Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.

Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

The dystrophin gene, located at Xp21, codifies dystrophin, which is part of a protein complex responsible for the membrane stability of muscle cells. Its absence on muscle causes Duchenne Muscular Dystrophy (DMD), a severe disorder, while a defect of muscle dystrophin causes Becker Muscular Dystrophy (DMB), a milder disease. The replacement of the defective muscle through stem cells transplantation is a possible future treatment for these patients. Our objective was to analyze the potential of CD34+ stem cells from umbilical cord blood to differentiate in muscle cells and express dystrophin, in vitro. Protein expression was analyzed by Immunofluorescence, Western Blotting (WB) and Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR). CD34+ stem cells and myoblasts from a DMD affected patient started to fuse with muscle cells immediately after co-cultures establishment. Differentiation in mature myotubes was observed after 15 days and dystrophin-positive regions were detected through Immunofluorescence analysis. However, WB or RT-PCR analysis did not detect the presence of normal dystrophin in co-cultures of CD34+ and DMD or DMB affected patients' muscle cells. In contrast, some CD34+ stem cells differentiated in dystrophin producers' muscle cells, what was observed by WB, reinforcing that this progenitor cell has the potential to originate muscle dystrophin in vitro, and not just in vivo like reported before.

Heparin and penicillamine–hypotaurine–epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development

Anti-parasitic and anti-viral immune responses in insects

Insects encounter many microorganisms in nature and to survive they have developed counter measures against the invading pathogens. In Drosophila melanogaster research on insect immunity has mainly been focused on infections by bacteria and fungi. We have explored the immune response against natural infections of the parasite Octosporea muscaedomesticae and the Drosophila C virus as compared to natural infections of bacteria and fungi. By using Affymetrix Drosophila GeneChips, we were able to obtain 48 genes uniquely induced after parasitic infection. It was also clearly shown that natural infections led to different results than when injecting the pathogens. In order to search for the ultimate role of the lepidopteran protein hemolin, we used RNA interference (RNAi). We could show that injection of double stranded RNA (dsRNA) of Hemolin in pupae of Hyalophora cecropia led to embryonic malformation and lethality and that there was a sex specific difference. We continued the RNAi investigation of hemolin in another lepidopteran species, Antheraea pernyi, and discovered that hemolin was induced by dsRNA per se. A similar induction of hemolin was seen after infection with baculovirus and we therefore performed in vivo experiments on baculovirus infected pupae. We could show that a low dose of dsHemolin prolonged the period before the A. pernyi pupae showed any symptoms of infection, while a high dose led to a more rapid onset of symptoms. By performing in silico analysis of the hemolin sequence from A. pernyi in comparison with other Hemolin sequences, it was possible to select a number of sites that either by being strongly conserved or variable could be important targets for future studies of hemolin function.

Embryonic and larval development of haliotis tuberculata coccinea reeve: an indexed micro-photographic sequence

[EN] First description of the complete embryo and larval development of the Canarian abalone (Haliotis tuberculata coccinea Reeve.) was conducted along 39 stages from fertilization to the appearance of the third tubule on the cephalic tentacles and illustrated in a microphotographic sequence. Eggs obtained by induced spawning with hydrogen peroxide from the GIA captive broodstock were stocked at a density of 10 eggs/mL and kept at 23 0.5 BC for 62 h until the formation of the third tubule. Live eggs and larvae were continuously observed on a 24 h basis at a 3400 magnification under transmitted light. At each stages, specific morphological features, illustrated by microscopic photographs, were described, as well as the time required for their apparition. Fertilized eggs diameter was 205 8 mm (mean SD), whereas length and width of larvae ready to undergo metamorphosis were 216.6 5.3 mmand 172 8.8 mm, respectively. Knowledge on the larval morphological development acquired through this study will contribute to the improvement of larval rearing techniques for this abalone species.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Induction Motor Diagnosis in Variable Speed Drives

Several diagnostic techniques are presented for the detection of electrical fault in induction motor variable speed drives. These techinques are developed taking into account the impact of the control system on machine variables and non stationary operating conditions.

Ein transgenes Mausmodell zur Untersuchung des Einflusses von Matrix Metalloproteinase-2 und -14 auf die Leber

MMP-2 and MMP-14 process extracellular matrix proteins,cytokines, growth factors and adhesion molecules to generatefragments with enhanced or reduced biological activity.In this study, a vectorsystem was developed for theconditional expression of MMP-2 and MMP-14 in the liver oftransgenic mice. For this vectorsystem the murine albuminpromotor was chosen together with the cre/lox system toachieve an inducible MMP-expression in the liver.Only one of the MMP-14 transgenic lines expressed highamounts of active MMP-14 protein after recombination of thelox-P sites. In these mice MMP-14 was able to activate MMP-2and MMP-13 in vivo. However, none of the livers of MMP-14overexpressing mice showed no differences in liverweight,amount of extracellular matrixproteins and rate ofproliferation, apoptosis and tumor-induction when comparedto the liver of wildtype mice.On the other hand overexpression of MMP-2 was embryoniclethal in all MMP-2 transgenic lines. After crossing theMMP-2 transgenic mice with cre deleter mice, a cre mediatedrecombination could be shown at day 6.5 post coitum (pc).Some of the double transgenic embryos of one of thetransgenic lines had severe deformations of the head,especially of the telencephalon and the mesencephalon.It could be shown in this study that disregulation of MMP-2in early embryonic development is lethal but anoverexpression of MMP-14 has no influence on the embryonicdevelopment or the homeostasis of the adult liver.With this conditional vectorsystem it is to possible studythe influnce of MMP-2 and MMP-14 on fibrogenesis,regeneration and tumorgenesis in the liver of mice.

The role of Interleukin-12 in liver inflammation

Chronic liver inflammation during viral hepatitis is a major health problem worldwide. The role of proinflammatory cytokines, like IL-12, in breaking hepatic immune tolerance, and inducing acute liver inflammation and virus clearance is not clear. Nor is clear its role in uncontrolled severe inflammatory response, leading to fulminant hepatitis and hepatic failure. This work, focused in the study of the role of endogenous produced IL-12 in inducing hepatic inflammatory responses, demonstrates: In vitro, using adenovirus coding for IL-12, that hepatocytes stimulate CD4+ T cells in a tolerogenic manner, and that endogenous IL-12 is able to switch the immune response into Th1; and in vivo, that endogenous IL-12 induces hepatocyte damage and virus elimination in mice infected with adenovirus. In addition, and in order to study in vivo the relevance of IL-12 in acute inflammation, conditional IL-12 transgenic mice expressing IL-12 in the liver after cre-recombinase mediated induction were generated. For this purpose, an IL-12 fusion protein was created, which demonstrated high levels of bioactivity. Induction of IL-12 expression during embryonic development was achieved by crossbreeding with Act-Cre transgenic mice; induction of IL-12 expression in adult mice was achieved by a plasmid coding for the cre-recombinase. This study demonstrates that after induction, IL-12 is expressed in the liver of the transgenic mice. It also demonstrates that hepatic expression of IL-12 induces splenomegaly and liver inflammation, characterized by large infiltrations in portal tracts and veins, associated with hepatic damage, necrosis areas and lethality. Furthermore, constitutive hepatic IL-12 expression does not lead to abortion, but to total lethality, short after delivery. In conclusion, in this study, a transgenic mouse model has been generated, in which the expression of active IL-12 in the liver can be induced at any time; this model will be very helpful for studying hepatic pathologies. This study has also demonstrated that hepatic produced IL-12 is able of breaking liver tolerance inducing inflammation, virus elimination, severe hepatocyte damage, and lethality. These findings suggest IL-12 as a key cytokine in acute liver inflammation and fulminant hepatic failure. 5.1 Future studies Once the importance of IL-12 in inducing hepatic inflammation and virus elimination was demonstrated in this study, understanding the mechanisms of the IL-12 induced liver damage, and more important, how to avoid it will be the main focus in the future. It is very important to achieve hepatic inflammation for a more effective and faster viral elimination, but avoiding the toxicity of IL-12, which leads to massive liver injury and lethality is obviously necessary to allow IL-12 as therapy. For that purpose, future studies will be mainly base on three different points: 1. The determination of different cell populations present in the hepatic infiltration, which of them are responsible for liver injury, and as well their state of activation. 2. The measure of other pro- and anti-inflammatory cytokines and chemokines, which can play a role in IL-12-induced liver inflammation and hepatocyte damage. For these purposes, specific blocking antibodies (anti TNF-alpha, anti IL-12, anti IFN-g) will be used. The study with different transgenic mice: TNF-alpha Receptor knockout, TGF-b, will also help in determining the role of those cytokines during IL-12-induced liver damage and lethality. 3. The establishing of liver pathology models (viral infection, tumours, auto-antigens) in mice. Induction of IL-12 at any time of the pathology development will help in clarifying the role of IL-12 in those models. Finally, the transgenic mice expressing IL-23 in the liver will be generated.

Evaluation of a viral vector system, based on a defective interfering RNA of tomato bushy stunt virus, for protein expression and the induction of gene silencing

A viral vector system was developed based on a DI-RNA, a sub-viral particle derived from TBSV-BS3-statice. This newly designed vector system was tested for its applicability in protein expression and induction of gene silencing. Two strategies were pursued. The first strategy was replication of the DI-RNA by a transgenically expressed TBSV replicase and the second was the replication by a so called helper virus. It could be demonstrated by northern blot analysis that the replicase, expressed by the transgenic N. benthamiana plant line TR4 or supplied by the helper virus, is able to replicate DI-RNA introduced into the plant cells. Various genes were inserted into different DI constructs in order to study the vector system with regard to protein expression. However, independent of how the replicase was provided no detectable amounts of protein were produced in the plants. Possible reasons for this failure are identified: the lack of systemic movement of the DI-RNA in the transgenic TR4 plants and the occurrence of deletions in the inserted genes in both systems. As a consequence the two strategies were considered unsuitable for protein expression. The DI-RNA vector system was able to induce silencing of transgenes as well as endogenous genes. Several different p19 deficient helper virus constructs were made to evaluate their silencing efficiency in combination with our DI-RNA constructs. However, it was found that our vector system can not compete with other existing VIGS (virus induced gene silencing) systems in this field. Finally, the influence of DI sequences on mRNA stability on transient GUS expression experiments in GUS silenced plants was evaluated. The GUS reporter gene system was found to be unsuitable for distinguishing between expression levels of wild type plants and GUS silenced transgenic plants. The results indicate a positive effect of the DI sequences on the level of protein expression and therefore further research into this area is recommended.

Tetracycline-inducible RNAi Knockdown of SCL (Stem Cell Leukaemia) in Mice

RNAi (RNA interference) is a powerful technology for sequence-specific targeting of mRNAs. This thesis was aimed at establishing conditions for conditional RNAi-mediated silencing first in vitro and subsequently also in transgenic mice. As a target the basic helix-loop-helix transcription factor encoding gene SCL (stem cell leukaemia also known as Tal-1 or TCL5) was used. SCL is a key regulator for haematopoietic development and ectopic expression of SCL is correlated with acute T-lymphoblastic leukaemias. Loss of SCL function studies demonstrated that ab initio deletion of SCL resulted in embryonic lethality around day E9 in gestation. To be able to conditionally inactivate SCL, RNAi technology was combined with the tetracycline-dependent regulatory system. This strategy allowed to exogenously control the induction of RNAi in a reversible fashion and consequently the generation of a completely switchable RNAi knockdown. First a suitable vector allowing for co-expression of tetracycline-controlled shRNAs (small hairpin RNAs) and constitutively active EGFP (enhanced green fluorescent protein) was generated. This novel vector, pRNAi-EGFP, was then evaluated for EGFP expression and tetracycline-mediated expression of shRNAs. Four sequences targeting different regions within the SCL mRNA were tested for their efficiency to specifically knockdown SCL. These experiments were performed in M1 murine leukaemia cells and subsequently in the HEK 293 cell line, expressing an engineered HA-tagged SCL protein. The second assay provided a solid experimental method for determining the efficiency of different SCL-siRNA knockdown constructs in tissue culture. Western blotting analyses revealed a down regulation of SCL protein for all four tested SCL-specific target sequences albeit with different knockdown efficiencies (between 25% and 100%). Furthermore, stringent tetracycline-dependent switchability of shRNA expression was confirmed by co-transfecting the SCL-specific pRNAi-EGFP vector (SCL-siRNA) together with the HA-tagged SCL expression plasmid into the HEK 293TR /T-REx cell line constitutively expressing the tetracycline repressor (TetR). These series of experiments demonstrated tight regulation of siRNA expression without background activity. To be able to control the SCL knockdown in vivo and especially to circumvent any possible embryonic lethality a transgenic mouse line with general expression of a tetracycline repressor was needed. Two alternative methods were used to generate TetR mice. The first approach was to co-inject the tetracycline-regulated RNAi vector together with a commercially available and here specifically modified T-REx expression vector (SCL-siRNA T-REx FRT LoxP mouse line). The second method involved the generation of a TetR expressor mouse line, which was then used for donating TetR-positive oocytes for pronuclear injection of the RNAi vector (SCL-siRNA T-REx mouse line). As expected, and in agreement with data from conditional Cre-controlled adult SCL knockout mice, post-transcriptional silencing of SCL by RNAi caused a shift in the maturation of red blood cell populations. This was shown in the bone marrow and peripheral blood by FACS analysis with the red blood cell-specific TER119 and CD71 markers which can be used to define erythrocyte differentiation (Lodish plot technique). In conclusion this study established conditions for effective SCL RNAi-mediated silencing in vitro and in vivo providing an important tool for further investigations into the role of SCL and, more generally, of its in vivo function in haematopoiesis and leukaemia. Most importantly, the here acquired knowledge will now allow the establishment of other completely conditional and reversible knockdown phenotypes in mice.

Untersuchungen zur Funktion und Regulation der Rezeptor-Tyrosin-Phosphatase RPTPlambda bei der Entwicklung des Mittel- und Hinterhirns

Der isthmische Organisator liegt an der Grenze zwischen dem sich entwickelnden Mittel- und Hinterhirn und kontrolliert Wachstum und Musterbildung dieser beiden Hirnregionen. In der vorliegenden Arbeit wird die räumliche und zeitliche Expression der Rezeptor-ähnlichen Protein Tyrosin Phosphatase lambda aus dem Huhn (cRPTPλ, auch als cRPTPψ bekannt) während der Entwicklung dieser Struktur beschrieben. Nach einer anfänglich weitläufigen Expression im kaudalen Vorderhirn und in der Mittelhirnregion, beschränkt sich die Expression von cRPTPλ zwischen dem embryonalen Tag E2 und E3.5 auf die ventrale Mittellinie des Neuralrohrs, den Bereich der späteren neuralen Retina und Linse und auf einen schmalen Ring anterior der isthmischen Einschnürung, welcher der molekularen Mittel- / Hinterhirngrenze (MHO) entspricht. Ab dem embryonalen Tag E3.5 wird RPTPλ dann auch im gesamten Mittelhirn gebildet. Um Hinweise auf die Funktion von cRPTPλ zu bekommen, wurde die Regulation dieses Moleküls untersucht. Die Expression von cRPTPλ am MHO wird von dem Fibroblasten Wachstumsfaktor Fgf8 und dem Transkriptionsfaktor Lmx1b, nicht aber von dem sezernierten Glykoprotein Wnt1 induziert. Der Transkriptionsfaktor En-1 unterdrückt die Expression von cRPTPλ am MHO. cRPTPλ-Expression im Mittelhirn wird negativ durch das sezernierte Protein Sonic Hedgehog reguliert, während Lmx1b und En-1 dort keinen Einfluss auf das Expressionsmuster von cRPTPλ haben. Fgf8 und Wnt1 sind maßgeblich an der Regulation von Wachstum und Musterbildung des embryonalen Mittelhirns beteiligt. Funktionelle Studien zu RPTPλ deuten darauf hin, dass dieses Protein als negativer Rückkopplungsmechanismus beider Signalwege wirken kann. RNAi- und Überexpressionsstudien am MHO lieferten Hinweise darauf, dass RPTPλ der Induktion der Wnt1-Expression durch Fgf8 entgegenwirkt. Dies scheint durch Interaktion noch unbekannter Faktoren mit der Juxtamembrandomäne von RPTPλ vermittelt zu werden. Auf das Expressionsmuster von Fgf8 selbst, oder einer Reihe anderer Faktoren, die ebenfalls von Fgf8 reguliert werden, hat RPTPλ allerdings keinen Einfluss. Des Weiteren konnte in dieser Arbeit gezeigt werden, dass eine „künstliche“ Aufrechterhaltung der Expression von cRPTPλ im Mittelhirn zwischen dem embryonalen Tag E2 und E3.5 zu einem stark verkleinerten Mesenzephalon führt. RPTPλ bindet in vivo an β-Catenin, ein zentrales Protein des kanonischen Wnt-Signalweges, und moduliert dadurch vermutlich das Wnt-Signal, welches seinerseits Proliferation im Mesenzephalon fördert. Durch diesen Mechanismus könnte cRPTPλ als „Bremse“ des kanonischen Wnt-Signalweges im Mittelhirn wirken.

Analyse der konditionell-induzierten Überexpression von Smad7 in epithelialen Geweben der adulten Maus

Smad7 ist eine inhibitorische Komponente der TGF-β- bzw. Activin-Signalweiterleitung und erfüllt eine wichtige Aufgabe bei deren Regulation. So führt eine konstitutive Überexpression von Smad7 in epithelialen Geweben zum Auftreten verschiedener Phänotypen, wie embryonaler bzw. perinataler Letalität, Hyperproliferation der Epidermis und Thymusatrophie. Auch die Entwicklung der T-Zellen im Thymus und epithelialer Anhangsgebilde wie z.B. von Haaren und Zähnen wird dadurch beeinträchtigt. In dieser Arbeit sollte nun in der adulten Maus der Effekt einer Überexpression von Smad7 in epithelialen Geweben untersucht werden. Zu diesem Zweck wurde ein, auf dem Cre/loxP-Prinzip beruhendes Transgensystem verwendet (K5-Smad7-tg und K14-creERT2), welches eine konditionell-induzierte Überexpression von Smad7 in epithelialen Zellen der adulten Maus erlaubte. Die so gezüchteten doppeltransgenen Tiere wiesen keine signifikanten Veränderungen gegenüber ihren wildtyp bzw. einfachtransgenen Geschwistertieren auf. Die Überexpression von Smad7 in epithelialen Geweben der adulten Maus zu einem Auftreten verschiedenster veränderter Phänotypen der Haut und deren Anhänge, sowie der Schneidezähne. Bei diesen Tieren konnte auch ein signifikanter Körpergewichtsverlust und eine Erhöhung der Mortalitätsrate beobachtet werden, welche sich im Verlauf nach erfolgter Rekombination einstellte. Weitere Analysen zeigten signifikante Veränderungen in der Haut und im Thymus. So konnte in der Haut eine Erhöhung der Proliferationsrate epidermaler Zellen, eine reduzierte Expression von Smad3 und im Thymus Veränderungen in der Gesamtzahl der lebenden T-Zellen und deren Differenzierung beobachtete werden. Mit dieser Arbeit konnte gezeigt werden, daß die Hemmung der Signalweiterleitung der TGF-β-Superfamilie, speziell von TGF-β und Activin, zu verschiedenen morphogenetischen Defekten der Haut und deren Anhänge, der Zähne und der T-Zellentwicklung im Thymus führt.