T cell division and death are segregated by mutation of TCRbeta chain constant domains.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Development of Self-Cleaning Box Culvert Design – Phase II, TR-619, 2013

Culverts are common means to convey flow through the roadway system for small streams. In general, larger flows and road embankment heights entail the use of multibarrel culverts (a.k.a. multi-box) culverts. Box culverts are generally designed to handle events with a 50-year return period, and therefore convey considerably lower flows much of the time. While there are no issues with conveying high flows, many multi-box culverts in Iowa pose a significant problem related to sedimentation. The highly erosive Iowa soils can easily lead to the situation that some of the barrels can silt-in early after their construction, becoming partially filled with sediment in few years. Silting can reduce considerably the capacity of the culvert to handle larger flow events. Phase I of this Iowa Highway Research Board project (TR-545) led to an innovative solution for preventing sedimentation. The solution was comprehensively investigated through laboratory experiments and numerical modeling aimed at screening design alternatives and testing their hydraulic and sediment conveyance performance. Following this study phase, the Technical Advisory Committee suggested to implement the recommended sediment mitigation design to a field site. The site selected for implementation was a 3-box culvert crossing Willow Creek on IA Hwy 1W in Iowa City. The culvert was constructed in 1981 and the first cleanup was needed in 2000. Phase II of the TR 545 entailed the monitoring of the site with and without the selfcleaning sedimentation structure in place (similarly with the study conducted in laboratory). The first monitoring stage (Sept 2010 to December 2012) was aimed at providing a baseline for the operation of the as-designed culvert. In order to support Phase II research, a cleanup of the IA Hwy 1W culvert was conducted in September 2011. Subsequently, a monitoring program was initiated to document the sedimentation produced by individual and multiple storms propagating through the culvert. The first two years of monitoring showed inception of the sedimentation in the first spring following the cleanup. Sedimentation continued to increase throughout the monitoring program following the depositional patterns observed in the laboratory tests and those documented in the pre-cleaning surveys. The second part of Phase II of the study was aimed at monitoring the constructed self-cleaning structure. Since its construction in December 2012, the culvert site was continuously monitored through systematic observations. The evidence garnered in this phase of the study demonstrates the good performance of the self-cleaning structure in mitigating the sediment deposition at culverts. Besides their beneficial role in sediment mitigation, the designed self-cleaning structures maintain a clean and clear area upstream the culvert, keep a healthy flow through the central barrel offering hydraulic and aquatic habitat similar with that in the undisturbed stream reaches upstream and downstream the culvert. It can be concluded that the proposed self-cleaning structural solution “streamlines” the area upstream the culvert in a way that secures the safety of the culvert structure at high flows while producing much less disturbance in the stream behavior compared with the current constructive approaches.

The contact region between three domains of the extracellular loop of ASIC1a is critical for channel function.

Acid-sensing ion channels are members of the epithelial Na(+) channel/degenerin family. They are neuronal nonvoltage-gated Na(+) channels that are activated by extracellular acidification. In this study, we investigated the role of a highly conserved region of the extracellular part of ASIC1a that forms the contact between the finger domain, the adjacent beta-ball, and the upper palm domain in ASIC1a. The finger domain contributes to the pH-dependent gating and is linked via this contact zone to the rest of the protein. We found that mutation to Cys of residues in this region led to decreased channel expression and current amplitudes. Exposure of the engineered Cys residues to Cd(2+) or to charged methane thiosulfonate sulfhydryl reagents further reduced current amplitudes. This current inhibition was not due to changes in acid-sensing ion channel pH dependence or unitary conductance and was likely due to a decrease of the probability of channel opening. For some mutants, the effect of sulfhydryl reagents depended on the pH of exposure in the range 7.4 to 6.8, suggesting that this zone undergoes conformational changes during inactivation. Our study identifies a region in ASIC1a whose integrity is required for normal channel function.

Lipid binding by the unique and SH3 domains of c-Src suggests a new regulation mechanism

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase,SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity.

Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.

Emergence of novel domains in proteins

Proteins are composed of a combination of discrete, well-defined, sequence domains, associated with specific functions that have arisen at different times during evolutionary history. The emergence of novel domains is related to protein functional diversification and adaptation. But currently little is known about how novel domains arise and how they subsequently evolve. To gain insights into the impact of recently emerged domains in protein evolution we have identified all human young protein domains that have emerged in approximately the past 550 million years. We have classified them into vertebrate-specific and mammalian-specific groups, and compared them to older domains. We have found 426 different annotated young domains, totalling 995 domain occurrences, which represent about 12.3% of all human domains. We have observed that 61.3% of them arose in newly formed genes, while the remaining 38.7% are found combined with older domains, and have very likely emerged in the context of a previously existing protein. Young domains are preferentially located at the N-terminus of the protein, indicating that, at least in vertebrates, novel functional sequences often emerge there. Furthermore, young domains show significantly higher non-synonymous to synonymous substitution rates than older domains using human and mouse orthologous sequence comparisons. This is also true when we compare young and old domains located in the same protein, suggesting that recently arisen domains tend to evolve in a less constrained manner than older domains. We conclude that proteins tend to gain domains over time, becoming progressively longer. We show that many proteins are made of domains of different age, and that the fastest evolving parts correspond to the domains that have been acquired more recently.

Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription.

The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAP(c), a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.

Fanconi anemia and genome stability : the role of FANCM

RÉSUMÉ: Le génome de toute cellule est susceptible d'être attaqué par des agents endogènes et exogènes. Afin de préserver l'intégrité génomique, les cellules ont développé des multitudes de mécanismes. La réplication de l'ADN, une étape importante durant le cycle cellulaire, constitue un stress et présente un danger important pour l'intégrité du génome. L'anémie de Fanconi est une maladie héréditaire rare dont les protéines impliquées semblent jouer un rôle crucial dans la réponse au stress réplicatif. La maladie est associée à une instabilité chromosomique ainsi qu'à une forte probabilité de développer des cancers. Les cellules des patients souffrant de l'anémie de Fanconi sont sensibles à des agents interférant avec la réplication de l'ADN, et plus particulièrement àdes agents qui fient les deux brins d'ADN d'une manière covalente. L'anémie de Fanconi est une maladie génétiquement hétérogène. Treize protéines ont pu être identifiées. Elles semblent figurer dans une même voie de signalisation qui est aussi connue sous le nom de « FA/BRCA pathway », car un des gènes est identique au gène BRCA2 (breast cancer susceptibility gene 2). Huit protéines forment un complexe nucléaire dont l'intégrité est nécessaire à la monoubiquitination de deux autres protéines, FANCD2 et FANCI, en réponse à un stress réplicatif. A ce jour, la fonction moléculaire des protéines du « FA/BRCA pathway »reste encore mal décrite. Au début de mon travail de thèse, nous avons donc décidé de purifier les protéines du complexe nucléaire et d'étudier leurs propriétés biochimiques. Nous avons tout d'abord étudié les cinq protéines connues à l'époque qui sont FANCA, FANCC, FANCE, FANCF et FANCG. Par la suite, nous avons étendu notre étude à des protéines découvertes plus récemment, FANCL, FANCM et FAAP24, en concentrant finalement notre travail sur la caractérisation de FANCM. FANCM, contrairement aux autres protéines du complexe, est constituée de deux domaines conservés suggérant un rôle important dans le métabolisme de l'ADN. Il s'agit d'un domaine « DEAH box hélicase »situé dans la partie N-terminale et d'un domaine « ERCC4 nuclease »situé dans la partie C-terminale de la protéine. Dans cette étude, nous avons purifié avec succès la protéine FANCM entière à partir d'un système hétérologue. Nous montrons que FANCM s'attache de manière spécifique à des jonctions de Holliday et des fourches de réplication. De plus, nous démontrons que FANCM peut déplacer le point de jonction de ces structures via son domaine hélicase de manière dépendante de l'ATP. FANCM est aussi capable de dissocier de grands intermédiaires de la recombinaison, via la migration de jonctions de Holliday à travers une région d'homologie de 2.6 kb. Tous ces résultats suggèrent que FANCM peut s'attacher spécifiquement à des fourches de réplication et à des jonctions de Holliday in vitro et que son domaine hélicase est associé à une activité migratoire efficace. Nous pensons que FANCM peut avoir un rôle direct sur les intermédiaires de réplication. Ceci est en accord avec l'idée que les protéines de l'anémie de Fanconi coordonnent la réparation de l'ADN au niveau des fourches de réplication arrêtées. Nos résultats donnent une première indication quant au rôle de FANCM dans la cellule et peuvent contribuer à élucider la fonction de cette voie de signalisation peu comprise jusqu'à présent. SUMMARY: The genome of every cell is subject to a constant offence by endogenous and exogenous agents. Not surprisingly; cells have evolved a multitude of mechanisms which aim at preserving genomic integrity. A key step during the life cycle of a cell, DNA replication itself, constitutes a special danger to the integrity of the genome. The proteins defective in the rare hereditary disease Fanconi anemia (FA) are suspected to play a crucial role in the cellular response to DNA replication stress. The disease is associated with chromosomal instability and pronounced cancer susceptibility. Cells from Fanconi anemia patients are sensitive to a variety of agents which interfere with DNA replication, DNA interstrand cross-linking agents being particularly threatening to their survival. Fanconi anemia is a genetically heterogeneous disease with 13 different proteins identified, which seem to work together in a common pathway. Since one of the FA genes is identical to the breast cancer susceptibility gene BRCA2, it is also referred to as the FA/BRCA pathway. Eight proteins form a nuclear complex, whose integriry is required for the monoubiquitination of two other FA proteins, FANCD2 and FANCI, in response to DNA replication stress. Despite intensive research, the function of the FA/BRCA pathway at a molecular level has remained largely elusive so far. At the beginning of my thesis, we therefore decided to purify the proteins of the FA core complex and to investigate their biochemical properties. We started with the five proteins which were known at that time, FANCA, FANCC, FANCE, FANCF, and FACG. Later on, we extended our studies to the newly discovered proteins FANCL, FANCM, and FAAP24, and eventually focused our work on the characterisation of FANCM. In contrast to the other core complex proteins, FANCM contains two conserved domains, which point to a role in DNA metabolism: an N-terminal DEAH box helicase domain and a C-terminal ERCC4 nuclease domain. In this study, we have successfully purified full-length FANCM from a recombinant source. We show that purified FANCM binds to branched DNA molecules, such as Holliday junctions and replication forks, with high specificity and affinity. In addition, we demonstrate that FANCM can translocate the junction point of branched DNA molecules due to its helicase domain in an ATPase-dependent manner. FANCM can even dissociate large recombination intermediates, via branch migration of Holliday junctions through a 2.6 kb region of homology. Taken together, our data suggest that FANCM can specifically bind to replication forks and Holliday junctions in vitro, and that its DEAH box helicase domain is associated with a potent branch migration activity. We propose that FANCM might have a direct role in the processing of DNA replication intermediates. This is consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks. Our findings provide a first hint as to the context in which FANCM might play a role in the cell. We are optimistic that they might be key to further elucidate the function of a pathway which is far from being understood.

Cathodic Protection for a Continuous Box Girder Bridge Deck, HR-553, 1993

Bridge deck deterioration due to corrosive effect of deicers on reinforcing steel is a major problem facing many agencies. Cathodic protection is one method used to prevent reinforcing steel corrosion. The application of a direct current to the embedded reinforcing steel and a sacrificial anode protects the steel from corrosion. This 1992 project involved placing an Elgard Titanium Anode Mesh Cathodic Protection System on a bridge deck. The anode was fastened to the deck after the Class A repair-work and the overlay was placed using the Iowa Low Slump Dense Concrete System. The system was set up initially at 1 mA/sq ft.

Development of Self-Cleaning Box Culvert Designs, TR-545, 2009

The main function of a roadway culvert is to effectively convey drainage flow during normal and extreme hydrologic conditions. This function is often impaired due to the sedimentation blockage of the culvert. This research sought to understand the mechanics of sedimentation process at multi-box culverts, and develop self-cleaning systems that flush out sediment deposits using the power of drainage flows. The research entailed field observations, laboratory experiments, and numerical simulations. The specific role of each of these investigative tools is summarized below: a) The field observations were aimed at understanding typical sedimentation patterns and their dependence on culvert geometry and hydrodynamic conditions during normal and extreme hydrologic events. b) The laboratory experiments were used for modeling sedimentation process observed insitu and for testing alternative self-cleaning concepts applied to culverts. The major tasks for the initial laboratory model study were to accurately replicate the culvert performance curves and the dynamics of sedimentation process, and to provide benchmark data for numerical simulation validation. c) The numerical simulations enhanced the understanding of the sedimentation processes and aided in testing flow cases complementary to those conducted in the model reducing the number of (more expensive) tests to be conducted in the laboratory. Using the findings acquired from the laboratory and simulation works, self-cleaning culvert concepts were developed and tested for a range of flow conditions. The screening of the alternative concepts was made through experimental studies in a 1:20 scale model guided by numerical simulations. To ensure the designs are effective, performance studies were finally conducted in a 1:20 hydraulic model using the most promising design alternatives to make sure that the proposed systems operate satisfactory under closer to natural scale conditions.

Mapping of the binding domains of the axon guidance receptor Unc5B to Netrin-1 and FLRT3

Anàlisi de les interaccions, a nivell neuronal, que tenen lloc durant el desenvolupament embrionari entre el receptor Unc5B (receptor present a la membrana) i les proteïnes Netrin-1 i FLRT3 (fibronectin and leucine-rich transmembrane proteins). La interacció entre aquest receptor i Netrin-1 ha estat profundament estudiada fins al moment, de manera que es coneix que aquesta promou una repulsió en la guia d’axons durant el desenvolupament embrionari. A més, la interacció està implicada en la senyalització per a diferents processos com l’angiogènesi i la supervivència cel·lular. Per altra banda, la interacció entre neurones Unc5B positives i FLRT3, promou un retard en la migració de les neurones. Diversos estudis demostren que aquest retard en la migració està relacionat amb certes patologies mentals.

Monte Carlo simulation of a clearance box monitor used for nuclear power plant decommissioning.

When decommissioning a nuclear facility it is important to be able to estimate activity levels of potentially radioactive samples and compare with clearance values defined by regulatory authorities. This paper presents a method of calibrating a clearance box monitor based on practical experimental measurements and Monte Carlo simulations. Adjusting the simulation for experimental data obtained using a simple point source permits the computation of absolute calibration factors for more complex geometries with an accuracy of a bit more than 20%. The uncertainty of the calibration factor can be improved to about 10% when the simulation is used relatively, in direct comparison with a measurement performed in the same geometry but with another nuclide. The simulation can also be used to validate the experimental calibration procedure when the sample is supposed to be homogeneous but the calibration factor is derived from a plate phantom. For more realistic geometries, like a small gravel dumpster, Monte Carlo simulation shows that the calibration factor obtained with a larger homogeneous phantom is correct within about 20%, if sample density is taken as the influencing parameter. Finally, simulation can be used to estimate the effect of a contamination hotspot. The research supporting this paper shows that activity could be largely underestimated in the event of a centrally-located hotspot and overestimated for a peripherally-located hotspot if the sample is assumed to be homogeneously contaminated. This demonstrates the usefulness of being able to complement experimental methods with Monte Carlo simulations in order to estimate calibration factors that cannot be directly measured because of a lack of available material or specific geometries.

Nondeclarative learning in children with Specific Language Impairment: Predicting regularities in the visuomotor, phonological, and cognitive domains.

Ullman (2004) suggested that Specific Language Impairment (SLI) results from a general procedural learning deficit. In order to test this hypothesis, we investigated children with SLI via procedural learning tasks exploring the verbal, motor, and cognitive domains. Results showed that compared with a Control Group, the children with SLI (a) were unable to learn a phonotactic learning task, (b) were able but less efficiently to learn a motor learning task and (c) succeeded in a cognitive learning task. Regarding the motor learning task (Serial Reaction Time Task), reaction times were longer and learning slower than in controls. The learning effect was not significant in children with an associated Developmental Coordination Disorder (DCD), and future studies should consider comorbid motor impairment in order to clarify whether impairments are related to the motor rather than the language disorder. Our results indicate that a phonotactic learning but not a cognitive procedural deficit underlies SLI, thus challenging Ullmans' general procedural deficit hypothesis, like a few other recent studies.