré que la nature et la composition des vecteurs lipidiques modulaient les interactions micelles-protéines. Ces travaux de thèse dégagent des perspectives prometteuses quant à l'avancement des connaissances et des mécanismes moléculaires régissant l'absorption intestinale de la vitamine D, et souligne le fait que nos choix alimentaires pourraient optimiser notre statut en vitamine D. / Overs the last past decades, vitamin D has been described as a micronutrient of interest, beneficial to human health, but the mechanisms governing its intestinal absorption have been poorly investigated. Indeed, it has long been assumed that vitamin D was absorbed by a passive process. We first showed that vitamin D absorption was not only passive but involved membrane transporters including such as SR-BI, CD36 and NPC1-L1 and that pharmaceutical compounds used to inhibit cholesterol absorption (ei: Ezetimibe - Ezetrol ®) could also reduce vitamin D absorption. Then, we have shown that dietary molecules such as phytosterols could limit its absorption, while some free fatty acids such as oleic acid could increase its bioavailability. These data suggest that other dietary lipids daily consumed with vitamin D could improve or conversely limit its absorption. Finally, we investigated real-time interactions between various lipid ligands and scavenger receptors such as SR-BI and CD36 using the technique of surface plasmon resonance. This highlighted the fact that mixed-micelle lipid composition could modulate their interactions with these proteins.This thesis bring promising prospects in the advancement of knowledge and molecular mechanisms regulating vitamin D intestinal absorption, and enlightens the fact that our food choices could lead to an optimization of our vitamin D status.

A novel bacterial expression system that is capable of producing high levels of soluble, stable, biologically active human vitamin D3 and estrogen receptors has been developed. The method utilizes ubiquitin fusion technology and a low temperature nalidixic acid induction of the lambda PL promoter. This system can produce large quantities of receptor antigen, but only a small fraction displays wild-type DNA and hormone binding properties. Therefore, the use of this system to overproduce receptors for crystallization studies is not practical. To overcome these problems, a 2 um based ubiquitin fusion system which allows regulated expression of the estrogen receptor in yeast (Saccharomyces cerevisiae) was developed. This system produces the estrogen receptor to a level of 0.2% of the total soluble protein. Moreover, this protein is undegradable, soluble, and biologically active. To test the transcriptional activity of the estrogen receptor produced in yeast, a cis-trans transcription assay was developed. This assay revealed that the transcriptional activity of the human estrogen receptor expressed in yeast was similar to that observed in transfected mammalian cells. This reconstituted estrogen transcription unit in Saccharomyces cerevisiae was utilized to examine the regulation of estrogen receptor functions by antiestrogens, to develop a random and rapid approach for identifying novel estrogen response elements, to characterize estrogen receptor variants cloned from human breast tumors, and to examine the effect of estrogen receptor on the regulation of osteocalcin gene.

No description available.

No description available.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 158-177.

Cardiovascular disease is a major cause of morbidity and mortality around the globe, which is linked to athero-thrombosis. The risk factors for atherothrombosis, thus cardiovascular disease is impaired anti-thrombotic and antiinflammatory functions of the endothelium. Thrombosis is a hallmark of cardiovascular disease/complications characterised by increased platelet activation and increased secretion of platelet micro-particles that induce angiogenesis. This study determined the role of platelet micro-particles derived microRNA in the regulation of angiogenesis and migration, with a focus on the regulation of thrombospondin-1 release by platelet micro-particles delivered Let- 7a. The role of thrombospondin-1 receptors (integrin beta-1 and integrin associated protein) and downstream caspase-3 activation were explored by Let-7a inhibition prior to PMP treatment. MicroRNA dependent modulation of proangiogenic proteins including monocyte chemoattractant protein-1 and placental growth factor, and recruitment of activating transcription factor-4 protein to their promoter regions were explored. Main findings are: 1. Platelet micro-particles induce angiogenesis, migration, and release of novel cytokine subsets specific to platelet micro-particle’s RNA content. 2. The targeting of thrombospondin-1 mRNA by platelet micro-particles’ transferred Let-7a chiefly modulate the angiogenic effect on endothelial cells. 3. The inhibition of thrombospondin-1 translation enable platelet micro-particles to increase angiogenesis and migration in the presence of functional integrin beta-1 and integrin associated protein, and reduced cleaving of caspase-3. 4. Platelet micro-particle modulate the transcription of monocyte chemoattractant protein-1 and placental growth factor in a Let-7a dependent manner. 5. Let-7a induce angiogenesis ii independent of other platelet micro-particle’s microRNAs. Platelet micro-particle derived Let-7a is a master regulator of endothelial cell function in this model, which presents an opportunity for the development of new biomarkers and therapeutic approaches in the management of cardiovascular disease. Future studies should aim to confirm these findings in-vivo.

Mares are seasonally polyestric. The breeding season in spring and summer and the winter anestrus are flanked by transitional periods. Endometrial diseases are a frequent cause of subfertility and have an economic impact on the horse breeding industry. They include different forms of endometrosis, endometritis, glandular maldifferentiation, and angiosis. Except for suppurative endometritis, these are subclinical and can only be diagnosed by the microscopic examination of an endometrial biopsy. Endometrosis is characterized by periglandular fibrosis and nonsuppurative endometritis by stromal infiltration with lymphocytes and plasma cells. The pathogenesis of endometrosis and nonsuppurative endometritis is still undetermined. Some mares are predisposed to persistent endometritis; this has likely a multifactorial etiology. Glandular differentiation has to be interpreted under consideration of the season. The presence of endometrial diseases is associated with alterations in the expression of several intra- and extracellular molecular markers. Some of them may have potential to be used as diagnostic biomarkers for equine endometrial health and disease. The aim of this review is to provide an overview on pathomorphological findings of equine endometrial diseases, to outline data on analyses of cellular and molecular mechanisms, and to discuss the impact of these data on reproduction and treatment.

Cardiovascular disease is a major cause of morbidity and mortality around the globe, which is linked to athero-thrombosis. The risk factors for atherothrombosis, thus cardiovascular disease is impaired anti-thrombotic and antiinflammatory functions of the endothelium. Thrombosis is a hallmark of cardiovascular disease/complications characterised by increased platelet activation and increased secretion of platelet micro-particles that induce angiogenesis. This study determined the role of platelet micro-particles derived microRNA in the regulation of angiogenesis and migration, with a focus on the regulation of thrombospondin-1 release by platelet micro-particles delivered Let- 7a. The role of thrombospondin-1 receptors (integrin beta-1 and integrin associated protein) and downstream caspase-3 activation were explored by Let-7a inhibition prior to PMP treatment. MicroRNA dependent modulation of proangiogenic proteins including monocyte chemoattractant protein-1 and placental growth factor, and recruitment of activating transcription factor-4 protein to their promoter regions were explored. Main findings are: 1. Platelet micro-particles induce angiogenesis, migration, and release of novel cytokine subsets specific to platelet micro-particle’s RNA content. 2. The targeting of thrombospondin-1 mRNA by platelet micro-particles’ transferred Let-7a chiefly modulate the angiogenic effect on endothelial cells. 3. The inhibition of thrombospondin-1 translation enable platelet micro-particles to increase angiogenesis and migration in the presence of functional integrin beta-1 and integrin associated protein, and reduced cleaving of caspase-3. 4. Platelet micro-particle modulate the transcription of monocyte chemoattractant protein-1 and placental growth factor in a Let-7a dependent manner. 5. Let-7a induce angiogenesis ii independent of other platelet micro-particle’s microRNAs. Platelet micro-particle derived Let-7a is a master regulator of endothelial cell function in this model, which presents an opportunity for the development of new biomarkers and therapeutic approaches in the management of cardiovascular disease. Future studies should aim to confirm these findings in-vivo.