Targeted Small Business Link, Summer 2011, Vol. 4, no. 3

Targeted Small Business News and information from the Iowa Department of Economic Development.

"In vitro" study of the molecular mechanism of the "E.Coli" Hsp 70/Hsp40/NEF chaperone system and its interactions with ?-sinuclein

SUMMARY Under stressful conditions, mutant or post-translationally modified proteins may spontaneously misfold and form toxie species, which may further assemble into a continuum of increasingly large and insoluble toxic oligomers that may further condense into less toxic, compact amyloids in the cell Intracellular accumulation of aggregated proteins is a common denominator of several neurodegenerative diseases. To cope with the cytotoxicity induced by abnormal, aggregated proteins, cells have evolved various defence mechanisms among which, the molecular chaperones Hsp70. Hsp70 (DnaK in E. coii) is an ATPase chaperone involved in many physiological processes in the cell, such as assisting de novo protein folding, dissociating native protein oligomers and serving as pulling motors in the import of polypeptides into organelles. In addition, Hsp70 chaperones can actively solubilize and reactivate stable protein aggregates, such as heat- or mutation-induced aggregates. Hsp70 requires the cooperation of two other co-chaperones: Hsp40 and NEF (Nucleotide exchange factor) to fulfil its unfolding activity. In the first experimental section of this thesis (Chapter II), we studied by biochemical analysis the in vitro interaction between recombinant human aggregated α-synuclein (a-Syn oligomers) mimicking toxic a-Syn oligomers species in PD brains, with a model Hsp70/Hsp40 chaperone system (the E. coii DnaK/DnaJ/GrpE). We found that chaperone-mediated unfolding of two denatured model enzymes were strongly affected by α-Syn oligomers but, remarkably, not by monomers. This in vitro observed dysfunction of the Hsp70 chaperone system resulted from the sequestration of the Hsp40 proteins by the oligomeric α-synuclein species. In the second experimental part (Chapter III), we performed in vitro biochemical analysis of the co-chaperone function of three E. coii Hsp40s proteins (DnaJ, CbpA and DjlA) in the ATP-fuelled DnaK-mediated refolding of a model DnaK chaperone substrate into its native state. Hsp40s activities were compared using dose-response approaches in two types of in vitro assays: refolding of heat-denatured G6PDH and DnaK-mediated ATPase activity. We also observed that the disaggregation efficiency of Hsp70 does not directly correlate with Hsp40 binding affinity. Besides, we found that these E. coii Hsp40s confer substrate specificity to DnaK, CbpA being more effective in the DnaK-mediated disaggregation of large G6PDH aggregates than DnaJ under certain conditions. Sensibilisées par différents stress ou mutations, certaines protéines fonctionnelles de la cellule peuvent spontanément se convertir en formes inactives, mal pliées, enrichies en feuillets bêta, et exposant des surfaces hydrophobes favorisant l'agrégation. Cherchant à se stabiliser, les surfaces hydrophobes peuvent s'associer aux régions hydrophobes d'autres protéines mal pliées, formant des agrégats protéiques stables: les amyloïdes. Le dépôt intracellulaire de protéines agrégées est un dénominateur commun à de nombreuses maladies neurodégénératives. Afin de contrer la cytotoxicité induite par les protéines agrégées, les cellules ont développé plusieurs mécanismes de défense, parmi lesquels, les chaperonnes moléculaires Hsp70. Hsp70 nécessite la collaboration de deux autres co-chaperonnes : Hsp40 et NEF pour accomplir son activité de désagrégation. Hsp70 (DnaK, chez E. coli) est impliquée par ailleurs dans d'autres fonctions physiologiques telles que l'assistanat de protéines néosynthétisées à la sortie du ribosome, ou le transport transmembranaire de polypeptides. Par ailleurs, les chaperonnes Hsp70 peuvent également solubiliser et réactiver des protéines agrégées à la suite d'un stress ou d'une mutation. Dans la première partie expérimentale de cette thèse (Chapter II), nous avons étudié in vitro l'interaction entre les oligomères d'a-synucleine, responsables entre autres, de la maladie de Parkinson, et le système chaperon Hsp70/Hsp40 (système Escherichia coli DnaK/DnaJ/GrpE). Nous avons démontré que contrairement aux monomères, les oligomères d'a-synucleine inhibaient le système chaperon lors du repliement de protéines agrégées. Cette dysfonction du système chaperon résulte de la séquestration des chaperonnes Hsp40 par les oligomères d'a-synucleine. La deuxième partie expérimentale (Chapitre III) est consacrée à une étude in vitro de la fonction co-chaperonne de trois Hsp40 d'is. coli (DnaJ, CbpA, et DjlA) lors de la désagrégation par DnaK d'une protéine pré-agrégée. Leurs activités ont été comparées par le biais d'une approche dose-réponse au niveau de deux analyses enzymatiques: le repliement de la protéine agrégée et l'activité ATPase de DnaK. Par ailleurs, nous avons mis en évidence que l'efficacité de désagrégation d'Hsp70 et l'affinité des chaperonnes Hsp40 vis-à-vis de leur substrat n'étaient pas corrélées positivement. Nous avons également montré que ces trois chaperonnes Hsp40 étaient directement impliquées dans la spécificité des fonctions accomplies par les chaperonnes Hsp70. En effet, DnaK en présence de CbpA assure la désagrégation de large agrégats protéiques avec une efficacité nettement plus accrue qu'en présence de DnaJ.

Ownership structure and innovation: Is there a real link?

This work focuses on the study of the relationship between ownership and control structure of the company and its innovative activity. Its aim consists of analysing the role that may be played by determinants within the company related to ownership structure when the decision to incur research and development activities is taken as well as on the output of this innovate process. Among these determinants we may think of issues such as who owns the firm and how the control of decision-making is distributed, the nature of this control and the level of concentration of ownership, among others. The study is carried out for the year 2001 using a representative sample of Spanish manufacturing industries.

Influence of the driving mechanism on the response of systems with athermal dynamics: The example of the random-field Ising model

We investigate the influence of the driving mechanism on the hysteretic response of systems with athermal dynamics. In the framework of local mean-field theory at finite temperature (but neglecting thermally activated processes), we compare the rate-independent hysteresis loops obtained in the random field Ising model when controlling either the external magnetic field H or the extensive magnetization M. Two distinct behaviors are observed, depending on disorder strength. At large disorder, the H-driven and M-driven protocols yield identical hysteresis loops in the thermodynamic limit. At low disorder, when the H-driven magnetization curve is discontinuous (due to the presence of a macroscopic avalanche), the M-driven loop is reentrant while the induced field exhibits strong intermittent fluctuations and is only weakly self-averaging. The relevance of these results to the experimental observations in ferromagnetic materials, shape memory alloys, and other disordered systems is discussed.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome.

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushing's syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate-activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid-excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid-induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.

Patterns of calcium-binding proteins in human inferior colliculus: identification of subdivisions and evidence for putative parallel systems.

The subdivisions of human inferior colliculus are currently based on Golgi and Nissl-stained preparations. We have investigated the distribution of calcium-binding protein immunoreactivity in the human inferior colliculus and found complementary or mutually exclusive localisations of parvalbumin versus calbindin D-28k and calretinin staining. The central nucleus of the inferior colliculus but not the surrounding regions contained parvalbumin-positive neuronal somata and fibres. Calbindin-positive neurons and fibres were concentrated in the dorsal aspect of the central nucleus and in structures surrounding it: the dorsal cortex, the lateral lemniscus, the ventrolateral nucleus, and the intercollicular region. In the dorsal cortex, labelling of calbindin and calretinin revealed four distinct layers.Thus, calcium-binding protein reactivity reveals in the human inferior colliculus distinct neuronal populations that are anatomically segregated. The different calcium-binding protein-defined subdivisions may belong to parallel auditory pathways that were previously demonstrated in non-human primates, and they may constitute a first indication of parallel processing in human subcortical auditory structures.

Targeted Small Business Link, Fall 2011

Targeted Small Business news from the Iowa Economic Development Authority

The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal.

Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum-sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen-responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen-limited growth in P. aeruginosa biofilms.

L'insertion sociale plurielle des femmes cadres supérieurs en Suisse : contribution à l'étude du bien-être subjectif au quotidien : approche qualitative intégrative

RESUME« L'insertion sociale plurielle des femmes cadres supérieurs en Suisse. Contribution à l'étude du bien-être subjectif au quotidien. Approche intégrative qualitative. »Depuis une quarantaine d'années, nombreux sont les travaux qui étudient les relations entre les différents milieux de vie au quotidien et, plus particulièrement, l'impact de leurs articulations singulières sur la santé. Nous y identifions deux axes principaux : l'un aborde ce phénomène selon une perspective de « conflit travail-famille » en termes de « stress », l'autre se focalise davantage sur la promotion du « bien-être » au travers d'une approche d'« équilibre travail-vie ». Or, l'ensemble de ces recherches considère comme «pathogènes» les désajustements, les contradictions et les tensions vécus au quotidien. Selon cette tendance, le « bien-être » apparaît comme un état d'équilibre ultime indépendant du contexte de vie du sujet. Toutefois, peu de recherches portent sur la dimension située du bien-être dans son rapport à l'activité concrète au sein des milieux deNotre étude s'intéresse à cette question auprès des femmes cadres supérieurs, selon une perspective critique et développementale en psychologie de la santé (Lyons & Chamberlain, 2006 ; Santiago-Delefosse, 2002,2011 ; Malrieu, 1989 ; Vygotski, 1985). En effet, cette population constitue un terrain privilégié pour comprendre le sens donné à l'activité à partir des contraintes, responsabilités et demandes perçues dans des contextes parfois contradictoires, et pour analyser le rôle de ces derniers dans un bien-être subjectif.En cohérence avec notre positionnement théorique, nous avons mené des entretiens qualitatifs focalisés sur l'activité quotidienne auprès de 20 femmes, et ceci en deux temps (T1-T2) (40 entretiens). Les résultats issus des analyses du contenu des discours permettent de définir le vécu de l'articulation des milieux de vie chez nos participantes, selon trois axes à la fois interdépendants et autonomes. Chaque axe se définit par une série de supports spécifiques jouant un rôle structurant dans leur bien-être subjectif. Ainsi, le premier axe se caractérise par des supports de maîtrise subjective, ainsi que par l'appropriation de contraintes sociales et corporelles, selon un rythme de l'activité soutenu. Le deuxième s'accompagne de supports qui favorisent la prise de distance au travers du relâchement du rythme et du lâcher prise de la maîtrise, par la création d'espaces personnels et sociaux « pour soi ». Enfin, le troisième porte sur le positionnement de soi par rapport à autrui en termes de « personnalisation ».Construits en rapport à la corporéité et à autrui au sein de contextes spécifiques, ces différents supports prennent leur sens au sein d'un système de pratiques global, unique pour chaque femme. Selon cette conception critique, le bien-être subjectif chez les femmes cadres se définit comme le fruit d'un processus mouvant issu des tensions vécues entre les trois axes de l'articulation des milieux de vie. Il est par conséquent social, corporel et psychologique.Nos résultats ouvrent des perspectives de recherche et d'intervention, notamment en santé et travail. Ces ouvertures sont orientées vers une approche intégrative en psychologie de la santé, c'est-à-dire, de la prise en compte dans des questions de santé et de bien-être du processus de construction du sujet en relation à son insertion sociale plurielle.ABSTRACT« Plural social participation among women senior managers in Switzerland. Contribution to the study of subjective well-being in everyday life. An integrative and qualitativeapproach.»For the last forty years, a large body of literature has studied the relationships between different social realms in everyday life and, more particularly, the impact of their singular intertwinements with health. We identify two main trends : The first one focuses on this phenomenon through the « work-family conflict » perspective in terms of « stress » whilst the second one is more concerned by the promotion of « well-being » through a « work-life balance » approach. However, both of these trends consider disadjustments, contradictions and tensions in everyday life as « pathogenic ». According to this conception, « well- being » appears as an ultimate state of balance which is indépendant from the subject's life context. Nevertheless, few studies have examined the situated dimension of well-being in its link to concrete activity in social realms.Our research is concerned with this issue among women senior managers from a critical and developmental perspective in Health Psychology (Lyons & Chamberlain, 2006 ; Santiago-Delefosse, 2002, 2011 ; Malrieu, 1989 ; Vygotski, 1985). In fact, this population represents a favourable field so as to study : how the meaning of daily activity is constructed accross different and often conflictive social realms ; to understand the many ways in which this population deals with perceived constraints, responsibilities and requests, and to analyse the role of situated plural activity in subjective well-being.Consitent with our theoretical framework, we have designed a qualitative method. We have conducted two-time (T1-T2) interviews with 20 women by focusing on their daily activity (40 interviews). The Thematic Content Analysis reveals that four different social realms are articulated among our participants through three main axes, which are at the same time interdependent and autonomous. Each one of these axis is defined by a certain number of specific supports that play an important role in these women's subjective well- being. The first axis is concerned by several supports that signify a « feeling of control » along with the appropriation of social and body constraints by means of a rapid pace of activity. The second one regards the use of supports that contribute to « put things into perspective » by means of the slowing down of the pace of activity and through letting go of the feeling of control. This mechanism includes the creation of personal and social spaces of « one's own ». The third axis is defined by the positioning of the self in regard to others through a process of « personnalization ».Developed in specific contexts through the intertwinnements between the body and social others, supports belonging to these axes acquire significance and meaning on the basis of their relationship to a global system of activities of which they are part. However, this articulation is uniquely defined for each participant. According to this critical approach, subjective well-being among women senior managers emerges as a meaningful and changing process, situated in a plural social context. This is, it appears as the result of conflictual interactions defining the three different axis that we have identified. Subjective well-being is hence a social, embodied and psychological phenomena that is closely linked to the articulation of different social realms. Our findings open new research and practice perspectives, especially concerning health and work issues. These perspectives convey an integrative approach in Health Psychology by considering health and well-being by taking into account the process of construction of the subject in regard to his or her plural social participation.

Role of the GNOM gene in Arabidopsis apical-basal patterning--From mutant phenotype to cellular mechanism of protein action.

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.

Noise-Driven Mechanism for Pattern Formation

We extend the mechanism for noise-induced phase transitions proposed by Ibañes et al. [Phys. Rev. Lett. 87, 020601 (2001)] to pattern formation phenomena. In contrast with known mechanisms for pure noise-induced pattern formation, this mechanism is not driven by a short-time instability amplified by collective effects. The phenomenon is analyzed by means of a modulated mean field approximation and numerical simulations.

Targeted Small Business Link, Winter 2011

Targeted small business news from the Iowa Economic Development Authority's Targeted Small Business Program

Bacterial-induced protection against allergy through a novel multi-component immunoregulatory mechanism

Airborne microbial products have been reported to promote immune responses that suppress asthma, yet how these beneficial effects take place remains controversial and poorly understood. We have found that pulmonary exposure with the bacterium Escherichia coli leads to a suppression of allergic airway inflammation, characterized by reduced airway-hyperresponsiveness, eosinophilia and cytokine production by T cells in the lung. This immune modulation was neither mediated by the induction of a Th1 response nor regulatory T cells; was dependent on TLR-4 but did not involve TLR-desensitization. Dendritic cell migration to the draining lymph nodes and subsequent activation of T cells was unaffected by prior exposure to E.coli indicating that the immunomodulation was limited to the lung environment. In non-treated control mice ovalbumin was primarily presented by airway CD11b+ CD11c+ DCs expressing high levels of MHC class II molecules whilst the DCs in E.coli-treated mice displayed a less activated phenotype and had impaired antigen presentation capacity. Consequently, in situ Th2 cytokine production by ovalbuminspecific effector T cells recruited to the airways was significantly reduced. The suppression of airways hyper responsiveness was mediated through the recruitment of IL-17-producing gd-T cells; however, the suppression of dendritic cells and T cells was mediated through a distinct mechanism that could not be overcome by the local administration of activated dendritic cells, or by the in vivo administration of TNF-alpha. Taken together, these data reveal a novel multi-component immunoregulatory pathway that acts to protect the airways from allergic inflammation.

Giant step bunching from self-organized coalescence of SrRuO3 islands

Step bunching develops in the epitaxy of SrRuO3 on vicinal SrTiO3(001) substrates. We have investigated the formation mechanisms and we show here that step bunching forms by lateral coalescence of wedgelike three-dimensional islands that are nucleated at substrate steps. After coalescence, wedgelike islands become wider and straighter with growth, forming a self-organized network of parallel step bunches with altitudes exceeding 30 unit cells, separated by atomically flat terraces. The formation mechanism of step bunching in SrRuO3, from nucleated islands, radically differs from one-dimensional models used to describe bunching in semiconducting materials. These results illustrate that growth phenomena of complex oxides can be dramatically different to those in semiconducting or metallic systems.