A two-phase composite in simple shear: Effective mechanical anisotropy development and localization potential

We present a combined shape and mechanical anisotropy evolution model for a two-phase inclusion-bearing rock subject to large deformation. A single elliptical inclusion embedded in a homogeneous but anisotropic matrix is used to represent a simplified shape evolution enforced on all inclusions. The mechanical anisotropy develops due to the alignment of elongated inclusions. The effective anisotropy is quantified using the differential effective medium (DEM) approach. The model can be run for any deformation path and an arbitrary viscosity ratio between the inclusion and host phase. We focus on the case of simple shear and weak inclusions. The shape evolution of the representative inclusion is largely insensitive to the anisotropy development and to parameter variations in the studied range. An initial hardening stage is observed up to a shear strain of gamma = 1 irrespective of the inclusion fraction. The hardening is followed by a softening stage related to the developing anisotropy and its progressive rotation toward the shear direction. The traction needed to maintain a constant shear rate exhibits a fivefold drop at gamma = 5 in the limiting case of an inviscid inclusion. Numerical simulations show that our analytical model provides a good approximation to the actual evolution of a two-phase inclusion-host composite. However, the inclusions develop complex sigmoidal shapes resulting in the formation of an S-C fabric. We attribute the observed drop in the effective normal viscosity to this structural development. We study the localization potential in a rock column bearing varying fraction of inclusions. In the inviscid inclusion case, a strain jump from gamma = 3 to gamma = 100 is observed for a change of the inclusion fraction from 20% to 33%.

Development and validation of a gas chromatography-negative chemical ionization tandem mass spectrometry method for the determination of ethyl glucuronide in hair and its application to forensic toxicology.

Ethyl glucuronide (EtG) is a minor and direct metabolite of ethanol. EtG is incorporated into the growing hair allowing retrospective investigation of chronic alcohol abuse. In this study, we report the development and the validation of a method using gas chromatography-negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS) for the quantification of EtG in hair. EtG was extracted from about 30 mg of hair by aqueous incubation and purified by solid-phase extraction (SPE) using mixed mode extraction cartridges followed by derivation with perfluoropentanoic anhydride (PFPA). The analysis was performed in the selected reaction monitoring (SRM) mode using the transitions m/z 347-->163 (for the quantification) and m/z 347-->119 (for the identification) for EtG, and m/z 352-->163 for EtG-d(5) used as internal standard. For validation, we prepared quality controls (QC) using hair samples taken post mortem from 2 subjects with a known history of alcoholism. These samples were confirmed by a proficiency test with 7 participating laboratories. The assay linearity of EtG was confirmed over the range from 8.4 to 259.4 pg/mg hair, with a coefficient of determination (r(2)) above 0.999. The limit of detection (LOD) was estimated with 3.0 pg/mg. The lower limit of quantification (LLOQ) of the method was fixed at 8.4 pg/mg. Repeatability and intermediate precision (relative standard deviation, RSD%), tested at 4 QC levels, were less than 13.2%. The analytical method was applied to several hair samples obtained from autopsy cases with a history of alcoholism and/or lesions caused by alcohol. EtG concentrations in hair ranged from 60 to 820 pg/mg hair.

IL-7/anti-IL-7 mAb complexes restore T cell development and induce homeostatic T Cell expansion without lymphopenia.

IL-7, a member of the common gamma-chain family of cytokines, is essential for B and T lymphocyte development and homeostasis of mature T cell subsets. Thus, naive and memory T cells are both dependent on IL-7 for survival and homeostatic proliferation under lymphopenic conditions. In line with prior findings with IL-2, we show in this study that the biological activity of IL-7 in vivo is greatly increased by association with anti-IL-7 mAb. Under in vivo conditions, IL-7/mAb complexes displayed 50- to 100-fold higher activity than free IL-7 and induced massive expansion of pre-B cells. IL-7/mAb complexes also increased thymopoiesis in normal mice and restored thymopoeisis in IL-7-deficient mice. For mature T cells, IL-7/mAb complexes induced marked homeostatic proliferation of both naive and memory CD4(+) and CD8(+) cell subsets even under normal T cell-replete conditions. Finally, IL-7/mAb complexes were able to enhance the magnitude of the primary response of Ag-specific naive CD8(+) cells. The strong stimulatory activity of IL-7/mAb complexes could be useful for treatment of immunodeficiency and cancer.

The role of the c-myc gene in pigment cell development and homeostasis

Résumé La dérégulation de c-Myc est un événement fréquent de la transformation cellulaire. Une régulation positive de cette oncoprotéine a été démontrée dans divers mélanomes cutanés primaires et métastatiques et est associée à un pronostic défavorable (Grover et al., 1996; Zhuang et al., 2008). c-Myc est considéré comme une molécule centrale impliquée dans plusieurs processus de l'homéostasie cellulaire. En raison de sa contribution importante dans la progression tumorale, la fonction de c-Myc a été étudiée intensément. Cependant nous connaissons peu le rôle de ce facteur de transcription dans l'embryogenèse et dans la spécification tissulaire. Un déficit total de c-Myc pendant l'embryogenèse conduit à la mort embryonnaire avant 10.5 jours de gestation. Cette mort est causée par de multiples imperfections du développement touchant la taille de l'embryon, le coeur, le péricarde, le tube neural et les cellules sanguines (Davis et al., 1993; Trumpp et al., 2001). Récemment, il a été montré que la plupart de ces anomalies sont secondaires et résultent d'une insuffisance du placenta dans les embryons c-myc-/- (Dubois et al., 2008). Sachant que c-Myc est important dans la maintenance des lignées de la crête neurale (Wei et al., 2007), nous nous sommes intéressés au rôle de c-Myc dans le développement des cellules pigmentaires et à leur homéostasie après la naissance. Un allèle floxé de c-myc (Trumpp et al., 2001) a été utilisé pour supprimer ce gène spécifiquement dans la lignée mélanocytaire à l'aide d'une souris transgénique Tyr::Cre (Delmas et al., 2003). L'ablation des deux allèles de c-myc dans les mélanocytes des souris c-myccKO conduit au phénotype de grisonnement des poils, observé directement après la naissance et associé à une diminution du nombre de mélanocytes dans le bulbe des follicules pileux. Les cellules pigmentaires restantes expriment les marqueurs mélanogéniques (Tyr, TRP-1, Dct and MITF) et semblent être fonctionnelles puisqu'elles peuvent produire et transférer la mélanine. De plus, la capacité de prolifération des mélanocytes déficients en c-Myc dans le bulbe des follicules pileux ne semble pas être affectée chez les nouveaux-nés. Les cellules souches mélanocytaires sont présentes, mais en nombre réduit, dans le bulge des follicules pileux à la fin de la morphogenèse chez les souris c-myccKO âgées de huit jours. Ces cellules sont maintenues sans changement durant le premier cycle pileux (vérifié à l'âge de trente jours), ce qui sous-entend que la fonction de c-Myc n'est pas nécessaire pour ce processus. Ceci explique pourquoi, en supposant que des cellules souches mélanocytaires fonctionnelles sont présentes dans la peau, nous n'observons pas de dilution de couleur de la robe liée à l'âge. Cependant, la présence de ces cellules souches mélanocytaires dans la peau c-myccKO ne suffit pas à assurer une quantité normale de mélanocytes différenciés dans le bulbe des follicules pileux. Cette population de cellules pigmentaires matures est sévèrement affectée par la suppression de c-Myc, ce qui contribue amplement au phénotype de grisonnement des poils. De plus, c-Myc paraît être important pour le développement des mélanocytes. Ainsi, le nombre de mélanoblastes diminue dans les embryons c-myccKO à partir du douzième jour de gestation. A treize jours de gestation, au stade où les mélanoblastes pénètrent dans l'épiderme et prolifèrent, les mélanoblastes déficients en c-Myc ne s'adaptent pas aux signaux de prolifération et se retrouvent en nombre réduit dans l'épiderme. Finalement, nous nous sommes intéressés, au rôle de N-Myc, un homologue proche de c-Myc, dans la lignée mélanocytaire. Nos expériences ont montré que. N-Myc était superflu pour le développement et l'homéostasie des mélanocytes, une seule copie du gène c-myc étant suffisante pour maintenir une pigmentation normale de la robe des souris c-mycc-myccKO/+~N_ myccKO/KO. Cependant, le rôle essentiel de N-Myc dans la maintenance des cellules mélanocytaires précurseurs apparaît lorsque c-Myc est absent, puisque la suppression simultanée des deux Myc résulte en une perte complète de la coloration de la robe. Ceci implique la présence d'un mécanisme compensatoire entre c- et N-Myc dans la lignée mélanocytaire, avec un rôle prédominant de c-Myc. Summary Deregulation of c-Myc is known to be a common event in cellular transformation. Upregulation of this oncoprotein was shown in a variety of primary and metastatic cutaneous melanomas and has been associated with a poor prognosis (Grover et al., 1996; Zhuang et al., 2008). c-myc is seen as a central molecule involved in many aspects of cellular homeostasis. c-Myc function has been intensively studied mostly because of its significant contribution to tumour progression. However little is known on the role of this transcription factor in embryogenesis and tissue specification. Complete loss of c-Myc during embryogenesis results in embryonic death before E10.5 due to multiple developmental defects including embryonic size, heart, pericardium, neural tube and blood cells (Davis et al., 1993; Trumpp et al., 2001). Recently it was discovered that most of these abnormalities are secondary and results of placental insufficiency in c-Myc-/- embryos (Dubois et al., 2008). Here, we focused on the role of c-Myc in pigment cell development and homeostasis after birth, knowing that c-Myc is important in the maintenance of neural crest lineages (Wei et al., 2007). A floxed allele of c-Myc (Trumpp et al., 2001) was used to specifically delete this gene in the melanocyte lineage using Tyr::Cre transgenic mice (Delmas et al., 2003). Removal of both c-Myc alleles in melanocytes of c-MyccKO mouse led to the grey hair phenotype which is seen directly after birth and was associated with a decrease in the melanocyte number in the bulb of the hair follicle. The remaining population of pigment cells express melanogenic markers (Tyr, TRP-1, Dct and MITF) and seem functionally normal since they can produce and transfer melanin. Furthermore proliferation capacity of c-Myc deficient melanocytes in the bulb of hair follicle seems not to be affected in newborn animals. Melanocyte stem cells (MSCs) are present but reduced in numbers in the bulge of the hair follicle at the end of morphogenesis in 8 days old c-MyccKO mice. These cells are maintained through the first hair cycle (as verified at P30) without any further changes, suggesting that c-Myc function is not required for this process. This explains why we did not detect any agerelated coat color dilution, assuming a presence of functional MSCs in the skin. Importantly, presence of MSCs in c-MyccKO skin was not sufficient for assuring a normal number of differentiated melanocytes in the bulb of the hair follicle. This population of mature pigmented cells is severely affected upon c-myc deletion thus largely contributing to the grey hair phenotype. Moreover, c-Myc appears to be important for melanocyte development. Thus, melanoblast number is affected in c-MyccKO embryos day 12 of gestation onwards. At E13.5, when melanoblasts enter the epidermis and proliferate, c-myc deficient melanoblasts failed to adapt to proliferation signals and are therefore reduced in number in the epidermis. Finally, we addressed the role of N-Myc, a closest homologue of c-Myc, in the melanocyte lineage. In these experiments, N-Myc was dispensable for melanocyte development and homeostasis, and even one copy of the c-myc gene was sufficient to maintain normal coat color pigmentation in c-mycc-mycCKO/+ ,N-myccKO/KO mice. However the crucial role of N-Myc in maintenance of melanocyte precursor cells became apparent when c-myc is eliminated since simultaneous deletion of both Myc results in complete loss of coat color pigmentation. This suggests compensatory mechanisms between c- and N-Myc with a predominant role of c-Myc in melanocyte lineage.

Characterization of BMP signaling in breast development and tumorigenesis

Résumé L'influence des hormones reproductives sur le développement du cancer du sein a été établie au travers de nombreuse études épidémiologiques. Nous avons précédemment démontré que le gène Wnt-4 est un médiateur essentiel de la progestérone dans le développement lobulo-alvéolaire de l'épithélium mammaire. De plus, le rôle de la voie de signalisation Wnt dans la tumorigénèse de la glande mammaire mutine est largement établi. Pour comprendre sa fonction dans le cancer du sein, nous avons activée cette voie en surexprimant le gène Wnt-1 dans des cellules épithéliales primaires de sein, au moyen d'un rétrovirus. Ceci a conduit à la transformation oncogénique de ces cellules et à l'obtention d'un modèle de carcinogénèse du sein dénommé Wnt-1 HMEC. L'analyse de l'expression des gènes induits par la surexpression de Wnt-1 dans ces cellules, a permis d'identifier les gènes BMP4 et 7. Alors que des analyses de RT-PCR ont montré leur forte expression dans les cellules Wnt-1-HMECs, la présence d'une grande quantité de la protéine BMP7 a été constatée dans les tumeurs dérivées de ces cellules. L'importante phosphorylation des Smad 1, 5, S dans les Wnt-1 HMECs indique l'activation de la voie BMP, possiblement due à la stimulation ce celle-ci par BMP7. L'activation de la voie Wnt par la ß-Caténine, conduit à la transcription de BMP7, identifiant ainsi ce gène comme un gène cible de la voie canonique. La pertinence de nos observations a par ailleurs été confirmée par le fait que BMP7 est surexprimé dans les tumeurs de seins humains. Afin d'élucider la fonction de la voie BMP dans le sein, nous avons utilisé le modèle mutin. L'expression du gène BMP7 dans les souris transgéniques MMTV Wnt-1 s'est avérée élevée, démontrant qu'il est aussi un gène cible de la voie Wnt in-vivo. L'expression de l'ARN messager .codant pour la protéine BMP7 est induite lors du développement lobulo-alvéolaire, qui se fait sous l'influence de la progestérone et de Wnt-4. Ensemble, ces observations corroborent le fait qu'une stimulation avec de la progestérone suffit à induire la transcription du gène dans les 24h. Nos résultats coïncident d'autre part avec le fait que BMP7 est exprimé dans la couche myoépithéliale de l'épithélium où la voie Wnt est activée. L'analyse de souris reportrices de l'activité de la voie BMP, suggère une activation dans la couche luminale de l'épithélium durant tout le développement de la glande mammaire. Curieusement, cette même voie est active dans le mésenchyme lors de la mammogénèse embryonnaire. Finalement, nos analyses d'immunofluorescence démontrent la capacité de prolifération des cellules ayant activé BMP, ainsi que leur nette ségrégation d'avec les cellules exprimant le récepteur à la progestérone. Nos résultats démontrent que le gène BMP7 est un gène cible de la voie Wnt canonique dans le sein. Son expression dans la couche myoépitheliale est induite par Wnt-4, lui-même sécrété par les cellules luminales sensibles à la progestérone. La sécrétion de la protéine BMP7 conduit finalement à l'activation de la voie BMP dans les cellules négatives pour le récepteur à la progestérone. Abstract Epidemiological studies highlight the repetitive exposure to circulating progesterone as a major risk in the development of breast cancer. Work in our laboratory showed that Wnt-4 is an essential mediator of progesterone-driven side-branch formation, while Wnt signaling has long been established as strongly oncogenic in the mouse mammary gland. To address the role of Wnt in breast tumorigenesis we activated the pathway in primary human breast epithelial cells by means of refroviral Wnt-1 expression. This resulted in a Wnt1-induced breast carcinogenesis model, being referred to as Wnt-1-HMECs. Gene expression profiling revealed the Bone Morphogenetic Protein 4 and 7 (BMP4 and 7) a mong the most upregulated gene by ectopic Wnt-1 expression in primary HMECs. RT-PCR analysis confirmed elevated BMP4 and 7 mRNA levels in Wnt-1-infected HMECs, as well as strong BMP7 expression in the tumors derived from these cells. Smad 1, 5, 8 phosphorylation was high in Wnt-1HMECs whereas below detection limit in primary HMECs suggesting that the increased expression of BMP-7 results in activation of downstream signaling. Ectopic expressíon of a stabilized form of ßcatenin in primary HMECs resulted in increased transcription of BMP-7 suggesting that it is a target of canonical Wnt signaling. The clinical relevance of our observations was confirmed by the finding of BMP7 being upregulated in human breast tumor samples. To elucidate the role of BMP ligands in the breast in-vivo, we made use of the mouse model. Expression of the BMP7 gene was found to be increased in MMTV-Wnt-1 transgenic animals, suggesting that BMP7 may also be a Wnt 1 target gene in vivo. Expression of BMP7 was upregulated in mid-pregnancy which coincides with progesterone/Wnt induced side branching. BMP7 was induced within 24 hours by progesterone. Consistent with it being a target of canonical Wnt signaling, we demonstrated preferential expression of this ligand in the myoepithelial cells, the target cells of Wnt signals. In-vivo analysis of BMP signaling using a reporter mouse revealed the activation of the pathway in the luminal layer of the epithelium throughout postnatal development. Interestingly, during embryonic mammogenesis the pathway was found to be active in the mesenchyme. Immunofluorescence studies demonstrated that cells with BMP activity can proliferate. They also revealed a clear segregation between progesterone receptor positive cells and cells with active BMP signaling. Together our observations suggest that BMP-7 is a canonical Wnt signaling target both in HMECs and in the mouse mammary gland in-vivo. It is expressed in the myoepithelium possibly in response to Wnt-4, which is secreted by steroid receptor positive cells in response to progesterone. BMP-7 in turn may impinge on lumina) epithelial cells and activate BMP signaling in PR negative cells.

A Swiss neighbourhood index of socioeconomic position: Development and Association with Mortality

Background Area-based measures of socioeconomic position (SEP) suitable for epidemiological research are lacking in Switzerland. The authors developed the Swiss neighbourhood index of SEP (Swiss-SEP). Methods Neighbourhoods of 50 households with overlapping boundaries were defined using Census 2000 and road network data. Median rent per square metre, proportion households headed by a person with primary education or less, proportion headed by a person in manual or unskilled occupation and the mean number of persons per room were analysed in principle component analysis. The authors compared the index with independent income data and examined associations with mortality from 2001 to 2008. Results 1.27 million overlapping neighbourhoods were defined. Education, occupation and housing variables had loadings of 0.578, 0.570 and 0.362, respectively, and median rent had a loading of −0.459. Mean yearly equivalised income of households increased from SFr42 000 to SFr72 000 between deciles of neighbourhoods with lowest and highest SEP. Comparing deciles of neighbourhoods with lowest to highest SEP, the age- and sex-adjusted HR was 1.38 (95% CI 1.36 to 1.41) for all-cause mortality, 1.83 (95% CI 1.71 to 1.95) for lung cancer, 1.48 (95% CI 1.44 to 1.51) for cardiovascular diseases, 2.42 (95% CI 1.94 to 3.01) for traffic accidents, 0.93 (95% CI 0.85 to 1.02) for breast cancer and 0.86 (95% CI 0.78 to 0.95) for suicide. Conclusions Developed using a novel approach to define neighbourhoods, the Swiss-SEP index was strongly associated with household income and some causes of death. It will be useful for clinical- and population-based studies, where individual-level socioeconomic data are often missing, and to investigate the effects on health of the socioeconomic characteristics of a place.

Functional plasticity of the LACK-reactive Vbeta4-Valpha8 CD4(+) T cells normally producing the early IL-4 instructing Th2 cell development and susceptibility to Leishmania major in BALB / c mice.

Early production of IL-4 by LACK-reactive Vbeta4-Valpha8 CD4(+) T cells instructs aberrant Th2 cell development and susceptibility to Leishmania major in BALB / c mice. This was demonstrated using Vbeta4(+)-deficient BALB / c mice as a result of chronic infection with MMTV (SIM), a mouse mammary tumor virus expressing a Vbeta4-specific superantigen. The early IL-4 response was absent in these mice which develop a Th1 response to L. major. Here, we studied the functional plasticity of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells using BALB/ c mice inoculated with L. major shortly after infection with MMTV (SIM), i. e. before deletion of Vbeta4(+) cells. These mice fail to produce the early IL-4 response to L. major and instead exhibit an IFN-gamma response that occurs within LACK-reactive Vbeta4-Valpha8 CD4(+) T cells. Neutralization of IFN-gamma restores the production of IL-4 by these cells. These data suggest that the functional properties of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells are not irreversibly fixed.

Hes1 is a critical but context-dependent mediator of canonical Notch signaling in lymphocyte development and transformation.

Although canonical Notch signaling regulates multiple hematopoietic lineage decisions including T cell and marginal zone B cell fate specification, the downstream molecular mediators of Notch function are largely unknown. We showed here that conditional inactivation of Hes1, a well-characterized Notch target gene, in adult murine bone marrow (BM) cells severely impaired T cell development without affecting other Notch-dependent hematopoietic lineages such as marginal zone B cells. Competitive mixed BM chimeras, intrathymic transfer experiments, and in vitro culture of BM progenitors on Delta-like-expressing stromal cells further demonstrated that Hes1 is required for T cell lineage commitment, but dispensable for Notch-dependent thymocyte maturation through and beyond the beta selection checkpoint. Furthermore, our data strongly suggest that Hes1 is essential for the development and maintenance of Notch-induced T cell acute lymphoblastic leukemia. Collectively, our studies identify Hes1 as a critical but context-dependent mediator of canonical Notch signaling in the hematopoietic system.

Aging preferentially affects molecular pathways implicated in development and disease of myelinating glial cells

The central and peripheral nervous systems are involved in multiple age-dependent neurological deficits that are often attributed to alterations in function of myelinating glial cells. However, the molecular events that underlie the age-related decline of glial cell function are unknown. We used Schwann cells as a model to study biological processes affected in glial cells by aging. We comprehensively profiled gene expression of the Schwann cellrich mouse sciatic nerve throughout life, from day of birth until senescence (840 days of age). We combined the aging data with the microarray transcriptional data obtained using nerves isolated from Schwann cell-specific neuropathy-inducing mutants MPZCre/+/Lpin1fE2−3/fE2−3 , MPZCre/+/ScapfE1/fE1 and Pmp22-null mice. The majority of age related transcripts were also affected in the analyzed mouse models of neuropathy (54.4%) and in development (59.5%) indicating a high level of overlapping in implicated molecular pathways. We observed that compared to peripheral nerve development, dynamically changing expression profiles in aging have opposite (anticorrelated) orientation while they copy the orientation of transcriptional changes observed in analyzed neuropathy models. Subsequent clustering and biological annotation of dynamically changing transcripts revealed that the processes most significantly deregulated in aging include inflammatory/immune response and lipid biosynthesis/metabolism. Importantly, the changes in these pathways were also observed in myelinated oligodendrocyte-rich optic nerves of aged mice, albeit with lower magnitude. This observation suggests that similar biological processes are affected in aging glial cells in central and peripheral nervous systems, however with different dynamics. Our data, which provide the first comprehensive comparison of molecular changes in glial cells in three distinct biological conditions comprising development, aging and disease, provide not only a new inside into the molecular alterations underlying neural system aging but also identify target pathways for potential therapeutic approaches to prevent or delay complications associated with age-related and inherited forms of neuropathies. *Current address: Department of Physiology, UCSF, San Francisco, CA, USA.

Guideline adaptation: an approach to enhance efficiency in guideline development and improve utilisation.

BACKGROUND: Developing and updating high-quality guidelines requires substantial time and resources. To reduce duplication of effort and enhance efficiency, we developed a process for guideline adaptation and assessed initial perceptions of its feasibility and usefulness. METHODS: Based on preliminary developments and empirical studies, a series of meetings with guideline experts were organised to define a process for guideline adaptation (ADAPTE) and to develop a manual and a toolkit made available on a website (http://www.adapte.org). Potential users, guideline developers and implementers, were invited to register and to complete a questionnaire evaluating their perception about the proposed process. RESULTS: The ADAPTE process consists of three phases (set-up, adaptation, finalisation), 9 modules and 24 steps. The adaptation phase involves identifying specific clinical questions, searching for, retrieving and assessing available guidelines, and preparing the draft adapted guideline. Among 330 registered individuals (46 countries), 144 completed the questionnaire. A majority found the ADAPTE process clear (78%), comprehensive (69%) and feasible (60%), and the manual useful (79%). However, 21% found the ADAPTE process complex. 44% feared that they will not find appropriate and high-quality source guidelines. DISCUSSION: A comprehensive framework for guideline adaptation has been developed to meet the challenges of timely guideline development and implementation. The ADAPTE process generated important interest among guideline developers and implementers. The majority perceived the ADAPTE process to be feasible, useful and leading to improved methodological rigour and guideline quality. However, some de novo development might be needed if no high quality guideline exists for a given topic.

Improving globally distributed software development and support processes - A workflow view

We propose a new approach and related indicators for globally distributed software support and development based on a 3-year process improvement project in a globally distributed engineering company. The company develops, delivers and supports a complex software system with tailored hardware components and unique end-customer installations. By applying the domain knowledge from operations management on lead time reduction and its multiple benefits to process performance, the workflows of globally distributed software development and multitier support processes were measured and monitored throughout the company. The results show that the global end-to-end process visibility and centrally managed reporting at all levels of the organization catalyzed a change process toward significantly better performance. Due to the new performance indicators based on lead times and their variation with fixed control procedures, the case company was able to report faster bug-fixing cycle times, improved response times and generally better customer satisfaction in its global operations. In all, lead times to implement new features and to respond to customer issues and requests were reduced by 50%.

Cryo-electron microscopy of vitreous sections of native biological cells and tissues : methodology and applications

Résumé L'eau est souvent considérée comme une substance ordinaire puisque elle est très commune dans la nature. En fait elle est la plus remarquable de toutes les substances. Sans l'eau la vie sur la terre n'existerait pas. L'eau représente le composant majeur de la cellule vivante, formant typiquement 70 à 95% de la masse cellulaire et elle fournit un environnement à d'innombrables organismes puisque elle couvre 75% de la surface de terre. L'eau est une molécule simple faite de deux atomes d'hydrogène et un atome d'oxygène. Sa petite taille semble en contradiction avec la subtilité de ses propriétés physiques et chimiques. Parmi celles-là, le fait que, au point triple, l'eau liquide est plus dense que la glace est particulièrement remarquable. Malgré son importance particulière dans les sciences de la vie, l'eau est systématiquement éliminée des spécimens biologiques examinés par la microscopie électronique. La raison en est que le haut vide du microscope électronique exige que le spécimen biologique soit solide. Pendant 50 ans la science de la microscopie électronique a adressé ce problème résultant en ce moment en des nombreuses techniques de préparation dont l'usage est courrant. Typiquement ces techniques consistent à fixer l'échantillon (chimiquement ou par congélation), remplacer son contenu d'eau par un plastique doux qui est transformé à un bloc rigide par polymérisation. Le bloc du spécimen est coupé en sections minces (d’environ 50 nm) avec un ultramicrotome à température ambiante. En général, ces techniques introduisent plusieurs artefacts, principalement dû à l'enlèvement d'eau. Afin d'éviter ces artefacts, le spécimen peut être congelé, coupé et observé à basse température. Cependant, l'eau liquide cristallise lors de la congélation, résultant en une importante détérioration. Idéalement, l'eau liquide est solidifiée dans un état vitreux. La vitrification consiste à refroidir l'eau si rapidement que les cristaux de glace n'ont pas de temps de se former. Une percée a eu lieu quand la vitrification d'eau pure a été découverte expérimentalement. Cette découverte a ouvert la voie à la cryo-microscopie des suspensions biologiques en film mince vitrifié. Nous avons travaillé pour étendre la technique aux spécimens épais. Pour ce faire les échantillons biologiques doivent être vitrifiés, cryo-coupées en sections vitreuse et observées dans une cryo-microscope électronique. Cette technique, appelée la cryo- microscopie électronique des sections vitrifiées (CEMOVIS), est maintenant considérée comme étant la meilleure façon de conserver l'ultrastructure de tissus et cellules biologiques dans un état très proche de l'état natif. Récemment, cette technique est devenue une méthode pratique fournissant des résultats excellents. Elle a cependant, des limitations importantes, la plus importante d'entre elles est certainement dû aux artefacts de la coupe. Ces artefacts sont la conséquence de la nature du matériel vitreux et le fait que les sections vitreuses ne peuvent pas flotter sur un liquide comme c'est le cas pour les sections en plastique coupées à température ambiante. Le but de ce travail a été d'améliorer notre compréhension du processus de la coupe et des artefacts de la coupe. Nous avons ainsi trouvé des conditions optimales pour minimiser ou empêcher ces artefacts. Un modèle amélioré du processus de coupe et une redéfinitions des artefacts de coupe sont proposés. Les résultats obtenus sous ces conditions sont présentés et comparés aux résultats obtenus avec les méthodes conventionnelles. Abstract Water is often considered to be an ordinary substance since it is transparent, odourless, tasteless and it is very common in nature. As a matter of fact it can be argued that it is the most remarkable of all substances. Without water life on Earth would not exist. Water is the major component of cells, typically forming 70 to 95% of cellular mass and it provides an environment for innumerable organisms to live in, since it covers 75% of Earth surface. Water is a simple molecule made of two hydrogen atoms and one oxygen atom, H2O. The small size of the molecule stands in contrast with its unique physical and chemical properties. Among those the fact that, at the triple point, liquid water is denser than ice is especially remarkable. Despite its special importance in life science, water is systematically removed from biological specimens investigated by electron microscopy. This is because the high vacuum of the electron microscope requires that the biological specimen is observed in dry conditions. For 50 years the science of electron microscopy has addressed this problem resulting in numerous preparation techniques, presently in routine use. Typically these techniques consist in fixing the sample (chemically or by freezing), replacing its water by plastic which is transformed into rigid block by polymerisation. The block is then cut into thin sections (c. 50 nm) with an ultra-microtome at room temperature. Usually, these techniques introduce several artefacts, most of them due to water removal. In order to avoid these artefacts, the specimen can be frozen, cut and observed at low temperature. However, liquid water crystallizes into ice upon freezing, thus causing severe damage. Ideally, liquid water is solidified into a vitreous state. Vitrification consists in solidifying water so rapidly that ice crystals have no time to form. A breakthrough took place when vitrification of pure water was discovered. Since this discovery, the thin film vitrification method is used with success for the observation of biological suspensions of. small particles. Our work was to extend the method to bulk biological samples that have to be vitrified, cryosectioned into vitreous sections and observed in cryo-electron microscope. This technique is called cryo-electron microscopy of vitreous sections (CEMOVIS). It is now believed to be the best way to preserve the ultrastructure of biological tissues and cells very close to the native state for electron microscopic observation. Since recently, CEMOVIS has become a practical method achieving excellent results. It has, however, some sever limitations, the most important of them certainly being due to cutting artefacts. They are the consequence of the nature of vitreous material and the fact that vitreous sections cannot be floated on a liquid as is the case for plastic sections cut at room temperature. The aim of the present work has been to improve our understanding of the cutting process and of cutting artefacts, thus finding optimal conditions to minimise or prevent these artefacts. An improved model of the cutting process and redefinitions of cutting artefacts are proposed. Results obtained with CEMOVIS under these conditions are presented and compared with results obtained with conventional methods.