Evolving perspectives on business and books: practitioner implications

The research subject of this Portfolio of Essays is my ‘apparatus of thought’ in the context of my professional development. I have examined theory and the application of theory to a professional life. I analyse how my use of theory has changed and enabled my professional development. Each of the three Essays takes a different perspective and applies the theories of adult mental development, business and firm growth as an analytical framework. Using Kegan’s theories of Adult Mental Development as an ‘apparatus of thought’, Essay One is my reflection on my professional development in the light of my training, education, and development. I describe how changes in my ‘apparatus of thought’, my meaning-making system, have enabled me to develop my professional practice. Exposure to the theories of Adult Mental Development have also enabled me to understand how my development has precipitated and necessitated my career changes. In Essay Two, moving from military aviation to book publishing was a major career change for me, enabled by a change in my meaning-making system. In the context of my professional development, I sought to change my practice so that I could make a more meaningful contribution to the firm. To achieve this, I directed my reading towards a deeper understanding of the nature of the firm and the impact of industrial change on the firm. Using the context of my professional environment in Essay Three, I show how my use of theory has developed. I describe how I sought to change working practices in the firm and discuss the impact this initiative had on my professional self. I use the ‘data of my experience’ to examine my theory of the business from a Penrosian perspective. The Penrosian perspective coupled with my exposure to theories of Adult Mental Development and the effects of a transformational education process enabled me to transition to a leadership role with an international online publisher.

The role of transformative thinking and practice in a property entrepreneur's response to the funding crisis since 2008

This portfolio of exploration explores the role of transformative thinking and practice in a property entrepreneur’s response to the financial crisis which swept over Ireland from 2008. The complexity of this challenge and the mental capacity to meet its demands is at the core of the exploration. This inquiry emerged from the challenges the financial crisis presented to my values, beliefs, assumptions, and theories, i.e. the interpretive lens through which I make meaning of my experiences. Given the issue identified, this inquiry is grounded in aspects of theories of constructive developmental psychology, applied developmental science, and philosophy. Integrating and linking these elements to business practice is the applied element of the Portfolio. As the 2008 crisis unfolded I realised I was at the limits of my way of knowing. I came to understand that the underlying structure of a way of knowing is the ‘subject-object relationship’ i.e. what a way of knowing can reflect upon, look at, have perspective on, in other words, make object, as against what is it embedded in, attached to, identified within, or subject to. My goal became enhancing my awareness of how I made meaning and how new insights, which would transform a way of knowing, are created. The focus was on enhancing my practice. This Portfolio is structured into three essays. Essay One reported on my self-reflection and external evaluation out of which emerged my developmental goals. In Essay Two I undertake a reading for change programme in which different meaning making systems were confronted in order to challenge me as a meaning maker. Essay Three reported on my experiment which concerned the question whether it was possible for me as a property entrepreneur, and for others alike, to retain bank finance in the face of the overwhelming objective of the bank to deleverage their balance sheet of property loans. The output of my research can be grouped into General Developmental and Specific Business Implications. Firstly I address those who are interested in a transformational-based response to the challenges of operating in the property sector in Ireland during a crisis. I outline the apparatus of thought that I used to create insight, and thus transform how I thought, these are Awareness, Subject-object separation, Exploring other’s perspectives from the position of incompleteness, Dialectical thinking and Collingwood’s Questioning activity. Secondly I set out my learnings from the crisis and their impact on entrepreneurial behaviour and the business of property development. I identify ten key insights that have emerged from leading a property company through the crisis. Many of these are grounded in common sense, however, in my experience these were, to borrow Shakespeare’s words, “More honor'd in the breach than the observance” in pre Crisis Ireland. Finally I set out a four-step approach for forging a strategy. This requires my peer practitioners to identify (i) what they are subject to, (ii) Assess the Opportunity or challenge in a Systemic Context, (iii) Explore Multiple Perspectives on the opportunity or Challenge with an Orientation to change how you know and (iv) Using the Questioning Activity to create Knowledge. Based on my experience I conclude that transformative thinking and practice is a key enabler for a property entrepreneur, in responding to a major collapse of traditional (bank debt) funding.

A corpus-driven error analysis of the oral and written production of Leaving Certificate Spanish learners

The Leaving Certificate (LC) is the national, standardised state examination in Ireland necessary for entry to third level education – this presents a massive, raw corpus of data with the potential to yield invaluable insight into the phenomena of learner interlanguage. With samples of official LC Spanish examination data, this project has compiled a digitised corpus of learner Spanish comprised of the written and oral production of 100 candidates. This corpus was then analysed using a specific investigative corpus technique, Computer-aided Error Analysis (CEA, Dagneaux et al, 1998). CEA is a powerful apparatus in that it greatly facilitates the quantification and analysis of a large learner corpus in digital format. The corpus was both compiled and analysed with the use of UAM Corpus Tool (O’Donnell 2013). This Tool allows for the recording of candidate-specific variables such as grade, examination level, task type and gender, therefore allowing for critical analysis of the corpus as one unit, as separate written and oral sub corpora and also of performance per task, level and gender. This is an interdisciplinary work combining aspects of Applied Linguistics, Learner Corpus Research and Foreign Language (FL) Learning. Beginning with a review of the context of FL learning in Ireland and Europe, I go on to discuss the disciplinary context and theoretical framework for this work and outline the methodology applied. I then perform detailed quantitative and qualitative analyses before going on to combine all research findings outlining principal conclusions. This investigation does not make a priori assumptions about the data set, the LC Spanish examination, the context of FLs or of any aspect of learner competence. It undertakes to provide the linguistic research community and the domain of Spanish language learning and pedagogy in Ireland with an empirical, descriptive profile of real learner performance, characterising learner difficulty.

Politics, Power, PISA: a genealogy of mathematics education policy at second level in Ireland at the beginning of the 21st century

This thesis traces a genealogy of the discourse of mathematics education reform in Ireland at the beginning of the twenty first century at a time when the hegemonic political discourse is that of neoliberalism. It draws on the work of Michel Foucault to identify the network of power relations involved in the development of a single case of curriculum reform – in this case Project Maths. It identifies the construction of an apparatus within the fields of politics, economics and education, the elements of which include institutions like the OECD and the Government, the bureaucracy, expert groups and special interest groups, the media, the school, the State, state assessment and international assessment. Five major themes in educational reform emerge from the analysis: the arrival of neoliberal governance in Ireland; the triumph of human capital theory as the hegemonic educational philosophy here; the dominant role of OECD/PISA and its values in the mathematics education discourse in Ireland; the fetishisation of western scientific knowledge and knowledge as commodity; and the formation of a new kind of subjectivity, namely the subjectivity of the young person as a form of human-capital-to-be. In particular, it provides a critical analysis of the influence of OECD/PISA on the development of mathematics education policy here – especially on Project Maths curriculum, assessment and pedagogy. It unpacks the arguments in favour of curriculum change and lays bare their ideological foundations. This discourse contextualises educational change as occurring within a rapidly changing economic environment where the concept of the State’s economic aspirations and developments in science, technology and communications are reshaping both the focus of business and the demands being put on education. Within this discourse, education is to be repurposed and its consequences measured against the paradigm of the Knowledge Economy – usually characterised as the inevitable or necessary future of a carefully defined present.

Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells.

The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission-fusion dynamics in pluripotent ESCs.

Souvenirs of conquest: Israeli occupations as tourist events

It is perhaps self-evident to suggest that military conquest shares something with tourism because both involve encounters with "strange" landscapes and people. Thus it may not surprise that the former sometimes borrows rhetorical strategies from the latter - strategies for rendering the strange familiar or for translating threatening images into benign ones. There have been numerous studies of this history of borrowing. Scholars have considered how scenes of battle draw tourist crowds, how soldiers' ways of seeing can resemble those of leisure travelers, how televised wars have been visually structured as tourist events (e.g., the 2003 U.S. invasion of Iraq), and how the spoils of war can function as a body of souvenirs. These lines of inquiry expand our understanding of tourism as a field of cultural practices and help us to rethink the parameters of militarism and warfare by suggesting ways they are entangled with everyday leisure practices. © 2008 Cambridge University Press.

Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.

Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.

Evaluation of Altered Kras Codon Bias and NOS Inhibition During Lung Tumorigenesis

The small GTPases HRAS, NRAS and KRAS are mutated in approximately one-third of all human cancers, rendering the proteins constitutively active and oncogenic. Lung cancer is the leading cause of cancer deaths worldwide, and more than 20% of human lung cancers harbor mutations in RAS, with 98% of those occurring in the KRAS isoform. While there have been many advances in the understanding of KRAS–driven lung tumorigenesis, it remains a therapeutic challenge. To further this understanding and assess novel approaches for treatment, I have investigated two aspects of Kras–driven tumorigenesis in the lung:

(I) Despite nearly identical protein sequences, the three RAS proto-oncogenes exhibit divergent codon usage. Of the three isoforms, KRAS contains the most rare codons resulting in lower levels of KRAS protein expression relative to HRAS and NRAS. To determine the consequences of rare codon bias during de novo tumorigenesis, we created a knock-in Krasex3op mouse in which synonymous mutations in exon 3 converted codons from rare to common. These mice had reduced tumor burden and fewer oncogenic mutations in the Krasex3op allele following carcinogen exposure. The reduction in tumorigenesis appeared to be a product of rare codons affecting both the oncogenic and non–oncogenic alleles. Converting rare codons to common codons yielded a more potent oncogenic allele that promoted growth arrest and enhanced tumor suppression by the non-oncogenic allele. Thus, rare codons play an integral role in Kras tumorigenesis.

(II) Lung cancer patients exhale higher levels of NO and iNOS-/- mice are resistant to chemically induced lung tumorigenesis. I hypothesize that NO promotes Kras–driven lung adenocarcinoma, and NOS inhibition may decrease Kras–driven lung tumorigenesis. To test this hypothesis, I assessed efficacy of the NOS inhibitor L–NAME in a genetically engineered mouse model of Kras-driven lung adenocarcinoma. Adenoviral Cre recombinase was delivered into the lungs intranasally, resulting in expression of oncogenic KrasG12D and dominant-negative Trp53R172H in lung epithelial cells. L–NAME treatment was provided in the water and continued until survival endpoints. In this model, L–NAME treatment decreased tumor growth and prolonged survival. These data establish a potential clinical role for NOS inhibition in lung cancer treatment.

The `Ulama' and the State: Negotiating Tradition, Authority and Sovereignty in Contemporary Pakistan

This dissertation is an account of how contemporary Pakistani ulama grapple with their political realities and the Islamic state of Pakistan. The central conceptual question that scaffolds my dissertation is: How do Pakistani ulama negotiate tradition, authority and sovereignty with the Islamic Republic of Pakistan? In engaging with this issue, this dissertation employs a methodology that weds ethnography with rigorous textual analysis. The ulama that feature in this study belong to a variety of sectarian persuasions. The Sunni ulama are Deobandi and Barelvi; the Shia ulama in this study are Ithna Ashari.

In assessing the relationship between Pakistani ulama and their nation-state, I assert that the ulama's dialectical engagements with the state are best understood as a dexterous navigation between affirmation, critique, contestation and cultivation. In proposing this manner of thinking about Pakistani ulama's engagements with their state, I provide a more detailed and nuanced view of the ulama-state relationship compared to earlier works. While emphasizing Pakistani ulama's vitality and their impact on their state, this dissertation also draws attention to the manners in which the state impacts the ulama. It theorizes the subject formation of the ulama and asserts the importance of understanding the ulama as formed not just by the ethico-legal tradition in which they are trained but also by the state apparatus.

Event memory: A theory of memory for laboratory, autobiographical, and fictional events.

An event memory is a mental construction of a scene recalled as a single occurrence. It therefore requires the hippocampus and ventral visual stream needed for all scene construction. The construction need not come with a sense of reliving or be made by a participant in the event, and it can be a summary of occurrences from more than one encoding. The mental construction, or physical rendering, of any scene must be done from a specific location and time; this introduces a "self" located in space and time, which is a necessary, but need not be a sufficient, condition for a sense of reliving. We base our theory on scene construction rather than reliving because this allows the integration of many literatures and because there is more accumulated knowledge about scene construction's phenomenology, behavior, and neural basis. Event memory differs from episodic memory in that it does not conflate the independent dimensions of whether or not a memory is relived, is about the self, is recalled voluntarily, or is based on a single encoding with whether it is recalled as a single occurrence of a scene. Thus, we argue that event memory provides a clearer contrast to semantic memory, which also can be about the self, be recalled voluntarily, and be from a unique encoding; allows for a more comprehensive dimensional account of the structure of explicit memory; and better accounts for laboratory and real-world behavioral and neural results, including those from neuropsychology and neuroimaging, than does episodic memory.

Exploring the effects of image persistence in low frame rate virtual environments

© 2015 IEEE.In virtual reality applications, there is an aim to provide real time graphics which run at high refresh rates. However, there are many situations in which this is not possible due to simulation or rendering issues. When running at low frame rates, several aspects of the user experience are affected. For example, each frame is displayed for an extended period of time, causing a high persistence image artifact. The effect of this artifact is that movement may lose continuity, and the image jumps from one frame to another. In this paper, we discuss our initial exploration of the effects of high persistence frames caused by low refresh rates and compare it to high frame rates and to a technique we developed to mitigate the effects of low frame rates. In this technique, the low frame rate simulation images are displayed with low persistence by blanking out the display during the extra time such image would be displayed. In order to isolate the visual effects, we constructed a simulator for low and high persistence displays that does not affect input latency. A controlled user study comparing the three conditions for the tasks of 3D selection and navigation was conducted. Results indicate that the low persistence display technique may not negatively impact user experience or performance as compared to the high persistence case. Directions for future work on the use of low persistence displays for low frame rate situations are discussed.

Micro-Anatomical Quantitative Imaging Towards Enabling Automated Diagnosis of Thick Tissues at the Point of Care

Histopathology is the clinical standard for tissue diagnosis. However, histopathology has several limitations including that it requires tissue processing, which can take 30 minutes or more, and requires a highly trained pathologist to diagnose the tissue. Additionally, the diagnosis is qualitative, and the lack of quantitation leads to possible observer-specific diagnosis. Taken together, it is difficult to diagnose tissue at the point of care using histopathology.

Several clinical situations could benefit from more rapid and automated histological processing, which could reduce the time and the number of steps required between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is need for rapid detection of residual cancer on the surface of tumor resection specimens during excisional surgeries, which is known as intraoperative tumor margin assessment. Additionally, rapid assessment of biopsy specimens at the point-of-care could enable clinicians to confirm that a suspicious lesion is successfully sampled, thus preventing an unnecessary repeat biopsy procedure. Rapid and low cost histological processing could also be potentially useful in settings lacking the human resources and equipment necessary to perform standard histologic assessment. Lastly, automated interpretation of tissue samples could potentially reduce inter-observer error, particularly in the diagnosis of borderline lesions.

To address these needs, high quality microscopic images of the tissue must be obtained in rapid timeframes, in order for a pathologic assessment to be useful for guiding the intervention. Optical microscopy is a powerful technique to obtain high-resolution images of tissue morphology in real-time at the point of care, without the need for tissue processing. In particular, a number of groups have combined fluorescence microscopy with vital fluorescent stains to visualize micro-anatomical features of thick (i.e. unsectioned or unprocessed) tissue. However, robust methods for segmentation and quantitative analysis of heterogeneous images are essential to enable automated diagnosis. Thus, the goal of this work was to obtain high resolution imaging of tissue morphology through employing fluorescence microscopy and vital fluorescent stains and to develop a quantitative strategy to segment and quantify tissue features in heterogeneous images, such as nuclei and the surrounding stroma, which will enable automated diagnosis of thick tissues.

To achieve these goals, three specific aims were proposed. The first aim was to develop an image processing method that can differentiate nuclei from background tissue heterogeneity and enable automated diagnosis of thick tissue at the point of care. A computational technique called sparse component analysis (SCA) was adapted to isolate features of interest, such as nuclei, from the background. SCA has been used previously in the image processing community for image compression, enhancement, and restoration, but has never been applied to separate distinct tissue types in a heterogeneous image. In combination with a high resolution fluorescence microendoscope (HRME) and a contrast agent acriflavine, the utility of this technique was demonstrated through imaging preclinical sarcoma tumor margins. Acriflavine localizes to the nuclei of cells where it reversibly associates with RNA and DNA. Additionally, acriflavine shows some affinity for collagen and muscle. SCA was adapted to isolate acriflavine positive features or APFs (which correspond to RNA and DNA) from background tissue heterogeneity. The circle transform (CT) was applied to the SCA output to quantify the size and density of overlapping APFs. The sensitivity of the SCA+CT approach to variations in APF size, density and background heterogeneity was demonstrated through simulations. Specifically, SCA+CT achieved the lowest errors for higher contrast ratios and larger APF sizes. When applied to tissue images of excised sarcoma margins, SCA+CT correctly isolated APFs and showed consistently increased density in tumor and tumor + muscle images compared to images containing muscle. Next, variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was further tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. The results indicate that SCA+CT can accurately delineate APFs in heterogeneous tissue, which is essential to enable automated and rapid surveillance of tissue pathology.

Two primary challenges were identified in the work in aim 1. First, while SCA can be used to isolate features, such as APFs, from heterogeneous images, its performance is limited by the contrast between APFs and the background. Second, while it is feasible to create mosaics by scanning a sarcoma tumor bed in a mouse, which is on the order of 3-7 mm in any one dimension, it is not feasible to evaluate an entire human surgical margin. Thus, improvements to the microscopic imaging system were made to (1) improve image contrast through rejecting out-of-focus background fluorescence and to (2) increase the field of view (FOV) while maintaining the sub-cellular resolution needed for delineation of nuclei. To address these challenges, a technique called structured illumination microscopy (SIM) was employed in which the entire FOV is illuminated with a defined spatial pattern rather than scanning a focal spot, such as in confocal microscopy.

Thus, the second aim was to improve image contrast and increase the FOV through employing wide-field, non-contact structured illumination microscopy and optimize the segmentation algorithm for new imaging modality. Both image contrast and FOV were increased through the development of a wide-field fluorescence SIM system. Clear improvement in image contrast was seen in structured illumination images compared to uniform illumination images. Additionally, the FOV is over 13X larger than the fluorescence microendoscope used in aim 1. Initial segmentation results of SIM images revealed that SCA is unable to segment large numbers of APFs in the tumor images. Because the FOV of the SIM system is over 13X larger than the FOV of the fluorescence microendoscope, dense collections of APFs commonly seen in tumor images could no longer be sparsely represented, and the fundamental sparsity assumption associated with SCA was no longer met. Thus, an algorithm called maximally stable extremal regions (MSER) was investigated as an alternative approach for APF segmentation in SIM images. MSER was able to accurately segment large numbers of APFs in SIM images of tumor tissue. In addition to optimizing MSER for SIM image segmentation, an optimal frequency of the illumination pattern used in SIM was carefully selected because the image signal to noise ratio (SNR) is dependent on the grid frequency. A grid frequency of 31.7 mm-1 led to the highest SNR and lowest percent error associated with MSER segmentation.

Once MSER was optimized for SIM image segmentation and the optimal grid frequency was selected, a quantitative model was developed to diagnose mouse sarcoma tumor margins that were imaged ex vivo with SIM. Tumor margins were stained with acridine orange (AO) in aim 2 because AO was found to stain the sarcoma tissue more brightly than acriflavine. Both acriflavine and AO are intravital dyes, which have been shown to stain nuclei, skeletal muscle, and collagenous stroma. A tissue-type classification model was developed to differentiate localized regions (75x75 µm) of tumor from skeletal muscle and adipose tissue based on the MSER segmentation output. Specifically, a logistic regression model was used to classify each localized region. The logistic regression model yielded an output in terms of probability (0-100%) that tumor was located within each 75x75 µm region. The model performance was tested using a receiver operator characteristic (ROC) curve analysis that revealed 77% sensitivity and 81% specificity. For margin classification, the whole margin image was divided into localized regions and this tissue-type classification model was applied. In a subset of 6 margins (3 negative, 3 positive), it was shown that with a tumor probability threshold of 50%, 8% of all regions from negative margins exceeded this threshold, while over 17% of all regions exceeded the threshold in the positive margins. Thus, 8% of regions in negative margins were considered false positives. These false positive regions are likely due to the high density of APFs present in normal tissues, which clearly demonstrates a challenge in implementing this automatic algorithm based on AO staining alone.

Thus, the third aim was to improve the specificity of the diagnostic model through leveraging other sources of contrast. Modifications were made to the SIM system to enable fluorescence imaging at a variety of wavelengths. Specifically, the SIM system was modified to enabling imaging of red fluorescent protein (RFP) expressing sarcomas, which were used to delineate the location of tumor cells within each image. Initial analysis of AO stained panels confirmed that there was room for improvement in tumor detection, particularly in regards to false positive regions that were negative for RFP. One approach for improving the specificity of the diagnostic model was to investigate using a fluorophore that was more specific to staining tumor. Specifically, tetracycline was selected because it appeared to specifically stain freshly excised tumor tissue in a matter of minutes, and was non-toxic and stable in solution. Results indicated that tetracycline staining has promise for increasing the specificity of tumor detection in SIM images of a preclinical sarcoma model and further investigation is warranted.

In conclusion, this work presents the development of a combination of tools that is capable of automated segmentation and quantification of micro-anatomical images of thick tissue. When compared to the fluorescence microendoscope, wide-field multispectral fluorescence SIM imaging provided improved image contrast, a larger FOV with comparable resolution, and the ability to image a variety of fluorophores. MSER was an appropriate and rapid approach to segment dense collections of APFs from wide-field SIM images. Variables that reflect the morphology of the tissue, such as the density, size, and shape of nuclei and nucleoli, can be used to automatically diagnose SIM images. The clinical utility of SIM imaging and MSER segmentation to detect microscopic residual disease has been demonstrated by imaging excised preclinical sarcoma margins. Ultimately, this work demonstrates that fluorescence imaging of tissue micro-anatomy combined with a specialized algorithm for delineation and quantification of features is a means for rapid, non-destructive and automated detection of microscopic disease, which could improve cancer management in a variety of clinical scenarios.

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

Silica polymer bonding of stressed silica grains: An early growth of intergranular tensile strength

© 2015 Elsevier Ltd. All rights reserved.Laboratory tests on microscale are reported in which millimeter-sized amorphous silica cubes were kept highly compressed in a liquid environment of de-ionized water solutions with different silica ion concentrations for up to four weeks. Such an arrangement simulates an early evolution of bonds between two sand grains stressed in situ. In-house designed Grain Indenter-Puller apparatus allowed measuring strength of such contacts after 3-4 weeks. Observations reported for the first time confirm a long-existing hypothesis that a stressed contact with microcracks generates silica polymers, forming a bonding structure between the grains on a timescale in the order of a few weeks. Such structure exhibits intergranular tensile force at failure of 1-1.5 mN when aged in solutions containing silica ion concentrations of 200-to 500-ppm. The magnitude of such intergranular force is 2-3 times greater than that of water capillary force between the same grains.

ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation.

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.