Behavioural analysis of mouse mutants with altered neuronal and glial genes

Im ersten Teil dieser Doktorarbeit beabsichtigte meine Arbeit, die funktionelle Beteiligung des CB1 Rezeptors, einer Hauptkomponente des neuronalen Endocannabinoid-Systems (ECS), an der Ausbildung von verschiedenen Verhaltensphänotypen mit Hilfe von konditionalen Mausmutanten, denen der CB1 Rezeptor auf verschiedenen neuronalen Unterpopulationen fehlt, aufzuschlüsseln und zu untersuchen. Verschiedene Verhaltensmodelle wurden hierzu getestet. Dabei lag der Fokus dieser Arbeit auf der CB1f/f;D1-Cre Mauslinie, welche der CB1 Rezeptor auf den D1 Rezeptor exprimierenden Neuronen des Striatums fehlt. Ich konnte zeigen, dass der Verlust des CB1 Rezeptors auf diesen Neuronen keinen Einfluss auf basale neurologische Funktionen, Gewicht, Bewegung, Exploration, Sozialverhalten, Angst und Stressbewältigung der Tiere hat, jedoch eine Beteiligung an der Entwicklung von Suchtverhalten gegeben ist. Bei Betrachtung des Kokain-induzierten Suchtverhaltens zeigten die konditionalen Mausmutanten eine reduzierte Suchtanfälligkeit sowohl im Vergleich zu Tieren mit einem totalen CB1 Rezeptor Verlust in allen Körperzellen, als auch zu genetisch unveränderten Kontrollmäusen beider Linien.rnDes Weiteren zeigen die Ergebnisse dieser Studie eine große, aber gegensätzliche Beteiligung des ECS bei der Regulation von Exploration in Abhängigkeit des Verlustes des CB1 Rezeptors auf GABAergen Neuronen des Vorderhirns und kortikalen glutamatergen Neuronen, jedoch nicht auf striatalen Neuronen alleine. Zusätzlich war ich in der Lage, die Wichtigkeit des genetischen Hintergrunds von Mauslinien nicht nur auf die Ausbildung von spezifischen Verhaltensphänotypen, sondern auch auf die Genexpression zu zeigen.rnIn dem zweiten Teil dieser Arbeit, in dem ich mich auf die Funktion von Gliazellen konzentrierte, wurden ebenfalls Mausmutanten in verschiedenen Verhaltensmodellen getestet. Ein genetisches Auslöschen des NG2 Glykoproteins in Gliazellen sorgt in den Knock-out Mäusen für ein schlechteres Hörvermögen und ein reduziertes Depressionsverhalten im Vergleich zu ihren Wildtyp-Kontrollmäusen. Interessanterweise zeigten diese Tiere auch eine reduzierte Empfänglichkeit bei chemisch induzierten epileptischen Krämpfen, was eine Rolle des NG2 Glykoproteins bei der Kontrolle der glutamatergen Homöostase vorschlägt, die wahrscheinlich durch Strukturänderungen der Neuron-Glia-Synapse verursacht wird. rn

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

Microscopic analysis of chromatin localization and dynamics in C. elegans

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system.

Analysis of clonality and antibiotic resistance among early clinical isolates of Enterococcus faecium in the United States.

BACKGROUND: The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments. METHODS: Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994, we determined the multilocus sequence type; the presence of 16 putative virulence genes (hyl(Efm), esp(Efm), and fms genes); resistance to ampicillin (AMP) and vancomycin (VAN); and high-level resistance to gentamicin and streptomycin. RESULTS: Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the United States. The earliest CC17 isolates were part of an outbreak that occurred in 1982 in Richmond, Virginia. The characteristics of CC17 isolates included increases in resistance to AMP, the presence of hyl(Efm) and esp(Efm), emergence of resistance to VAN, and the presence of at least 13 of 14 fms genes. Eight of 41 of the early isolates with resistance to AMP, however, were not in CC17. CONCLUSIONS: Although not all early US AMP isolates were clonally related, E. faecium CC17 isolates have been circulating in the United States since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment.

Analysis of a human placental lactogen gene promoter

Human placental lactogen (hPL) and human growth hormone (hGH) comprise a multigene family that share $>$90% nucleic acid sequence homology including 500 bp of 5$\sp\prime$ flanking sequence. Despite these similarities, hGH is produced in the anterior pituitary while hPL is expressed in the placenta. For most genes studied to date, regulation of expression occurs by alterations at the level of transcriptional initiation. Nuclear proteins bind specific DNA sequences in the promoter to regulate gene expression. In this study, the hPL$\sb3$ promoter was analyzed for DNA sequences that contribute to its expression. The interaction between the hPL$\sb3$ promoter and nuclear proteins was examined using nuclear extracts from placental and non-placental cells.^ To identify regulatory elements in the promoter of the hPL$\sb3$ gene, 5$\sp\prime$ deletion mutants were constructed by cleaving 1200 bp of upstream sequence with various restriction enzymes. These DNA fragments were ligated 5$\sp\prime$ to a promoterless bacterial gene chloramphenicol acetyltransferase (CAT) and transfected into JEG-3 cells, a human placental choriocarcinoma cell line. The level of CAT activity reflects the ability of the promoter mutants to activate transcription. Deletion of the sequence between $-$142 bp and $-$129 bp, relative to the start of transcription, resulted in an 8-fold decrease in CAT activity. Nuclear proteins from JEG-3, HeLa, and HepG2 (human liver cells), formed specific binding complexes with this region of the hPL$\sb3$ promoter, as shown by gel mobility shift assay. The $-$142 bp to $-$129 bp region contains a sequence similar to that of a variant binding site for the transcription factor Sp1. Sp1-like proteins were identified by DNA binding assay, in the nuclear extracts of the three cell lines. A series of G nucleotides in the hPL$\sb3$ promoter regulatory region were identified by methylation interference assay to interact with the DNA-binding proteins and the pattern obtained is similar to that for other Sp1 binding sites that have been studied. This suggests that hPL$\sb3$ may be transcriptionally regulated by Sp1 or a Sp1-like transacting factor. ^

Analysis of mutations in $\beta$-tubulin genes affecting tubulin stability and assembly

Cmd4 is a colcemid-sensitive CHO cell line that is temperature sensitive for growth and expresses an altered $\beta$-tubulin, $\beta\sb1$. One revertant of this cell line, D2, exhibits a further alteration in $\beta\sb1$ resulting in an acidic shift in its isoelectric point and a decrease in its molecular weight to 40 kD, as measured by two dimensional gel electrophoresis. This $\beta$-tubulin variant has been shown to be assembly-defective and unstable. Characterization of the mutant $\beta\sb1$ in D2 by high pressure liquid chromatography (HPLC) revealed the loss of methionine containing tryptic peptides 7,8,9, and 10. Southern analysis of the genomic DNA digested with several different restriction enzymes resulted in the appearance of new restriction fragments 250 base pairs shorter than the corresponding fragments from the wild-type $\beta\sb1$-tubulin gene. Northern analysis on mRNA from D2 revealed two new message products that also differed by 250 bases from the corresponding wild type $\beta$-tubulin transcripts. To precisely define the region of the alteration, cloning and sequencing of the mutant and wild type genomic $\beta$-tubulin genes were conducted. A size-selected EcoRI genomic library was prepared using the Stratagene lambda Zap II phage cloning system. Using subclones of CHO $\beta$-tubulin cDNA as probes, a 2.5 kb wild type clone and a 2.3 kb mutant clone were identified from this library. Each of these was shown to contain a portion of the gene extending from intron 3 through the end of the coding sequence in exon 4 and into the 3$\sp\prime$ untranslated region on the basis of alignment with the published human $\beta$-tubulin sequence. Sequencing of the mutant 2.3 kb clone revealed that the mutation is due to a 246 base pair internal deletion in exon 4 (base pair 756-1001) that encodes amino acids 253-334. This deletion results in the loss of a putative binding site for GTP which could potentially explain the phenotype of this mutant $\beta$-tubulin. Also sequence comparison of the 3$\sp\prime$ untranslated region between different species revealed the conservation of 200 base pairs with 78% homology. It is proposed that this region could play an important role in the regulation of $\beta$-tubulin gene expression. ^

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

How comparable are parallel corpora? Measuring the distribution of general vocabulary and connectives

In this paper, we question the homogeneity of a large parallel corpus by measuring the similarity between various sub-parts. We compare results obtained using a general measure of lexical similarity based on χ2 and by counting the number of discourse connectives. We argue that discourse connectives provide a more sensitive measure, revealing differences that are not visible with the general measure. We also provide evidence for the existence of specific characteristics defining translated texts as opposed to non-translated ones, due to a universal tendency for explicitation.

CYTOGENETIC AND DNA CONTENT ANALYSIS IN MULTIPLE MYELOMA

In this dissertation, the cytogenetic characteristics of bone marrow cells from 41 multiple myeloma patients were investigated. These cytogenetic data were correlated with the total DNA content as measured by flow cytometry. Both the cytogenetic information and DNA content were then correlated with clinical data to determine if diagnosis and prognosis of multiple myeloma could be improved.^ One hundred percent of the patients demonstrated abnormal chromosome numbers per metaphase. The average chromosome number per metaphase ranged from 42 to 49.9, with a mean of 44.99. The percent hypodiploidy ranged from 0-100% and the percent hyperdiploidy from 0-53%. Detailed cytogenetic analyses were very difficult to perform because of the paucity of mitotic figures and the poor chromosome morphology. Thus, detailed chromosome banding analysis on these patients was impossible.^ Thirty seven percent of the patients had normal total DNA content, whereas 63% had abnormal amounts of DNA (one patient with less than normal amounts and 25 patients with greater than normal amounts of DNA).^ Several clinical parameters were used in the statistical analyses: tumor burden, patient status at biopsy, patient response status, past therapy, type of treatment and percent plasma cells. Only among these clinical parameters were any statistically significant correlations found: pretreatment tumor burden versus patient response, patient biopsy status versus patient response and past therapy versus patient response.^ No correlations were found between percent hypodiploid, diploid, hyperdiploid or DNA content, and the patient response status, nor were any found between those patients with: (a) normal plasma cells, low pretreatment tumor mass burden and more than 50% of the analyzed metaphases with 46 chromosomes; (b) normal amounts of DNA, low pretreatment tumor mass burden and more than 50% of the metaphases with 46 chromosomes; (c) normal amounts of DNA and normal quantities of plasma cells; (d) abnormal amounts of DNA, abnormal amounts of plasma cells, high pretreatment tumor mass burden and less than 50% of the metaphases with 46 chromosomes.^ Technical drawbacks of both cytogenetic and DNA content analysis in these multiple myeloma patients are discussed along with the lack of correlations between DNA content and chromosome number. Refined chromosome banding analysis awaits technical improvements before we can understand which chromosome material (if any) makes up the "extra" amounts of DNA in these patients. None of the correlations tested can be used as diagnostic or prognostic aids for multiple myeloma. ^

Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

Policy analysis of shifting to the state-run Texas women's health program

During the 82nd Texas legislature, state leaders passed a provision stating that healthcare providers, who perform, promote, or affiliate with providers who perform or promote elective abortion services may not be eligible to participate in the Texas Medicaid Women's Health Program (WHP). The federal government reacted to this new provision by vowing to eliminate its 90% share of program support on the grounds that the provision violated a patient's freedom to choose a provider; a right protected by the Social Security Act. Texas leaders stated that the Women's Health Program would continue without federal support, financed exclusively with state funds.^ The following policy analysis compares the projected impact of the current Medicaid Women's Health Program to the proposed state-run program using the criteria-alternative matrix framework. The criteria used to evaluate the program alternatives include population affected, unintended pregnancy and abortion impact, impact on cervical cancer rate, and state-level government expenditures. Each criterion was defined by selected measures. The population affected was measured by the number of women served in the programs. Government expenditures were measured in terms of payments for program costs, Medicaid delivery costs, and cervical cancer diagnostic costs. Unintended pregnancy impact was measured by the number of projected unplanned pregnancies and abortions under each alternative. The impact on cervical cancer was projected in terms of the number of new cervical cancer cases under each alternative. Differences in the projections with respect to each criterion were compared to assess the impact of shifting to the state-only policy.^ After examining program alternatives, it is highly recommended that Texas retain the Medicaid WHP. If the state does decide to move forward with the state-run WHP, it is recommended that the program run at its previous capacity. Furthermore, for the purpose of addressing the relatively high cervical cancer incidence rate in Texas, incorporating HPV vaccination coverage for women ages 18-26 as part of the Women's Health Program is recommended.^

Combined metabolome and proteome analysis of the mantle tissue from Pacific oyster Crassostrea gigas exposed to elevated pCO2

Ocean acidification (OA) has been found to affect an array of normal physiological processes in mollusks, especially posing a significant threat to the fabrication process of mollusk shell. In the current study, the impact of exposure to elevated pCO2 condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO2 could affect energy metabolism in oyster C. gigas, marked by differentially altered ATP, succinate, MDH, PEPCK and ALDH levels. Moreover, the up-regulated calponin-2, tropomyosins and myosin light chains indicated that elevated pCO2 probably caused disturbances in cytoskeleton structure in mantle tissue of oyster C. gigas. This work demonstrated that a combination of proteomics and metabolomics could provide important insights into the effects of OA at molecular levels.

Post-decentralization regional economies and actors -- putting the capacity of Local governments to the test

Decentralization in Indonesia was introduced institutionally in 2001, with a democratization drive promoted by international donors and by the intention of the new government to clear away the centralistic image of Soeharto. Decentralization has had some effects on regional economies and on local government administration. Compared to the period before decentralization, the share of gross regional domestic product and local government finance has increased in Java, though investment and bank borrowing have expanded to the outer islands. In qualitative aspects, decentralization has transferred not only administrative authority but also many new vested interests from the center to regions. Local governments have become more extensive economic actors in regional economies. Regional economic actors now compete actively for such vested interests and have missed the opportunity to create market-friendly regional economies. The government sector should not be a mere rent-seeking economic actor, but should play a role as a facilitator promoting private sector activities in regional economies.

Application of the Input-Output Decomposition Technique to China's Regional Economies

Structural decomposition techniques based on input-output table have become a widely used tool for analyzing long term economic growth. However, due to limitations of data, such techniques have never been applied to China's regional economies. Fortunately, in 2003, China's Interregional Input-Output Table for 1987 and Multi-regional Input-Output Table for 1997 were published, making decomposition analysis of China's regional economies possible. This paper first estimates the interregional input-output table in constant price by using an alternative approach: the Grid-Search method, and then applies the standard input-output decomposition technique to China's regional economies for 1987-97. Based on the decomposition results, the contributions to output growth of different factors are summarized at the regional and industrial level. Furthermore, interdependence between China's regional economies is measured and explained by aggregating the decomposition factors into the intraregional multiplier-related effect, the feedback-related effect, and the spillover-related effect. Finally, the performance of China's industrial and regional development policies implemented in the 1990s is briefly discussed based on the analytical results of the paper.

A generic framework for context-sensitive analysis of modular programs

Context-sensitive analysis provides information which is potentially more accurate than that provided by context-free analysis. Such information can then be applied in order to validate/debug the program and/or to specialize the program obtaining important improvements. Unfortunately, context-sensitive analysis of modular programs poses important theoretical and practical problems. One solution, used in several proposals, is to resort to context-free analysis. Other proposals do address context-sensitive analysis, but are only applicable when the description domain used satisfies rather restrictive properties. In this paper, we argüe that a general framework for context-sensitive analysis of modular programs, Le., one that allows using all the domains which have proved useful in practice in the non-modular setting, is indeed feasible and very useful. Driven by our experience in the design and implementation of analysis and specialization techniques in the context of CiaoPP, the Ciao system preprocessor, in this paper we discuss a number of design goals for context-sensitive analysis of modular programs as well as the problems which arise in trying to meet these goals. We also provide a high-level description of a framework for analysis of modular programs which does substantially meet these objectives. This framework is generic in that it can be instantiated in different ways in order to adapt to different contexts. Finally, the behavior of the different instantiations w.r.t. the design goals that motivate our work is also discussed.