Assembly and mechanism of bacterial twin-arginine translocation systems

The bacterial twin-arginine translocation (Tat) pathway is able to export pre-folded cofactor containing proteins across the cytoplasmic membrane. Tat substrates bear cleavable N-terminal signal peptides that are characterized by the presence of a critical and highly conserved twin-arginine motif which lends the Tat pathway its name. In Escherichia coli and many other Gram-negative bacteria, three integral membrane proteins: TatA, TatB and TatC are essential for Tat-dependent translocation. In contrast Bacillus subtilis possesses a simpler TatAC system which lacks the TatB component. In E. coli the TatA protein assembles into homo-oligomeric complexes that vary considerably in size. The TatA proteins found in B. subtilis do not exhibit the same degree of heterogeneity and this suggested mechanistic differences between the Tat pathways of Gram-negative and Gram-positive bacteria. How the Tat system works is still poorly understood, and the work presented in this thesis sought to gain insights into the assembly and mechanism of E. coli and B. subtilis Tat pathways. This work focused on the study of two previously uncharacterized components: the E. coli TatA paralog TatE subunit and B. subtilis TatAc subunit. In this thesis the purification and characterization of E. coli TatE complexes is reported. Using analytical gel filtration chromatography, blue-native gelelectrophoresis (BN-PAGE) and single-particle analysis of purified TatE complexes, it was found that the TatE complexes are more discrete than the highly heterogeneous TatA complexes. This finding, together with the ability of TatE to support the translocation of the 90-kDa TorA protein, suggested alternative translocation models in which single TatE complexes do not contribute the bulk of the translocation channel, similar to the B. subtilis model. In addition, co-purification and BN-PAGE experiments demonstrated for the first time that TatE interacts with TatA to form TatAE mixed complexes in the membrane, and reveals a completely novel form of Tat complex that might be functionally significant. A soluble population of TatE was also identified in E. coli cell extracts, and phase separation experiments using Triton X-114 suggested it may be mis-localized. In a separate set of studies, the ability of the B. subtilis TatAc protein to form active translocases in combination with the B. subtilis TatCd or TatCy proteins was investigated for the first time. The TatAcCd and TatAcCy mixed translocases were able to translocate several E. coli Tat substrates including, TorA, AmiA and AmiC. Finally BN-PAGE and gel filtration chromatography showed that the TatAcCd and TatAcCy complexes were significantly smaller than the previously described E. coli TatABC substrate-binding complex

Teaching and Care: Cripping Fieldwork in Teacher Education

Providing teacher candidates early and ongoing opportunities to learn their profession by participating in school settings is often posed as a way to improve their preparedness for becoming teachers. Two problems of “fieldwork,” however, are the limited access to settings in which inclusive education is practiced and the milieu of special education in the US that emphasizes ableist assertions of independence, support, and conventional notions of care, especially for youth characterized as intellectually disabled. We present an overview of the establishment of a “cripped” fieldwork experience for early program teacher candidates enrolled in a required undergraduate course. By engaging in qualitative narrative analysis of candidates’ journals, we report preliminary findings on evolving notions of care related to disability and education in self-reported field-based learning

Pavement Information Modelling (PIM): Best Practice to Build a Digital Repository for Roads Asset Management

The application of BIM methods and tools plays a key role in transportation infrastructure asset management. Road pavements represent one of the main components of the asset, which greatly influences safety and quality of service for users. The work presented herein exploited the potentialities of BIM processes and methods for management of road pavement structures. The specific goal was to define best practice for development of a methodological framework for Pavement Information Modelling (PIM). The starting point of the process was the identification of the specific BIM use, as intended by Kreider and Messner [1]. In this case, the BIM use identified concerned the 3rd (3D), 4th (4D), and 5th (5D) dimensions of BIM. The adopted approach had the aim to define the steps to build PIM based on geometrical and structural parameters to be used as a database for different kinds of maintenance strategies. Within this context, the main objectives of the study can be summarized as follows:(1)Define the steps to develop a PIM including all the relevant information to be stored for management purposes, from data collection to data restitution,(2)Define a best practice for the integration among BIM tools and road pavement management methods in order to obtain a digital repository for predictive maintenance strategies,(3)Define a planning and cost database for the different technologies and materials involved in the different maintenance strategies.From a practical point of view, the methodological framework was divided into three main categories (Figure 1) dealing with data: (i) data collection and input definition, which includes the analysis of available data and the BIM tools to be used to develop specific workflows; (ii) data processing, by dividing the workflows and related tasks in sub-sections for the fulfilment of the previously enounced objectives; (iii) data output, by defining the final result of each workflow.QC 20230706</p

chemical and spectroscopic characterization of insoluble and soluble humic acid fractions at different ph values

Background: Humic acids (HA) are organic molecules with complex structure and function and variable properties. They are insoluble in strongly acid pH conditions. At present, it is not clear how much is the amount of HA in solution at the pH of natural soils nor are known the characteristics of the different soluble fractions and their possible association with the inorganic phase of soil. The scope of this work was to characterize the soluble and insoluble fractions obtained by acidifying Na humate solution at pH values 3, 5, and 7 and to compare these fractions with the HA obtained at pH 1. At each pH, the precipitate and the soluble fractions were separated and characterized by elemental analysis, total acidity and carboxylic group content, infrared, and 13 C NMR and 1 H NMR spectroscopy. Results: The HA fraction insoluble at pH 1 had a high acid group content and aromaticity but a low content of Oand N-containing groups. At pH 3, a fraction with nearly the same characteristics was obtained. At pH 1 and pH 3, the inorganic phase bound to the insoluble humic material was largely constituted by clay minerals and some Al and Fe hydroxides. The soluble fractions at pH values 1 and 3 were very poor and they were composed of a silica gel-like phase associated with polar organic material rich in carboxylic and metal-carboxylate groups. At pH values 5 and 7, only a small fraction of the Na humates precipitated. The fractions remaining in the solution were mainly composed of organic material particularly rich in aromatic and aliphatic groups, while the inorganic phase contained phyllosilicates. The fractions insoluble at pH values 5 and 7 contained a large amount of inorganic material that consisted mainly of phyllosilicates. Conclusions: The soluble fractions obtained at pH values 5 and 7 represent the humic component that in environmental situations would be dissolved in the soil solution. Our findings could be very useful for a more detailed investigation into the way HA influence plant metabolism under environmental-like conditions, both as regards pH conditions and interaction with the mineral fraction

Evaluation of the anti-rutting potential of polymer-modified binders by means of creep-recovery shear tests

In the experimental study reported in this paper a creep-recovery shear test method was used to evaluate the anti-rutting potential of different polymer-modified bituminous binders. The effects of several factors related to modification were investigated, such as polymer type (SBS, Styrene-Butadiene-Styrene vs. EVA, Ethylene-Vinyl-Acetate), composition (styrene content), structure (linear vs. radial SBS) and dosage (3-6% by weight of the base bitumen). The effect of short-term aging was also considered by comparing binder response before and after treatment with the Rolling Thin-Film Oven Test (RTFOT). Following previous work on the development of the test protocol, experimental results were analyzed by referring to shear modulus curves G(t) and to permanent compliance (JP), obtained by dividing residual strain at the end of the unloading phase by the stress applied during creep loading. Results indicated the effectiveness of the proposed method in discriminating between the behavior of the different polymer-modified binders and in capturing the effects caused by the factors considered in the investigation. Reliable rankings of the binders were established and were explained by referring to the specific behavior of employed modifiers

Investigating cohesive healing of asphalt binders by means of a dissipated energy approach

The paper reports the results of an experimental investigation in which the cohesive healing properties of different types of asphalt binder were evaluated by means of the dissipated energy ratio approach. A specifically designed testing methodology was proposed which involves comparing the response of binders subjected to continuous oscillatory shear loading carried out without rest periods and with single rest periods introduced at predefined levels of damage A rheological parameter (Healing Ratio) was introduced to quantify the magnitude of healing occurring during rest time and to rank the consequent healing potential of binders. Obtained results indicate that the investigated binders did not completely recover their original fatigue resistance after rest time, confirming the existence of some intrinsic irreversible damage, the amount of which depends on the total damage experienced before load removal. Experimental results also indicate that healing performance of binders can be significantly enhanced by polymer modification. Keywords: Healing, Fatigue, Dissipated energy ratio, Asphalt binder, Polymer modificatio

In Order to Escort Her

In Order To Escort Her is a thirty-five minute hybrid-documentary in which Lila, my great-aunt, converses with her niece, my mother, about various supernatural creature encounters and spirit visitations from relatives. This is interspersed with a “visitation” from another close relative, communicating from a different time and space dimension. By weaving together supernatural stories and matter-of-fact accounts of resilience and survival, a magical world is created where boundaries between the material and the invisible realms blur. The main thematic concerns of this film are love, care, presence in joy amidst terror, and the journey of the soul