Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

A Heterozygous Deletion Mutation in the Cardiac Sodium Channel Gene SCN5A with Loss- and Gain-of-Function Characteristics Manifests as Isolated Conduction Disease, without Signs of Brugada or Long QT Syndrome.

BACKGROUND: The SCN5A gene encodes for the α-subunit of the cardiac sodium channel NaV1.5, which is responsible for the rapid upstroke of the cardiac action potential. Mutations in this gene may lead to multiple life-threatening disorders of cardiac rhythm or are linked to structural cardiac defects. Here, we characterized a large family with a mutation in SCN5A presenting with an atrioventricular conduction disease and absence of Brugada syndrome. METHOD AND RESULTS: In a large family with a high incidence of sudden cardiac deaths, a heterozygous SCN5A mutation (p.1493delK) with an autosomal dominant inheritance has been identified. Mutation carriers were devoid of any cardiac structural changes. Typical ECG findings were an increased P-wave duration, an AV-block I° and a prolonged QRS duration with an intraventricular conduction delay and no signs for Brugada syndrome. HEK293 cells transfected with 1493delK showed strongly (5-fold) reduced Na(+) currents with altered inactivation kinetics compared to wild-type channels. Immunocytochemical staining demonstrated strongly decreased expression of SCN5A 1493delK in the sarcolemma consistent with an intracellular trafficking defect and thereby a loss-of-function. In addition, SCN5A 1493delK channels that reached cell membrane showed gain-of-function aspects (slowing of the fast inactivation, reduction in the relative fraction of channels that fast inactivate, hastening of the recovery from inactivation). CONCLUSION: In a large family, congregation of a heterozygous SCN5A gene mutation (p.1493delK) predisposes for conduction slowing without evidence for Brugada syndrome due to a predominantly trafficking defect that reduces Na(+) current and depolarization force.

Modelling tree population dynamics at the alpine and boreal tree-line ecotones in response to climate and land-use change

Summary Ecotones are sensitive to change because they contain high numbers of species living at the margin of their environmental tolerance. This is equally true of tree-lines, which are determined by attitudinal or latitudinal temperature gradients. In the current context of climate change, they are expected to undergo modifications in position, tree biomass and possibly species composition. Attitudinal and latitudinal tree-lines differ mainly in the steepness of the underlying temperature gradient: distances are larger at latitudinal tree-lines, which could have an impact on the ability of tree species to migrate in response to climate change. Aside from temperature, tree-lines are also affected on a more local level by pressure from human activities. These are also changing as a consequence of modifications in our societies and may interact with the effects of climate change. Forest dynamics models are often used for climate change simulations because of their mechanistic processes. The spatially-explicit model TreeMig was used as a base to develop a model specifically tuned for the northern European and Alpine tree-line ecotones. For the latter, a module for land-use change processes was also added. The temperature response parameters for the species in the model were first calibrated by means of tree-ring data from various species and sites at both tree-lines. This improved the growth response function in the model, but also lead to the conclusion that regeneration is probably more important than growth for controlling tree-line position and species' distributions. The second step was to implement the module for abandonment of agricultural land in the Alps, based on an existing spatial statistical model. The sensitivity of its most important variables was tested and the model's performance compared to other modelling approaches. The probability that agricultural land would be abandoned was strongly influenced by the distance from the nearest forest and the slope, bath of which are proxies for cultivation costs. When applied to a case study area, the resulting model, named TreeMig-LAb, gave the most realistic results. These were consistent with observed consequences of land-abandonment such as the expansion of the existing forest and closing up of gaps. This new model was then applied in two case study areas, one in the Swiss Alps and one in Finnish Lapland, under a variety of climate change scenarios. These were based on forecasts of temperature change over the next century by the IPCC and the HadCM3 climate model (ΔT: +1.3, +3.5 and +5.6 °C) and included a post-change stabilisation period of 300 years. The results showed radical disruptions at both tree-lines. With the most conservative climate change scenario, species' distributions simply shifted, but it took several centuries reach a new equilibrium. With the more extreme scenarios, some species disappeared from our study areas (e.g. Pinus cembra in the Alps) or dwindled to very low numbers, as they ran out of land into which they could migrate. The most striking result was the lag in the response of most species, independently from the climate change scenario or tree-line type considered. Finally, a statistical model of the effect of reindeer (Rangifer tarandus) browsing on the growth of Pinus sylvestris was developed, as a first step towards implementing human impacts at the boreal tree-line. The expected effect was an indirect one, as reindeer deplete the ground lichen cover, thought to protect the trees against adverse climate conditions. The model showed a small but significant effect of browsing, but as the link with the underlying climate variables was unclear and the model was not spatial, it was not usable as such. Developing the TreeMig-LAb model allowed to: a) establish a method for deriving species' parameters for the growth equation from tree-rings, b) highlight the importance of regeneration in determining tree-line position and species' distributions and c) improve the integration of social sciences into landscape modelling. Applying the model at the Alpine and northern European tree-lines under different climate change scenarios showed that with most forecasted levels of temperature increase, tree-lines would suffer major disruptions, with shifts in distributions and potential extinction of some tree-line species. However, these responses showed strong lags, so these effects would not become apparent before decades and could take centuries to stabilise. Résumé Les écotones son sensibles au changement en raison du nombre élevé d'espèces qui y vivent à la limite de leur tolérance environnementale. Ceci s'applique également aux limites des arbres définies par les gradients de température altitudinaux et latitudinaux. Dans le contexte actuel de changement climatique, on s'attend à ce qu'elles subissent des modifications de leur position, de la biomasse des arbres et éventuellement des essences qui les composent. Les limites altitudinales et latitudinales diffèrent essentiellement au niveau de la pente des gradients de température qui les sous-tendent les distance sont plus grandes pour les limites latitudinales, ce qui pourrait avoir un impact sur la capacité des espèces à migrer en réponse au changement climatique. En sus de la température, la limite des arbres est aussi influencée à un niveau plus local par les pressions dues aux activités humaines. Celles-ci sont aussi en mutation suite aux changements dans nos sociétés et peuvent interagir avec les effets du changement climatique. Les modèles de dynamique forestière sont souvent utilisés pour simuler les effets du changement climatique, car ils sont basés sur la modélisation de processus. Le modèle spatialement explicite TreeMig a été utilisé comme base pour développer un modèle spécialement adapté pour la limite des arbres en Europe du Nord et dans les Alpes. Pour cette dernière, un module servant à simuler des changements d'utilisation du sol a également été ajouté. Tout d'abord, les paramètres de la courbe de réponse à la température pour les espèces inclues dans le modèle ont été calibrées au moyen de données dendrochronologiques pour diverses espèces et divers sites des deux écotones. Ceci a permis d'améliorer la courbe de croissance du modèle, mais a également permis de conclure que la régénération est probablement plus déterminante que la croissance en ce qui concerne la position de la limite des arbres et la distribution des espèces. La seconde étape consistait à implémenter le module d'abandon du terrain agricole dans les Alpes, basé sur un modèle statistique spatial existant. La sensibilité des variables les plus importantes du modèle a été testée et la performance de ce dernier comparée à d'autres approches de modélisation. La probabilité qu'un terrain soit abandonné était fortement influencée par la distance à la forêt la plus proche et par la pente, qui sont tous deux des substituts pour les coûts liés à la mise en culture. Lors de l'application en situation réelle, le nouveau modèle, baptisé TreeMig-LAb, a donné les résultats les plus réalistes. Ceux-ci étaient comparables aux conséquences déjà observées de l'abandon de terrains agricoles, telles que l'expansion des forêts existantes et la fermeture des clairières. Ce nouveau modèle a ensuite été mis en application dans deux zones d'étude, l'une dans les Alpes suisses et l'autre en Laponie finlandaise, avec divers scénarios de changement climatique. Ces derniers étaient basés sur les prévisions de changement de température pour le siècle prochain établies par l'IPCC et le modèle climatique HadCM3 (ΔT: +1.3, +3.5 et +5.6 °C) et comprenaient une période de stabilisation post-changement climatique de 300 ans. Les résultats ont montré des perturbations majeures dans les deux types de limites de arbres. Avec le scénario de changement climatique le moins extrême, les distributions respectives des espèces ont subi un simple glissement, mais il a fallu plusieurs siècles pour qu'elles atteignent un nouvel équilibre. Avec les autres scénarios, certaines espèces ont disparu de la zone d'étude (p. ex. Pinus cembra dans les Alpes) ou ont vu leur population diminuer parce qu'il n'y avait plus assez de terrains disponibles dans lesquels elles puissent migrer. Le résultat le plus frappant a été le temps de latence dans la réponse de la plupart des espèces, indépendamment du scénario de changement climatique utilisé ou du type de limite des arbres. Finalement, un modèle statistique de l'effet de l'abroutissement par les rennes (Rangifer tarandus) sur la croissance de Pinus sylvestris a été développé, comme première étape en vue de l'implémentation des impacts humains sur la limite boréale des arbres. L'effet attendu était indirect, puisque les rennes réduisent la couverture de lichen sur le sol, dont on attend un effet protecteur contre les rigueurs climatiques. Le modèle a mis en évidence un effet modeste mais significatif, mais étant donné que le lien avec les variables climatiques sous jacentes était peu clair et que le modèle n'était pas appliqué dans l'espace, il n'était pas utilisable tel quel. Le développement du modèle TreeMig-LAb a permis : a) d'établir une méthode pour déduire les paramètres spécifiques de l'équation de croissance ä partir de données dendrochronologiques, b) de mettre en évidence l'importance de la régénération dans la position de la limite des arbres et la distribution des espèces et c) d'améliorer l'intégration des sciences sociales dans les modèles de paysage. L'application du modèle aux limites alpines et nord-européennes des arbres sous différents scénarios de changement climatique a montré qu'avec la plupart des niveaux d'augmentation de température prévus, la limite des arbres subirait des perturbations majeures, avec des glissements d'aires de répartition et l'extinction potentielle de certaines espèces. Cependant, ces réponses ont montré des temps de latence importants, si bien que ces effets ne seraient pas visibles avant des décennies et pourraient mettre plusieurs siècles à se stabiliser.

Generation and characterization of a Notch1 signaling-specific reporter mouse line.

Signaling through the Notch1 receptor is essential for the control of numerous developmental processes during embryonic life as well as in adult tissue homeostasis and disease. Since the outcome of Notch1 signaling is highly context-dependent, and its precise physiological and pathological role in many organs is unclear, it is of great interest to localize and identify the cells that receive active Notch1 signals in vivo. Here, we report the generation and characterization of a BAC-transgenic mouse line, N1-Gal4VP16, that when crossed to a Gal4-responsive reporter mouse line allowed the identification of cells undergoing active Notch1 signaling in vivo. Analysis of embryonic and adult N1-Gal4VP16 mice demonstrated that the activation pattern of the transgene coincides with previously observed activation patterns of the endogenous Notch1 receptor. Thus, this novel reporter mouse line provides a unique tool to specifically investigate the spatial and temporal aspects of Notch1 signaling in vivo. genesis 50:700-710, 2012. © 2012 Wiley Periodicals, Inc.

Association of pharmacogenetic markers with premature discontinuation of first-line anti-HIV therapy: an observational cohort study.

BACKGROUND: Poor tolerance and adverse drug reactions are main reasons for discontinuation of antiretroviral therapy (ART). Identifying predictors of ART discontinuation is a priority in HIV care. METHODS: A genetic association study in an observational cohort to evaluate the association of pharmacogenetic markers with time to treatment discontinuation during the first year of ART. Analysis included 577 treatment-naive individuals initiating tenofovir (n = 500) or abacavir (n = 77), with efavirenz (n = 272), lopinavir/ritonavir (n = 184), or atazanavir/ritonavir (n = 121). Genotyping included 23 genetic markers in 15 genes associated with toxicity or pharmacokinetics of the study medication. Rates of ART discontinuation between groups with and without genetic risk markers were assessed by survival analysis using Cox regression models. RESULTS: During the first year of ART, 190 individuals (33%) stopped 1 or more drugs. For efavirenz and atazanavir, individuals with genetic risk markers experienced higher discontinuation rates than individuals without (71.15% vs 28.10%, and 62.5% vs 14.6%, respectively). The efavirenz discontinuation hazard ratio (HR) was 3.14 (95% confidence interval (CI): 1.35-7.33, P = .008). The atazanavir discontinuation HR was 9.13 (95% CI: 3.38-24.69, P < .0001). CONCLUSIONS: Several pharmacogenetic markers identify individuals at risk for early treatment discontinuation. These markers should be considered for validation in the clinical setting.

Phase I study of sorafenib combined with radiation therapy and temozolomide as first-line treatment of high-grade glioma.

BACKGROUND: Sorafenib (Sb) is a multiple kinase inhibitor targeting both tumour cell proliferation and angiogenesis that may further act as a potent radiosensitizer by arresting cells in the most radiosensitive cell cycle phase. This phase I open-label, noncontrolled dose escalation study was performed to determine the safety and maximum tolerated dose (MTD) of Sb in combination with radiation therapy (RT) and temozolomide (TMZ) in 17 patients with newly diagnosed high-grade glioma. METHODS: Patients were treated with RT (60 Gy in 2 Gy fractions) combined with TMZ 75 mg m(-2) daily, and Sb administered at three dose levels (200 mg daily, 200 mg BID, and 400 mg BID) starting on day 8 of RT. Thirty days after the end of RT, patients received monthly TMZ (150-200 mg m(-2) D1-5/28) and Sb (400 mg BID). Pharmacokinetic (PK) analyses were performed on day 8 (TMZ) and on day 21 (TMZ&Sb) (Clinicaltrials ID: NCT00884416). RESULTS: The MTD of Sb was established at 200 mg BID. Dose-limiting toxicities included thrombocytopenia (two patients), diarrhoea (one patient) and hypercholesterolaemia (one patient). Sb administration did not affect the mean area under the curve(0-24) and mean Cmax of TMZ and its metabolite 5-amino-imidazole-4-carboxamide (AIC). Tmax of both TMZ and AIC was delayed from 0.75 (TMZ alone) to 1.5 h (combined TMZ/Sb). The median progression-free survival was 7.9 months (95% confidence interval (CI): 5.4-14.55), and the median overall survival was 17.8 months (95% CI: 14.7-25.6). CONCLUSIONS: Although Sb can be combined with RT and TMZ, significant side effects and moderate outcome results do not support further clinical development in malignant gliomas. The robust PK data of the TMZ/Sb combination could be useful in other cancer settings.

90Yttrium-Ibritumomab Tiuxetan Consolidation of First Remission in Advanced-Stage Follicular Non-Hodgkin Lymphoma: Updated Results After a Median Follow-Up of 7.3 Years From the International, Randomized, Phase III First-Line Indolent Trial.

PURPOSE Updated results are presented after a median follow-up of 7.3 years from the phase III First-Line Indolent Trial of yttrium-90 ((90)Y) -ibritumomab tiuxetan in advanced-stage follicular lymphoma (FL) in first remission. PATIENTS AND METHODS Patients with CD20(+) stage III or IV FL with complete response (CR), unconfirmed CR (CRu), or partial response (PR) after first-line induction treatment were randomly assigned to (90)Y-ibritumomab consolidation therapy (rituximab 250 mg/m(2) days -7 and 0, then (90)Y-ibritumomab 14.8 MBq/kg day 0; maximum 1,184 MBq) or no further treatment (control). Primary end point was progression-free survival (PFS) from date of random assignment. Results For 409 patients available for analysis ((90)Y-ibritumomab, n = 207; control, n = 202), estimated 8-year overall PFS was 41% with (90)Y-ibritumomab versus 22% for control (hazard ratio [HR], 0.47; P < .001). For patients in CR/CRu after induction, 8-year PFS with (90)Y-ibritumomab was 48% versus 32% for control (HR, 0.61; P = .008), and for PR patients, it was 33% versus 10% (HR, 0.38; P < .001). For (90)Y-ibritumomab consolidation, median PFS was 4.1 years (v 1.1 years for control; P < .001). Median time to next treatment (TTNT) was 8.1 years for (90)Y-ibritumomab versus 3.0 years for control (P < .001) with approximately 80% response rates to second-line therapy in either arm, including autologous stem-cell transplantation. No unexpected toxicities emerged during long-term follow-up. Estimated between-group 8-year overall survival rates were similar. Annualized incidence rate of myelodysplastic syndrome/acute myeloblastic leukemia was 0.50% versus 0.07% in (90)Y-ibritumomab and control groups, respectively (P = .042). CONCLUSION (90)Y-ibritumomab consolidation after achieving PR or CR/CRu to induction confers 3-year benefit in median PFS with durable 19% PFS advantage at 8 years and improves TTNT by 5.1 years for patients with advanced FL.

Assessing the Glaucoma Quality of Life-15 (GCL-15) Questionnaire, Goldmann Tonometry and Moorfields Motion Displacement Test in Glaucoma Case Finding

Purpose: To compare the performance Glaucoma Quality of Life-15 (GQL-15) Questionnaire, intraocular pressure measurement (IOP Goldmann tonometry) and a measure of visual field loss using Moorfields Motion Displacement Test (MDT) in detecting glaucomatous eyes from a self referred population. Methods: The GQL-15 has been suggested to correlate with visual disability and psychophysical measures of visual function in glaucoma patients. The Moorfields MDT is a multi location perimetry test with 32 white line stimuli presented on a grey background on a standard laptop computer. Each stimulus is displaced between computer frames to give the illusion of "apparent motion". Participants (N=312, 90% older than 45 years; 20.5% family history of glaucoma) self referred to an advertised World Glaucoma Day (March 2009) Jules Gonin Eye Hospital, Lausanne Switzerland. Participants underwent a clinical exam (IOP, slit lamp, angle and disc examination by a general ophthalmologist), 90% completed a GQL-15 questionnaire and over 50% completed a MDT test in both eyes. Those who were classified as abnormal on one or more of the following (IOP >21 mmHg/ GQL-15 score >20/ MDT score >2/ clinical exam) underwent a follow up clinical examination by a glaucoma specialist including imaging and threshold perimetry. After the second examination subjects were classified as "healthy"(H), "glaucoma suspect" (GS) (ocular hypertension and/or suspicious disc, angle closure with SD) or "glaucomatous" (G). Results: One hundred and ten subjects completed all 4 initial examinations; of these 69 were referred to complete the 2nd examination and were classified as; 8 G, 24 GS, and 37 H. MDT detected 7/8 G, and 7/24 GS, with false referral rate of 3.8%. IOP detected 2/8 G and 8/24 GS, with false referral rate of 8.9%. GQL-15 detected 4/8 G, 16/24 GS with a false referral rate of 42%. Conclusions: In this sample of participants attending a self referral glaucoma detection event, the MDT performed significantly better than the GQL-15 and IOP in discriminating glaucomatous patients from healthy subjects. Further studies are required to assess the potential of the MDT as a glaucoma screening tool.

Recovery of postoperative visual loss following treatment of severe anaemia.

A 29-year-old pregnant woman noted acute visual loss following emergent Caesarean section complicated by excessive uterine bleeding. Postoperative visual acuity was count fingers in both eyes. Funduscopic changes were consistent with a diagnosis of anaemia-associated ischaemic optic neuropathy and retinopathy. One month later, because of persistent anaemia and poor visual recovery, blood transfusion was given. Following transfusion, the patient's vision improved over the next 6 months. In an otherwise healthy patient, visual loss associated with postoperative blood loss may still be partially reversible with correction of the anaemia, even after a delayed period of time.

Sorafenib fourth-line treatment in imatinib-, sunitinib-, and nilotinib-resistant metastatic GIST : a retrospective analysis : 10564

Background: Gastrointestinal stromal tumors (GISTs) are rare mesenchymal tumors usually caused by mutations in the KIT or PDGFRA gene. Advanced disease generally cannot be cured by surgery nor by tyrosine kinase inhibitors (TKI), but TKIs have considerably improved outcome for patients (pts) with advanced GIST. Patients failing TKI treatment with imatinib (IM), sunitinib (SU) or nilotinib (NI) have a poor prognosis. Sorafenib is a multi kinase inhibitor that blocks not only receptor tyrosine kinases such as KIT, VEGFR and PDGFR but also serine/threonine kinases along the RAS/RAF/MEK/ERK pathway. Recently, clinical activity of sorafenib in third-line treatment in patients with GIST after IM and SU failure has been shown (Wiebe et al. ASCO 2008, #10502). Methods: We report herein preliminary data of 32 pts treated with sorafenib in nine European centers. Centers were selected based on their previous and known experience in GIST and reported all pts treated. Pts received sorafenib after failure of IM, SU and NI in fourth-line treatment. Baseline characteristics and treatment details have been retrieved via questionary. Results: Median age at sorafenib treatment start was 62 years (range 33-81 y), and the majority of pts were male (63 %). Primary tumor site was gastric or small intestine in 25% and 41% of pts, respectively. All pts had failed IM, SU, NI. 19 % of pts achieved partial remission and 44% disease stabilization. Approximately half of the pts had an improvement of symptoms and/or performance. Half of the pts were on treatment longer than 4 months (actuarial data) and 41% of pts continue to receive sorafenib. Median progression-free survival is 20 weeks and median overall survival 42 weeks (Kaplan-Meier), at a median follow-up of 22 weeks (range 3-54). Conclusions: This is the largest series assessing efficacy of sorafenib fourth-line treatment for IM, SU and NI refractory GIST reported yet. Sorafenib displays significant clinical activity in this heavily pretreated group of patients.

Breast cancer and melanoma cell line identification by FTIR imaging after formalin-fixation and paraffin-embedding.

Over the past few decades, Fourier transform infrared (FTIR) spectroscopy coupled to microscopy has been recognized as an emerging and potentially powerful tool in cancer research and diagnosis. For this purpose, histological analyses performed by pathologists are mostly carried out on biopsied tissue that undergoes the formalin-fixation and paraffin-embedding (FFPE) procedure. This processing method ensures an optimal and permanent preservation of the samples, making FFPE-archived tissue an extremely valuable source for retrospective studies. Nevertheless, as highlighted by previous studies, this fixation procedure significantly changes the principal constituents of cells, resulting in important effects on their infrared (IR) spectrum. Despite the chemical and spectral influence of FFPE processing, some studies demonstrate that FTIR imaging allows precise identification of the different cell types present in biopsied tissue, indicating that the FFPE process preserves spectral differences between distinct cell types. In this study, we investigated whether this is also the case for closely related cell lines. We analyzed spectra from 8 cancerous epithelial cell lines: 4 breast cancer cell lines and 4 melanoma cell lines. For each cell line, we harvested cells at subconfluence and divided them into two sets. We first tested the "original" capability of FTIR imaging to identify these closely related cell lines on cells just dried on BaF2 slides. We then repeated the test after submitting the cells to the FFPE procedure. Our results show that the IR spectra of FFPE processed cancerous cell lines undergo small but significant changes due to the treatment. The spectral modifications were interpreted as a potential decrease in the phospholipid content and protein denaturation, in line with the scientific literature on the topic. Nevertheless, unsupervised analyses showed that spectral proximities and distances between closely related cell lines were mostly, but not entirely, conserved after FFPE processing. Finally, PLS-DA statistical analyses highlighted that closely related cell lines are still successfully identified and efficiently distinguished by FTIR spectroscopy after FFPE treatment. This last result paves the way towards identification and characterization of cellular subtypes on FFPE tissue sections by FTIR imaging, indicating that this analysis technique could become a potential useful tool in cancer research.

On the problematic of reserving and stochastic loss models

Abstract Traditionally, the common reserving methods used by the non-life actuaries are based on the assumption that future claims are going to behave in the same way as they did in the past. There are two main sources of variability in the processus of development of the claims: the variability of the speed with which the claims are settled and the variability between the severity of the claims from different accident years. High changes in these processes will generate distortions in the estimation of the claims reserves. The main objective of this thesis is to provide an indicator which firstly identifies and quantifies these two influences and secondly to determine which model is adequate for a specific situation. Two stochastic models were analysed and the predictive distributions of the future claims were obtained. The main advantage of the stochastic models is that they provide measures of variability of the reserves estimates. The first model (PDM) combines one conjugate family Dirichlet - Multinomial with the Poisson distribution. The second model (NBDM) improves the first one by combining two conjugate families Poisson -Gamma (for distribution of the ultimate amounts) and Dirichlet Multinomial (for distribution of the incremental claims payments). It was found that the second model allows to find the speed variability in the reporting process and development of the claims severity as function of two above mentioned distributions' parameters. These are the shape parameter of the Gamma distribution and the Dirichlet parameter. Depending on the relation between them we can decide on the adequacy of the claims reserve estimation method. The parameters have been estimated by the Methods of Moments and Maximum Likelihood. The results were tested using chosen simulation data and then using real data originating from the three lines of business: Property/Casualty, General Liability, and Accident Insurance. These data include different developments and specificities. The outcome of the thesis shows that when the Dirichlet parameter is greater than the shape parameter of the Gamma, resulting in a model with positive correlation between the past and future claims payments, suggests the Chain-Ladder method as appropriate for the claims reserve estimation. In terms of claims reserves, if the cumulated payments are high the positive correlation will imply high expectations for the future payments resulting in high claims reserves estimates. The negative correlation appears when the Dirichlet parameter is lower than the shape parameter of the Gamma, meaning low expected future payments for the same high observed cumulated payments. This corresponds to the situation when claims are reported rapidly and fewer claims remain expected subsequently. The extreme case appears in the situation when all claims are reported at the same time leading to expectations for the future payments of zero or equal to the aggregated amount of the ultimate paid claims. For this latter case, the Chain-Ladder is not recommended.

Multicenter phase II trial of gefitinib first-line therapy followed by chemotherapy in advanced non-small-cell lung cancer (NSCLC): SAKK protocol 19/03.

BACKGROUND: Gefitinib is active in patients with pretreated non-small-cell lung cancer (NSCLC). We evaluated the activity and toxicity of gefitinib first-line treatment in advanced NSCLC followed by chemotherapy at disease progression. PATIENTS AND METHODS: In all, 63 patients with chemotherapy-naive stage IIIB/IV NSCLC received gefitinib 250 mg/day. At disease progression, gefitinib was replaced by cisplatin 80 mg/m(2) on day 1 and gemcitabine 1250 mg/m(2) on days 1, 8 for up to six 3-week cycles. Primary end point was the disease stabilization rate (DSR) after 12 weeks of gefitinib. RESULTS: After 12 weeks of gefitinib, the DSR was 24% and the response rate (RR) was 8%. Median time to progression (TtP) was 2.5 months and median overall survival (OS) 11.5 months. Never smokers (n = 9) had a DSR of 56% and a median OS of 20.2 months; patients with epidermal growth factor receptor (EGFR) mutation (n = 4) had a DSR of 75% and the median OS was not reached after the follow-up of 21.6 months. In all, 41 patients received chemotherapy with an overall RR of 34%, DSR of 71% and median TtP of 6.7 months. CONCLUSIONS: First-line gefitinib monotherapy led to a DSR of 24% at 12 weeks in an unselected patients population. Never smokers and patients with EGFR mutations tend to have a better outcome; hence, further trials in selected patients are warranted.

Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice.

Mitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur in Candida glabrata. Cells with mitochondrial DNA deficiency (so-called "petite mutants") upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of such C. glabrata mutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and related C. glabrata isolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genes C. glabrata CDR1 (CgCDR1), CgCDR2, and CgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even with in vitro growth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays during in vitro growth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selected in vivo under azole therapy, even if strongly affecting in vitro growth characteristics, can confer a selective advantage under host conditions, allowing the C. glabrata mutant to be more virulent than wild-type isolates.

Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.