Transgenic mice as a tool for depression research: examples from the endocannabinoid and the corticotropin-releasing hormone system

Major depression belongs to the most serious and widespread psychiatric disorders in today’s society. There is a great need for the delineation of the underlying molecular mechanisms as well as for the identification of novel targets for its treatment. In this thesis, transgenic mice of the endocannabinoid and the corticotropin-releasing hormone (CRH) system were investigated to determine the putative role of these systems for depression-like phenotypes in mice. In the first part of the thesis, we found that the endocannabinoid system was prominently involved in a brain region-specific and temporally controlled manner in acute as well as in chronic stress processing. Genetic deletion in combination with pharmacological intervention revealed the importance of a fully functional endocannabinoid system for efficient neuroendocrine and behavioral stress coping. Accordingly, cannabinoid type 1 (CB1) receptor-deficient mice displayed several depression-like symptoms and molecular alterations, including “behavioral despair”, stress hormone hypersecretion and decreased glucocorticoid receptor and brain-derived neurotrophic factor expression in the hippocampus. However, the endocannabinoid system was dispensable for the efficacy of currently used antidepressant drugs. To facilitate future endocannabinoid research, a transgenic mouse was generated, which overexpressed the CB1 receptor protein fused to a fluorescent protein. In the second part of the thesis, conditional brain region-specific CRH overexpressing mice were evaluated as a model for pathological chronic CRH hyperactivation. Mutant mice showed aberrant neuroendocrine and behavioral stress coping and hyperarousal due to CRH-induced activation of the noradrenergic system in the brain. Mutant mice appeared to share similarities with naturally occurring endogenous CRH activation in wild-type mice and were sensitive to acute pharmacological blockade of CRH receptor type 1 (CRH-R1). Thus, CRH overexpressing mice serve as an ideal in vivo tool to evaluate the efficacy of novel CRH-R1 antagonists. Together, these findings highlight the potential of transgenic mice for the understanding of certain endo-phenotypes (isolated symptoms) of depression and their molecular correlates.

LCA-GIS Integrated approach for a Sustainable Land Use Under Energy Crops

In genere, negli studi di vocazionalità delle colture, vengono presi in considerazione solo variabili ambientali pedo-climatiche. La coltivazione di una coltura comporta anche un impatto ambientale derivante dalle pratiche agronomiche ed il territorio può essere più o meno sensibile a questi impatti in base alla sua vulnerabilità. In questo studio si vuole sviluppare una metodologia per relazionare spazialmente l’impatto delle colture con le caratteristiche sito specifiche del territorio in modo da considerare anche questo aspetto nell’allocazione negli studi di vocazionalità. LCA è stato utilizzato per quantificare diversi impatti di alcune colture erbacee alimentari e da energia, relazionati a mappe di vulnerabilità costruite con l’utilizzo di GIS, attraverso il calcolo di coefficienti di rischio di allocazione per ogni combinazione coltura-area vulnerabile. Le colture energetiche sono state considerate come un uso alternativo del suolo per diminuire l’impatto ambientale. Il caso studio ha mostrato che l’allocazione delle colture può essere diversa in base al tipo e al numero di impatti considerati. Il risultato sono delle mappe in cui sono riportate le distribuzioni ottimali delle colture al fine di minimizzare gli impatti, rispetto a mais e grano, due colture alimentari importanti nell’area di studio. Le colture con l’impatto più alto dovrebbero essere coltivate nelle aree a vulnerabilità bassa, e viceversa. Se il rischio ambientale è la priorità, mais, colza, grano, girasole, e sorgo da fibra dovrebbero essere coltivate solo nelle aree a vulnerabilità bassa o moderata, mentre, le colture energetiche erbacee perenni, come il panico, potrebbero essere coltivate anche nelle aree a vulnerabilità alta, rappresentando cosi una opportunità per aumentare la sostenibilità di uso del suolo rurale. Lo strumento LCA-GIS inoltre, integrato con mappe di uso attuale del suolo, può aiutare a valutarne il suo grado di sostenibilità ambientale.

Using national FADN database to describe Italian farms and arable fields. A comparison of sustainability level between organic versus conventional regimes.

The study defines a new farm classification and identifies the arable land management. These aspects and several indicators are taken into account to estimate the sustainability level of farms, for organic and conventional regimes. The data source is Italian Farm Account Data Network (RICA) for years 2007-2011, which samples structural and economical information. An environmental data has been added to the previous one to better describe the farm context. The new farm classification describes holding by general informations and farm structure. The general information are: adopted regime and farm location in terms of administrative region, slope and phyto-climatic zone. The farm structures describe the presence of main productive processes and land covers, which are recorded by FADN database. The farms, grouped by homogeneous farm structure or farm typology, are evaluated in terms of sustainability. The farm model MAD has been used to estimate a list of indicators. They describe especially environmental and economical areas of sustainability. Finally arable lands are taken into account to identify arable land managements and crop rotations. Each arable land has been classified by crop pattern. Then crop rotation management has been analysed by spatial and temporal approaches. The analysis reports a high variability inside regimes. The farm structure influences indicators level more than regimes, and it is not always possible to compare the two regimes. However some differences between organic and conventional agriculture have been found. Organic farm structures report different frequency and geographical location than conventional ones. Also different connections among arable lands and farm structures have been identified.

Analisi di morfologie carsiche o crio-carsiche in relazione alla mobilizzazione di fluidi nella regione di Arabia Terra, Marte.

This thesis tries to interpret the origin and evolution of karst-like forms present in Arabia Terra, a region of Mars that develops in the equatorial zone of the planet. The work has been carried out specifically in the craters Crommelin (4o 91’ N-10o 51’ E), 12000088 (3o 48’ N-1o 30’ E), NE 12000088 (4° 20’ N-2° 50’ E), C "2" (3° 54’ N-1° W), and in their surrounding areas. These craters contain layered deposits characterized by a high albedo and on which erosion is very pronounced. The area containing the craters is a plateau that has the same characteristics of albedo and texture. The preliminary morphological study has made use of instrumentation such as the Mars Reconnaissance Orbiter (MRO), in particular HiRISE images (High Resolution Imaging Science Experiment), CTX (Context Camera) and CRISM (Compact Reconnaissance Imaging Spectrometers for Mars). A regional geomorphological map has been drawn up containing the main morphotypes, and detailed geomorphological maps were prepared for different karst-like morphologies. The analysis of spectral data collected from CRISM instrumentation has allowed to identify the footprint of sulphate minerals in the external area. Data were collected for morphometric negative forms (karst-like) and positive forms (mud volcanoes, dikes and pingos). For the analysis of the relief forms DTMs (Digital Terrain Models) produced by the union of stereographic CTX couples or HiRISE were used. From the analysis of high-resolution images morphological footprints similar to periglacial environments have been identified, including the presence of patterned ground and polygonal cracks found all over the area of investigation, and relief structures similar to pingos present in the crater C "2". These observations allow us to imagine a geological past with a cold climate at the equator able to freeze the few fluids present in the Martian arid terrain. The development of karst-like landforms, on the other hand, can be attributed to a subsequent improval of the weather conditions that led to a normal climate regime for the equatorial areas, resulting in the degradation of the permafrost. The melt waters have thus allowed the partial dissolution of the sulphate layers. The karst-like forms look rather fresh suggesting them to be not that old.

Reference values of mechanical and thermal pain tests in a pain-free population

Quantitative sensory tests are widely used in human research to evaluate the effect of analgesics and explore altered pain mechanisms, such as central sensitization. In order to apply these tests in clinical practice, knowledge of reference values is essential. The aim of this study was to determine the reference values of pain thresholds for mechanical and thermal stimuli, as well as withdrawal time for the cold pressor test in 300 pain-free subjects. Pain detection and pain tolerance thresholds to pressure, heat and cold were determined at three body sites: (1) lower back, (2) suprascapular region and (3) second toe (for pressure) or the lateral aspect of the leg (for heat and cold). The influences of gender, age, height, weight, body-mass index (BMI), body side of testing, depression, anxiety, catastrophizing and parameters of Short-Form 36 (SF-36) were analyzed by multiple regressions. Quantile regressions were performed to define the 5th, 10th and 25th percentiles as reference values for pain hypersensitivity and the 75th, 90th and 95th percentiles as reference values for pain hyposensitivity. Gender, age and/or the interaction of age with gender were the only variables that consistently affected the pain measures. Women were more pain sensitive than men. However, the influence of gender decreased with increasing age. In conclusion, normative values of parameters related to pressure, heat and cold pain stimuli were determined. Reference values have to be stratified by body region, gender and age. The determination of these reference values will now allow the clinical application of the tests for detecting abnormal pain reactions in individual patients.

MicroRNA-381 Represses ID1 and is Deregulated in Lung Adenocarcinoma

MicroRNAs are small, noncoding RNAs that suppress gene expression by binding to the 3' untranslated region (UTR) and thereby repress translation or decrease messenger RNA stability. Inhibitor of differentiation 1 (ID1) is a putative stem-cell gene involved in invasion and angiogenesis. We previously showed that ID1 is regulated by Src kinases, overexpressed in human lung adenocarcinoma, and targeted by Src-dependent microRNAs. The current study focused on the association between miR-381 and ID1 in lung adenocarcinoma.

PBDEs in the atmosphere over the Asian marginal seas, and the Indian and Atlantic oceans

Air samples were collected from Jan 16 to Mar 14, 2008 onboard the Oceanic II- The Scholar Ship which navigated an east–west transect from Shanghai to Cape Verde, and polybrominated diphenyl ethers (PBDEs) were analyzed in these samples. PBDE concentrations in the atmosphere over the open seas were influenced by proximity to source areas and land, and air mass origins. The concentrations of Σ21PBDEs over the East and South China Seas, the Bay of Bengal and the Andaman Sea, the Indian Ocean, and the Atlantic Ocean were 10.8 ± 6.13, 3.22 ± 1.57, 5.12 ± 3.56, and 2.87 ± 1.81 pg m−3, respectively. BDE-47 and -99 were the dominant congeners in all the samples, suggesting that the widely used commercial penta-BDE products were the original sources. Over some parts of Atlantic and Indian Ocean, daytime concentrations of BDE-47 and BDE-99 were higher than the concentrations at night. The strong atmospheric variability does not always coincide with a diurnal cycle, but the variability in air concentrations in such remote areas of the ocean remains strong. No significant trends were found for each of PBDE congener with latitude.

Impact of weather and climate on the incidence of acute coronary syndromes

BACKGROUND: First investigations of the interactions between weather and the incidence of acute myocardial infarctions date back to 1938. The early observation of a higher incidence of myocardial infarctions in the cold season could be confirmed in very different geographical regions and cohorts. While the influence of seasonal variations on the incidence of myocardial infarctions has been extensively documented, the impact of individual meteorological parameters on the disease has so far not been investigated systematically. Hence the present study intended to assess the impact of the essential variables of weather and climate on the incidence of myocardial infarctions. METHODS: The daily incidence of myocardial infarctions was calculated from a national hospitalization survey. The hourly weather and climate data were provided by the database of the national weather forecast. The epidemiological and meteorological data were correlated by multivariate analysis based on a generalized linear model assuming a log-link-function and a Poisson distribution. RESULTS: High ambient pressure, high pressure gradients, and heavy wind activity were associated with an increase in the incidence of the totally 6560 hospitalizations for myocardial infarction irrespective of the geographical region. Snow- and rainfall had inconsistent effects. Temperature, Foehn, and lightning showed no statistically significant impact. CONCLUSIONS: Ambient pressure, pressure gradient, and wind activity had a statistical impact on the incidence of myocardial infarctions in Switzerland from 1990 to 1994. To establish a cause-and-effect relationship more data are needed on the interaction between the pathophysiological mechanisms of the acute coronary syndrome and weather and climate variables.

Development of a physically-based method for delineation of hydrologically homogeneous regions and flood quantile estimation in ungauged basins via the index flood method

Regional flood frequency techniques are commonly used to estimate flood quantiles when flood data is unavailable or the record length at an individual gauging station is insufficient for reliable analyses. These methods compensate for limited or unavailable data by pooling data from nearby gauged sites. This requires the delineation of hydrologically homogeneous regions in which the flood regime is sufficiently similar to allow the spatial transfer of information. It is generally accepted that hydrologic similarity results from similar physiographic characteristics, and thus these characteristics can be used to delineate regions and classify ungauged sites. However, as currently practiced, the delineation is highly subjective and dependent on the similarity measures and classification techniques employed. A standardized procedure for delineation of hydrologically homogeneous regions is presented herein. Key aspects are a new statistical metric to identify physically discordant sites, and the identification of an appropriate set of physically based measures of extreme hydrological similarity. A combination of multivariate statistical techniques applied to multiple flood statistics and basin characteristics for gauging stations in the Southeastern U.S. revealed that basin slope, elevation, and soil drainage largely determine the extreme hydrological behavior of a watershed. Use of these characteristics as similarity measures in the standardized approach for region delineation yields regions which are more homogeneous and more efficient for quantile estimation at ungauged sites than those delineated using alternative physically-based procedures typically employed in practice. The proposed methods and key physical characteristics are also shown to be efficient for region delineation and quantile development in alternative areas composed of watersheds with statistically different physical composition. In addition, the use of aggregated values of key watershed characteristics was found to be sufficient for the regionalization of flood data; the added time and computational effort required to derive spatially distributed watershed variables does not increase the accuracy of quantile estimators for ungauged sites. This dissertation also presents a methodology by which flood quantile estimates in Haiti can be derived using relationships developed for data rich regions of the U.S. As currently practiced, regional flood frequency techniques can only be applied within the predefined area used for model development. However, results presented herein demonstrate that the regional flood distribution can successfully be extrapolated to areas of similar physical composition located beyond the extent of that used for model development provided differences in precipitation are accounted for and the site in question can be appropriately classified within a delineated region.

Severe traumatic brain injury in Switzerland - feasibility and first results of a cohort study

BACKGROUND: We aimed to study the incidence and outcome of severe traumatic brain injury (TBI) in Switzerland and to test the feasibility of a large cohort study with case identification in the first 24 hours and 6-month follow-up. METHODS: From January to June 2005, we consecutively enrolled and followed up all persons with severe TBI (Abbreviated Injury Score of the head region >3 and Glasgow Coma Scale <9) in the catchment areas of 3 Swiss medical centres with neurosurgical facilities. The primary outcome was the Extended Glasgow Outcome Scale (GOSE) after 6 months. Secondary outcomes included survival, Functional Independence Mea - sure (FIM), and health-related quality of life (SF-12) at defined time-points up to 6 months after injury. RESULTS: We recruited 101 participants from a source population of about 2.47 million (ie, about 33% of Swiss population). The incidence of severe TBI was 8.2 per 100,000 person-years. The overall case fatality was 70%: 41 of 101 persons (41%) died at the scene of the accident. 23 of 60 hospitalised participants (38%) died within 48 hours, and 31 (53%) within 6 months. In all hospitalised patients, the median GOSE was 1 (range 1-8) after 6 months, and was 6 (2-8) in 6-month survivors. The median total FIM score was 125 (range 18-126); median-SF-12 component mea - sures were 44 (25-55) for the physical scale and 52 (32-65) for the mental scale. CONCLUSIONS: Severe TBI was associated with high case fatality and considerable morbidity in survivors. We demonstrated the feasibility of a multicentre cohort study in Switzerland with the aim of identifying modifiable determinants of outcome and improving current trauma care.

Molecular cytogenetics and gene mapping in sheep (Ovis aries, 2n = 54)

The development of a completely annotated sheep genome sequence is a key need for understanding the phylogenetic relationships and genetic diversity among the many different sheep breeds worldwide and for identifying genes controlling economically and physiologically important traits. The ovine genome sequence assembly will be crucial for developing optimized breeding programs based on highly productive, healthy sheep phenotypes that are adapted to modern breeding and production conditions. Scientists and breeders around the globe have been contributing to this goal by generating genomic and cDNA libraries, performing genome-wide and trait-associated analyses of polymorphism, expression analysis, genome sequencing, and by developing virtual and physical comparative maps. The International Sheep Genomics Consortium (ISGC), an informal network of sheep genomics researchers, is playing a major role in coordinating many of these activities. In addition to serving as an essential tool for monitoring chromosome abnormalities in specific sheep populations, ovine molecular cytogenetics provides physical anchors which link and order genome regions, such as sequence contigs, genes and polymorphic DNA markers to ovine chromosomes. Likewise, molecular cytogenetics can contribute to the process of defining evolutionary breakpoints between related species. The selective expansion of the sheep cytogenetic map, using loci to connect maps and identify chromosome bands, can substantially contribute to improving the quality of the annotated sheep genome sequence and will also accelerate its assembly. Furthermore, identifying major morphological chromosome anomalies and micro-rearrangements, such as gene duplications or deletions, that might occur between different sheep breeds and other Ovis species will also be important to understand the diversity of sheep chromosome structure and its implications for cross-breeding. To date, 566 loci have been assigned to specific chromosome regions in sheep and the new cytogenetic map is presented as part of this review. This review will also summarize the current cytogenomic status of the sheep genome, describe current activities in the sheep cytogenomics research sector, and will discuss the cytogenomics data in context with other major sheep genomics projects.

Targeted radiotherapy of prostate cancer with a gastrin-releasing peptide receptor antagonist is effective as monotherapy and in combination with rapamycin

UNLABELLED The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. METHODS To determine the effect of treatment with rapamycin and radiotherapy with a novel (177)Lu-labeled GRPr antagonist ((177)Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and (177)Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on (177)Lu-RM2 tumor uptake, in vivo small-animal PET studies with (68)Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of (177)Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with (177)Lu-RM2 alone or after pretreatment with rapamycin. RESULTS Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with (177)Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of (177)Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. CONCLUSION Radiotherapy using a (177)Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

AGROBACTERIUM VIRB10 CONTRIBUTIONS TO TYPE IV SUBSTRATE SECRETION, T-PILUS BIOGENESIS, AND OUTER MEMBRANE PORE FORMATION

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) identify residues/domains contributing to VirB10 stability, oligomerization, and function, and (iii) monitor structural changes accompanying energy activation or substrate translocation. These studies were aided by recent structural resolution of a periplasmic domain of a VirB10 homolog and a ‘core’ complex composed of homologs of VirB10 and two outer membrane associated subunits, VirB7 and VirB9. By use of the substituted cysteine accessibility method (SCAM), I confirmed the bitopic topology of VirB10. Through phenotypic studies of Ala-Cys insertion mutations, I identified “uncoupling” mutations in the TM and β-barrel domains that blocked T-pilus assembly but permitted substrate transfer. I showed that cysteine replacements in the C-terminal periplasmic domain yielded a variety of phenotypes in relation to protein accumulation, oligomerization, substrate transfer, and T-pilus formation. By SCAM, I also gained further evidence that VirB10 adopts different structural states during machine biogenesis. Finally, I showed that VirB10 supports substrate transfer even when its TM domain is extensively mutagenized or substituted with heterologous TM domains. By contrast, specific residues most probably involved in oligomerization of the TM domain are required for biogenesis of the T-pilus.

The physical map of {\it Escherichia coli\/} K-12 strain MG1655: Construction, comparison with other strains and implications for mechanisms controlling oligonucleotide frequency

Complete NotI, SfiI, XbaI and BlnI cleavage maps of Escherichia coli K-12 strain MG1655 were constructed. Techniques used included: CHEF pulsed field gel electrophoresis; transposon mutagenesis; fragment hybridization to the ordered $\lambda$ library of Kohara et al.; fragment and cosmid hybridization to Southern blots; correlation of fragments and cleavage sites with EcoMap, a sequence-modified version of the genomic restriction map of Kohara et al.; and correlation of cleavage sites with DNA sequence databases. In all, 105 restriction sites were mapped and correlated with the EcoMap coordinate system.^ NotI, SfiI, XbaI and BlnI restriction patterns of five commonly used E. coli K-12 strains were compared to those of MG1655. The variability between strains, some of which are separated by numerous steps of mutagenic treatment, is readily detectable by pulsed-field gel electrophoresis. A model is presented to account for the difference between the strains on the basis of simple insertions, deletions, and in one case an inversion. Insertions and deletions ranged in size from 1 kb to 86 kb. Several of the larger features have previously been characterized and some of the smaller rearrangements can potentially account for previously reported genetic features of these strains.^ Some aspects of the frequency and distribution of NotI, SfiI, XbaI and BlnI cleavage sites were analyzed using a method based on Markov chain theory. Overlaps of Dam and Dcm methylase sites with XbaI and SfiI cleavage sites were examined. The one XbaI-Dam overlap in the database is in accord with the expected frequency of this overlap. The occurrence of certain types of SfiI-Dcm overlaps are overrepresented. Of the four subtypes of SfiI-Dcm overlap, only one has a partial inhibitory effect on the activity of SfiI. Recognition sites for all four enzymes are rarer than expected based on oligonucleotide frequency data, with this effect being much stronger for XbaI and BlnI than for NotI and SfiI. The latter two enzyme sites are rare mainly due to apparent negative selection against GGCC (both) and CGGCCG (NotI). The former two enzyme sites are rare mainly due to effects of the VSP repair system on certain di-tri- and tetranucleotides, most notably CTAG. Models are proposed to explain several of the anomalies of oligonucleotide distribution in E. coli, and the biological significance of the systems that produce these anomalies is discussed. ^