The Francesca Da Rimini episode in drama.

Thesis (M.A.)--Boston Universit

Modulators of transit peptide activity in targeting and translocation of precursors into plastides : the mature domain, lipids and Toc-Tic components

The import of nuclear-encoded precursor proteins into chloroplasts occurs via a cleavable, N-terminal targeting sequence known as the transit peptide. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SStp) and three Cterminal deletion mutants (Δ52, Δ67, and Δ74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by inhibition of import of 35S-prSSU to show that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Import studies demonstrated that prSSU and Δ52 were processed and accumulated within the chloroplast, whereas Δ67 and Δ74 were rapidly degraded. Import-competent proteins were also able to induce anion channel closure for PIRAC (Protein Import Related Iconic C,/u\u3ehannel). Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not affect the mean duration of channel closure. In addition the same proteins, as well as the precursors to the 33 and 23 kD subunits of the oxygen evolving complex of photosystem II, prOE33 and prOE23, respectively, were used in liposome dye release assays to investigate the interaction between precursors and chloroplast outer membrane lipids. Chloroplast precursor proteins do interact with liposomes mimicking the chloroplast outer envelope lipids through a process mediated through the transit peptide and requiring the presence of non-bilayer forming lipids and anionic lipids. The interaction of the transit peptide with liposomes involves electrostatic interactions between the peptide and the anionic lipids in the liposome. From this study, two additional precursors were shown to be membrane active, prOE33 and prOE23 Finally to investigate the in vivo activity of prSSU transit peptide, we designed green fluorescent protein (GPP) chimeras with the precursor to the small subunit of Rubisco (prSSU-GFP). GFP alone or prSSU-GFP could be expressed in a transient assay in onion epidermal cells. These experiments lay the groundwork for expression of mutant precursors fused to GFP in either onion epidermal to explore the effect mutations in the transit peptide have on targeting and import relative to the wildtype precursor

Root Hair Single Cell Type Specific Profiles of Gene Expression and Alternative Polyadenylation Under Cadmium Stress

Transcriptional networks are tightly controlled in plant development and stress responses. Alternative polyadenylation (APA) has been found to regulate gene expression under abiotic stress by increasing the heterogeneity at mRNA 3′-ends. Heavy metals like cadmium pollute water and soil due to mining and industry applications. Understanding how plants cope with heavy metal stress remains an interesting question. The Arabidopsis root hair was chosen as a single cell model to investigate the functional role of APA in cadmium stress response. Primary root growth inhibition and defective root hair morphotypes were observed. Poly(A) tag (PAT) libraries from single cell types, i.e., root hair cells, non-hair epidermal cells, and whole root tip under cadmium stress were prepared and sequenced. Interestingly, a root hair cell type-specific gene expression under short term cadmium exposure, but not related to the prolonged treatment, was detected. Differentially expressed poly(A) sites were identified, which largely contributed to altered gene expression, and enriched in pentose and glucuronate interconversion pathways as well as phenylpropanoid biosynthesis pathways. Numerous genes with poly(A) site switching were found, particularly for functions in cell wall modification, root epidermal differentiation, and root hair tip growth. Our findings suggest that APA plays a functional role as a potential stress modulator in root hair cells under cadmium treatment

Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport

Liquid Chromatography Mass Spectrometry-Based Proteomics: Biological and Technological Aspects

Mass spectrometry-based proteomics has become the tool of choice for identifying and quantifying the proteome of an organism. Though recent years have seen a tremendous improvement in instrument performance and the computational tools used, significant challenges remain, and there are many opportunities for statisticians to make important contributions. In the most widely used "bottom-up" approach to proteomics, complex mixtures of proteins are first subjected to enzymatic cleavage, the resulting peptide products are separated based on chemical or physical properties and analyzed using a mass spectrometer. The two fundamental challenges in the analysis of bottom-up MS-based proteomics are as follows: (1) Identifying the proteins that are present in a sample, and (2) Quantifying the abundance levels of the identified proteins. Both of these challenges require knowledge of the biological and technological context that gives rise to observed data, as well as the application of sound statistical principles for estimation and inference. We present an overview of bottom-up proteomics and outline the key statistical issues that arise in protein identification and quantification.Comment: Published in at http://dx.doi.org/10.1214/10-AOAS341 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org

Normalization and missing value imputation for label-free LC-MS analysis

Shotgun proteomic data are affected by a variety of known and unknown systematic biases as well as high proportions of missing values. Typically, normalization is performed in an attempt to remove systematic biases from the data before statistical inference, sometimes followed by missing value imputation to obtain a complete matrix of intensities. Here we discuss several approaches to normalization and dealing with missing values, some initially developed for microarray data and some developed specifically for mass spectrometry-based data

Validity and sensitivity of a human cranial finite element model: Implications for comparative studies of biting performance

Finite element analysis (FEA) is a modelling technique increasingly used in anatomical studies investigating skeletal form and function. In the case of the cranium this approach has been applied to both living and fossil taxa to (for example) investigate how form relates to function or infer diet or behaviour. However, FE models of complex musculoskeletal structures always rely on simplified representations because it is impossible completely to image and represent every detail of skeletal morphology, variations in material properties and the complexities of loading at all spatial and temporal scales. The effects of necessary simplifications merit investigation. To this end, this study focuses on one aspect, model geometry, which is particularly pertinent to fossil material where taphonomic processes often destroy the finer details of anatomy or in models built from clinical CTs where the resolution is limited and anatomical details are lost. We manipulated the details of a finite element (FE) model of an adult human male cranium and examined the impact on model performance. First, using digital speckle interferometry, we directly measured strains from the infraorbital region and frontal process of the maxilla of the physical cranium under simplified loading conditions, simulating incisor biting. These measured strains were then compared with predicted values from FE models with simplified geometries that included modifications to model resolution, and how cancellous bone and the thin bones of the circum-nasal and maxillary regions were represented. Distributions of regions of relatively high and low principal strains and principal strain vector magnitudes and directions, predicted by the most detailed FE model, are generally similar to those achieved in vitro. Representing cancellous bone as solid cortical bone lowers strain magnitudes substantially but the mode of deformation of the FE model is relatively constant. In contrast, omitting thin plates of bone in the circum-nasal region affects both mode and magnitude of deformation. Our findings provide a useful frame of reference with regard to the effects of simplifications on the performance of FE models of the cranium and call for caution in the interpretation and comparison of FEA results

Root Hair Single Cell Type Specific Profiles of Gene Expression and Alternative Polyadenylation Under Cadmium Stress

Transcriptional networks are tightly controlled in plant development and stress responses. Alternative polyadenylation (APA) has been found to regulate gene expression under abiotic stress by increasing the heterogeneity at mRNA 3′-ends. Heavy metals like cadmium pollute water and soil due to mining and industry applications. Understanding how plants cope with heavy metal stress remains an interesting question. The Arabidopsis root hair was chosen as a single cell model to investigate the functional role of APA in cadmium stress response. Primary root growth inhibition and defective root hair morphotypes were observed. Poly(A) tag (PAT) libraries from single cell types, i.e., root hair cells, non-hair epidermal cells, and whole root tip under cadmium stress were prepared and sequenced. Interestingly, a root hair cell type-specific gene expression under short term cadmium exposure, but not related to the prolonged treatment, was detected. Differentially expressed poly(A) sites were identified, which largely contributed to altered gene expression, and enriched in pentose and glucuronate interconversion pathways as well as phenylpropanoid biosynthesis pathways. Numerous genes with poly(A) site switching were found, particularly for functions in cell wall modification, root epidermal differentiation, and root hair tip growth. Our findings suggest that APA plays a functional role as a potential stress modulator in root hair cells under cadmium treatment