<p>Picropodophyllin (PPP) is an analogue of the anti tumour lignan podophyllotoxin with the unique ability to selectively inhibit the receptor of Insulin like growth factor 1(IGF-1). IGF-1 is believed to play an important part in development of the endometrium facing implantation. With PPP treated mice, studies can be made to measure gene expression from tissue of both treated and untreated mice to compare the role of IGF-1 regarding ovulation, implantation and receptivity. The aim of this study was to analyze gene expression of some steroid hormone receptors and cytokines in ovaries from mice treated with PPP. In this study, seven mice were treated with PPP at different times and tissue was collected. PCR-primers for cDNA sequences of estrogene receptor α, estrogene receptor β, progesterone receptor A, progesterone receptor B, growth hormone receptor, interleukin 1 α, interleukin 1 β, tumour necrosis factor α and androgen receptor were used. Real Time PCR was run with the samples and gene expression was measured. The results of this study showed that the inhibition of IGF-1 receptor interacted with IGF-1 which lead to altered levels of estrogene receptor alpha, progesterone receptor, growth hormone receptor and androgen receptor that can decrease ovulation. The results also showed the differences in gene products between treated and untreated samples, suggesting that IGF-1 plays an important role regarding ovulation.</p> / <p>Studier med hjälp av den selektiva insulinlika tillväxtfaktor 1 receptorn (IGF-1R) antagonisten; picropodof?phyllin (PPP), hur samspelet mellan livmoderslemhinnan och implantationsprocessen, samt hur ovulationen påverkas av insulinlika tillväxtfaktorn 1 (IGF-1) kan nu utföras. IGF-1 tros ha en viktig roll för den reproduktiva processen, där den påverkar ovulation, implantation och embryoutveckling. IGF-familjen består av tre ligander; insulin, IGF-1 och IGF-2. IGF transporteras bundet till bindarprotein (IGFBP). Medlemmarna i IGF receptorfamiljen kan binda IGF-1, IGF-2 och insulin fast med olika affinitet. PPP som är en cykloligan, är en analog från podofyllotoxin och fungerar som en syntetisk IGF-1 receptorantagonist, som selektivt inhiberar receptorns aktivitet. PPP tros även kunna nedreglera genexpression av receptorn. Tre tidigare projektarbeten har utförts på vävnader från möss injicerade med PPP. Tyngdpunkterna i dessa arbeten har legat på immunhistokemiska studier av IGF-1 i reproduktionsorgan från möss, uttryck av IGF-1, dess receptor och bindarprotein 1 i ovarier och uterus efter behandling med PPP. I denna studie användes vävnad samt cDNA från sju möss behandlade med PPP, i olika stadier av reproduktionen samt även icke behandlade möss. Studiens syfte var att med sanntids-PCR jämföra genuttryck från östrogenreceptor α och β, progesteronreceptor A och B, tillväxthormonreceptor, Interleukin 1 α och β, ’tumor necrosis’ faktor α samt androgenreceptor i vävnad från PPP-behandlade och obehandlade möss och genom de erhållna resultaten från ovarievävnaden utläsa effekten på ovulationen och från uterusvävnaden effekten på implantation och receptivitet. Studieresultaten visade att IGF-1s frånvaro gav förändrade nivåer av genprodukter, som medförde minskad ovulationen. Studien visade att IGF-1s roll vid ovulationen var väsentlig.</p>

The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields. To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Picropodophyllin (PPP) is an analogue of the anti tumour lignan podophyllotoxin with the unique ability to selectively inhibit the receptor of Insulin like growth factor 1(IGF-1). IGF-1 is believed to play an important part in development of the endometrium facing implantation. With PPP treated mice, studies can be made to measure gene expression from tissue of both treated and untreated mice to compare the role of IGF-1 regarding ovulation, implantation and receptivity. The aim of this study was to analyze gene expression of some steroid hormone receptors and cytokines in ovaries from mice treated with PPP. In this study, seven mice were treated with PPP at different times and tissue was collected. PCR-primers for cDNA sequences of estrogene receptor α, estrogene receptor β, progesterone receptor A, progesterone receptor B, growth hormone receptor, interleukin 1 α, interleukin 1 β, tumour necrosis factor α and androgen receptor were used. Real Time PCR was run with the samples and gene expression was measured. The results of this study showed that the inhibition of IGF-1 receptor interacted with IGF-1 which lead to altered levels of estrogene receptor alpha, progesterone receptor, growth hormone receptor and androgen receptor that can decrease ovulation. The results also showed the differences in gene products between treated and untreated samples, suggesting that IGF-1 plays an important role regarding ovulation. / Studier med hjälp av den selektiva insulinlika tillväxtfaktor 1 receptorn (IGF-1R) antagonisten; picropodof?phyllin (PPP), hur samspelet mellan livmoderslemhinnan och implantationsprocessen, samt hur ovulationen påverkas av insulinlika tillväxtfaktorn 1 (IGF-1) kan nu utföras. IGF-1 tros ha en viktig roll för den reproduktiva processen, där den påverkar ovulation, implantation och embryoutveckling. IGF-familjen består av tre ligander; insulin, IGF-1 och IGF-2. IGF transporteras bundet till bindarprotein (IGFBP). Medlemmarna i IGF receptorfamiljen kan binda IGF-1, IGF-2 och insulin fast med olika affinitet. PPP som är en cykloligan, är en analog från podofyllotoxin och fungerar som en syntetisk IGF-1 receptorantagonist, som selektivt inhiberar receptorns aktivitet. PPP tros även kunna nedreglera genexpression av receptorn. Tre tidigare projektarbeten har utförts på vävnader från möss injicerade med PPP. Tyngdpunkterna i dessa arbeten har legat på immunhistokemiska studier av IGF-1 i reproduktionsorgan från möss, uttryck av IGF-1, dess receptor och bindarprotein 1 i ovarier och uterus efter behandling med PPP. I denna studie användes vävnad samt cDNA från sju möss behandlade med PPP, i olika stadier av reproduktionen samt även icke behandlade möss. Studiens syfte var att med sanntids-PCR jämföra genuttryck från östrogenreceptor α och β, progesteronreceptor A och B, tillväxthormonreceptor, Interleukin 1 α och β, ’tumor necrosis’ faktor α samt androgenreceptor i vävnad från PPP-behandlade och obehandlade möss och genom de erhållna resultaten från ovarievävnaden utläsa effekten på ovulationen och från uterusvävnaden effekten på implantation och receptivitet. Studieresultaten visade att IGF-1s frånvaro gav förändrade nivåer av genprodukter, som medförde minskad ovulationen. Studien visade att IGF-1s roll vid ovulationen var väsentlig.

Microplitis croceipes is a solitary endoparasitic wasp that oviposits in the hemocoel of Heleothis virescens larvae. Upon parasitization, the host larvaes physiology is altered; resulting in a compromised immune system and a decrease in the production of some vital proteins resulting in a terminal post-wandering prepupal state. Teratocytes, cells derived from the extraembryonic serosa of the parasitic wasp, mimic symptoms of parasitization when injected into host larvae, independent of other factors like polydnavirus and venom. Some of the inhibition of protein synthesis can be attributed to proteins secreted by the teratocytes (teratocyte secretory proteins or TSP). A fraction of TSP between 330 kDa inhibits protein synthesis in vivo, in the in vitro fat body and testes assays, and in the rabbit reticulocyte lysate and wheat germ extract assays. This fraction, however, has no effect on nucleic acid synthesis. Its effect on protein synthesis is dose dependent and exposure time sensitive. A 13.9 kDa protein isolated from TSP and expressed in a baculovirus system seems primarily responsible for the inhibition. Although TSP14 production was low, it did bind to the cell surface, enter the cell, and inhibit protein synthesis as the 330 kDa factor did.

abstract: Here I document the breadth of the CAP (Cysteine-RIch Secretory Proteins (CRISP), Antigen 5 (Ag5), and the Pathogenesis-Related 1 (PR)) protein superfamily and trace some of the major events in the evolution of this family with particular focus on vertebrate CRISP proteins. Specifically, I sought to study the origin of these CAP subfamilies using both amino acid sequence data and gene structure data, more precisely the positions of exon/intron borders within their genes. Counter to current scientific understanding, I find that the wide variety of CAP subfamilies present in mammals, where they were originally discovered and characterized, have distinct homologues in the invertebrate phyla contrary to the common assumption that these are vertebrate protein subfamilies. In addition, I document the fact that primitive eukaryotic CAP genes contained only one exon, likely inherited from prokaryotic SCP-domain containing genes which were, by nature, free of introns. As evolution progressed, an increasing number of introns were inserted into CAP genes, reaching 2 to 5 in the invertebrate world, and 5 to 15 in the vertebrate world. Lastly, phylogenetic relationships between these proteins appear to be traceable not only by amino acid sequence homology but also by preservation of exon number and exon borders within their genes. / Dissertation/Thesis / Masters Thesis Biology 2016

Fehlerhafte sekretorische Proteine werden über einen speziellen Abbauweg, die ER-assoziierte Proteindegradation (ERAD), mit Lysin48-verknüpften Ubiquitinketten polyubiquitiniert und dem proteolytischen Abbau am 26S Proteasom zugeführt. In der Hefe Saccharomyces cerevisiae bilden die beiden ER-membranständigen RING-Ubiquitinligasen Hrd1 und Doa10 zentrale Komponenten im Ubiquitinierungsprozess. Das lösliche zytosolische Ubiquitin-konjugierende Enzym Ubc7, welches mit beiden Ligasen bei der Polyubiquitinierung von Substratproteinen zusammenwirkt, wird über den membranverankerten Co-Faktor Cue1 an die ER-Membran rekrutiert. Die in dieser Arbeit dargestellten Ergebnisse belegen zwei weitere Funktionen für Cue1 im Ubiquitinierungsprozess: Die Bindung von Ubc7 an einen carboxyterminalen Bereich in Cue1 führt zur Stimulation der Ubiquitinierungsaktivität von Ubc7 mit den RING-Ligasen. Darüber hinaus bewirkt die Ubiquitin-bindende CUE-Domäne in Cue1 eine Steigerung der Länge der Ubiquitinketten und deren Syntheserate, was zum effektiven Abbau einiger ER-membrangebundener Substratproteine beiträgt. Die durch Ubc7 synthetisierten Lysin48-verknüpften Ubiquitinketten werden in Abhängigkeit eines schleifenförmigen sauren Bereichs in Ubc7 gebildet. Entfernen dieses Bereichs resultiert im Abbruch der Ubiquitinierung nach Konjugation eines Monoubiquitins auf dem Substrat. An der Hrd1-Ligase werden durch Ubc7 polyubiquitinierte Proteine umgehend zum Proteasom transferiert. Für den Doa10-abhängigen Substratabbau ist die Funktion eines weiteren Ubiquitin-konjugierenden Enzyms, Ubc6, notwendig. Die hier gezeigten Daten weisen auf eine Ubc6-abhängige Verknüpfung von Ubiquitinmolekülen in einer Lysin11-abhängigen Weise hin. Eine Inhibition der Synthese Lysin11-verknüpfter Ubiquitinketten hatte jedoch keinen Effekt auf den Abbau von Substratproteinen. Stattdessen wurde der Abbau von Ubc6 selbst durch Unterbindung der Bildung Lysin27-verknüpfter Ubiquitinketten verhindert. / Aberrant secretory proteins are removed from the cell in a process termed „endoplasmic reticulum-associated protein degradation" (ERAD), as it screens the endoplasmic reticulum for unwanted polypeptides and triggers their elimination via the 26S proteasome. To this end, client proteins of the ERAD pathway are polyubiquitinated with lysine48-linked ubiquitin chains at the ER membrane. Two ER membrane-integrated RING ubiquitin ligases, Hrd1 and Doa10, constitute central components of the ubiquitination machinery in Saccharomyces cerevisiae. To polyubiquitinate substrate proteins, both ligases interact with the ubiquitin-conjugating enzyme Ubc7. Since Ubc7 itself is a soluble cytosolic protein, it is recruited to the ER-membrane by is anchoring factor Cue1. Results in this study reveal two additional functions of Cue1 in the ubiquitination reaction: First, binding of Ubc7 to the Cue1-carboxyterminus stimulates the ubiquitin chain formation by Ubc7 and the ligases. Second, the CUE domain within Cue1 increases the chain length and accelerates the synthesis of the polyubiquitin chain, which results in efficient degradation of certain substrate proteins. Formation of lysine48-linked ubiquitin chains by Ubc7 depends on an acidic loop within Ubc7. Deletion of this structure leads to inhibition of ubiquitin chain elongation after the initial substrate monoubiquitination. Client proteins, ubiquitinated by Ubc7 and Hrd1, are immediately transferred to the proteasome. For Doa10-dependent substrate degradation, the activity of another ubiquitin-conjugating enzyme, Ubc6, is required. Data shown here indicate a function of Ubc6 in the formation of lysine11-linked polyubiquitin, since mutation of this lysine residue resulted in the prevention of ubiquitin chain synthesis. However, expression of this ubiquitin mutant had no effect on substrate degradation. Moreover, the proteolysis of Ubc6 itself is inhibited by prevention of lysin27-linked polyubiquitin chain formation.

The central role played by the Endoplasmic Reticulum (ER) in the correct folding and assembly of secretary and membrane proteins cannot be overstated. As the first compartment in the secretary pathway, it is responsible for the synthesis, modification and targeting of proteins to their proper destinations within the secretary pathway and the extracellular space. Protein folding in this specialized compartment is dynamic and involves a host of molecular chaperones and folding catalysts. Once inside the ER lumen, proteins fold into their native conformation and undergo a multitude of post-translational modifications, including N-linked glycosylation and disulfide oxidation. The proper conformational maturation of nascent proteins that traverse the secretary pathway is both aided and monitored by a complex process termed ER quality control. A variety of quality control mechanisms that rely on the chaperone systems operate in the ER. These act in close concert with the molecular machinery involved in degradation of non-native proteins to maintain homeostasis. The common goal of these mechanisms is to prevent expression and secretion of misfolded proteins. As a general rule, only those proteins that have successfully completed their folding and passed a stringent selection process are allowed to exit the ER on their way to their final destinations. The importance of the normal functioning of the ER is underlined by the fact that disruption in protein folding, resulting in ER stress, has now been identified as the biochemical basis of many ER storage diseases including Diabetes mellitus, Endocrinopathies and Hemophilia A. Processing events occurring inside the ER lumen are known to influence the efficiency of protein secretion. Vastly different rates of exocytose observed among secretary proteins have been found to correlate with the rate of exit from the ER. One such example is the interesting secretion property exhibited by Retinol Binding Protein (RBP) The principal carrier of retinol (Vitamin A) in plasma. RBP is a single domain protein consisting of three intramolecular disulfide bonds and helps transport retinol from the liver stores to the various target tissues in the body. Availability of its ligand, retinol, while not affecting its synthesis, is known to be the major factor in regulating RBP secretion from the liver. In the absence of retinol, apo-RBP has been shown to be retained in the ER by a hitherto unclear mechanism. Like most other secretary proteins, RBP is co-translationally targeted to the ER lumen, where it undergoes disulfide oxidation as the only modification. It has been shown to form a complex with another secretary protein, Transthyretin (TTR) in the ER and this complex formation is thought to prevent premature glomerular filtration of the otherwise small RBP with its bound retinol. Despite attaining a mature conformation, apo-RBP is not secreted and awaits conversion to its ligand-bound, holo form in order to exit the ER. It is widely believed that ligand binding may relieve this retention of RBP from the ER quality control machinery. However the precise mechanisms that mediate and regulate RBP folding, ligand binding, TTR assembly and secretion are not clearly understood. Though the folding and secretion properties of RBP have been described in HepG2 cells, its interactions with the ER resident chaperones have not been addressed. Apart from being an important cell biological question, the study of RBP assumes a lot of significance with its recent emergence as a key player in the pathogenesis of type 2 diabetes mellitus. It has been proposed that lowering of serum RBP levels could be a new strategy for treating type 2 diabetes mellitus. The present study was undertaken with the intention of analyzing the oxidative folding of RBP in the ER more closely. A systematic approach aimed at understanding the early events associated with folding and maturation of RBP, with particular emphasis on the role of ER-resident chaperones and the quality control machinery, is likely to provide interesting insights into the mechanisms involved in its ligand dependent secretion. Reconstitution of RBP biogenesis in a cell free system. The folding of RBP in cells is extremely quick with rapid oxidation kinetics. This makes it difficult to systematically analyze the early folding events in cultured cells. It was necessary to make use of a simplified system that would faithfully recapitulate the folding process in the ER. Therefore, a cell free translation system consisting of rabbit reticulocyte lysate and canine pancreatic microcosms as a source of ER-derived membranes was developed. This system affords the advantage of easy manipulation while still preserving the overall environment that prevails in the ER of intact cells. Extensive biochemical and functional characterization of the isolated microcosms was carried out and in vitro translation and microsomal translocation of RBP was established. Though initially confined to studies on membrane insertion and core glycosylate, the cell free system supplemented with microcosms has subsequently been used to analyze folding and assembly of a number of secretary and membrane proteins. A similar strategy has been adopted in the present study of RBP folding and maturation. Oxidative folding of RBP in isolated microcosms: Delineation of its disulfide oxidation pathway Using glutathione (GSSG) as the oxidant, co- and posttranslational disulfide oxidation of RBP was carried out in isolated microcosms. The ability to manipulate the redox status of this cell free system has helped to considerably slow down the oxidative folding of RBP so that a more careful analysis of the folding process could be performed. RBP was found to undergo oxidative folding with a t1/2 of 30 minutes and folding proceeded through at least one disulfide-bonded intermediate. Non-reducing SDS PAGE was used to resolve the folding intermediates. The pattern of oxidation was in good agreement with that reported earlier in HepG2 cells. No significant effect of retinol was observed on either the folding kinetics or the pattern of disulfide oxidation of RBP in isolated microsomes.A DTT sensitivity assay, used to probe the conformational maturity of folding RBP, revealed that RBP was capable of maturing into a DTT-resistant conformation in isolated microsomes. With the aid of disulfide mutants, the probable disulfide oxidation pathway of RBP in the ER has been determined. Single and double disulfide mutants of RBP were generated by site-directed mutagenesis and their posttranslational oxidation patterns were analyzed and compared with that of the wild type protein. Based on the results obtained, it was clear that the folding intermediate was made up of one of the two big disulfide loops and that the presence of both these loops was essential for RBP to fold into a fully oxidized, compact form. It has not been possible to determine the contribution of the third, smallest disulfide loop to the oxidative folding of RBP. Molecular events associated with the early oxidative folding of RBP To gain insights into the possible role of ER chaperones in the oxidative folding of RBP, the oligomeric state of folding RBP was analyzed by velocity sedimentation and chemical crosslinking assays. Velocity sedimentation analysis revealed that the reduced form of RBP was present in a large complex of size >100 S20,W. Upon disulfide oxidation, it readily dissociated from the complex and assumed a monomeric state. This was evident even during co-translational oxidation which suggested that RBP transiently associated with the large complex during its oxidative folding. Dynamic nature of this complex indicated that this could be a folding complex containing the chaperone machinery of the ER. These results were also supported by crosslinking analysis performed in unbroken microsomes using the homo-bifunctional crosslinker, DSP. The early folding forms of RBP could be crosslinked to a large complex while upon disulfide oxidation, RBP matured to its monomeric form and was no longer crosslinkable. Sedimentation and crosslinking analyses of the RBP disulfide mutants revealed that while the double disulfide mutant remained irreversibly associated with the large complex, the single mutants were released upon acquiring one of the two big disulfide loops. This suggested that despite the lack of one of the two major disulfides, these mutants were considered ‘folded’ by the quality control machinery in the ER while the double mutant probably resembled a molten globule state and was therefore considered ‘unfolded’ and irreversibly retained. Results from crosslinking analysis in microsomes not engaged in active translation suggested that chaperones of the ER were organized in a complex constitutively thereby lending support to the concept of ER-matrix, a large network of luminal proteins consisting of ER chaperones and accessory factors. Given this scenario, it is not unlikely that newly synthesized protein substrates transiently associate with this large pre-existing complex of chaperones and dissociate during late stages of their maturation. Conclusion In all, this study provides significant insights into some of the early events associated with the oxidative folding of RBP in the ER. The delineation of the disulfide oxidation pathway of RBP has been possible. The results obtained from this study suggest that RBP probably dissociates from the quality control quite early during its folding process and this step in its maturation might not be influenced by retinol. The stimulus for its ligant dependent secretion is likely to operate at a later stage of its sojourn in the ER, possibly consequent to positive cues from accessory binding factors such as TTR. Lastly, Perservation of the ER microenvironment in isolated microsomes, as evidenced from this study, augurs well for the use of this system to analyze mechanisms underlying folding, maturation, secretion and/or retention of secretory proteins.

Prostate cancer remains one of the most commonly diagnosed cancers in men and is a leading cause of cancer death. While great success has been achieved at curing early stage prostate cancer, limited success has been obtained when treating late-stage hormone independent prostate cancer. This is due to the increased propensity for skeletal and non-skeletal metastases. Thus development of novel effective therapeutic modalities against late stage prostate cancer is of critical importance. / Towards these objectives, I have focused my attention on the role of prostate secretory protein (PSP-94) which is expressed in normal individuals and in patients with early stage prostate cancer. Using our well established in vivo models of prostate cancer, I have evaluated the ability of PSP-94 and its amino acids 31-45 required (PCK3145) to decrease tumor growth and skeletal metastases in vivo and evaluated the potential mechanism(s) associated with PCK3145 anti-cancer actions. / Prostatic cancer can also develop as a result of epigenetic activation of tumor promoting genes. To evaluate the role of methylation in prostate cancer, late stage prostate cancer cells were treated with the universal methylating agent S-adenosylmethionine (SAM) and an anti-sense oligonucleotide directed against MBD2 (AS). Scrambled oligonucleotide was included as a control (S). Both SAM and MBD2-AS resulted in inhibition in uPA, MMP-2 and VEGF production leading to decreased tumor cell invasive capacity. However, SAM and MBD2-AS were not able to either further repress partially methylated genes (GSTP1) or reactivate already methylated genes (AR). Furthermore, SAM and MBD2-AS treatment resulted in significant reduction in tumor growth in vivo . Immunohistochemical and RT-PCR analyses carried out on SAM and MBD2-AS tumors revealed decreased protein and mRNA expression of uPA and MMP-2 which was partially due to increased methylation of the respective promoters even after 10 weeks post in vitro treatment as analyzed by bisulfate sequencing. In addition decreased levels of angiogenesis and tumor survival markers were observed. / Collectively, these studies are aimed at the development of novel reliable approached to diagnose and treat advanced, hormone refractory prostate cancer to reduce tumor associated morbidity and mortality.

No description available.

Der Einzeller Eimeria tenella zählt zu den hochpathogenen Parasiten des Haushuhns und ist Verursacher der Blinddarm-Kokzidiose, eine Erkrankung, die zu hohen ökonomischen Belastungen in der Geflügelindustrie führt. Zur Zeit existiert noch kein Impfstoff, der auf Basis von einzelnen Antigenen wirksam ist. Insbesondere die Identifizierung und Charakterisierung von sekretorischen Proteinen, welche an der Parasit-Wirtszell-Interaktion beteiligt sind, könnte einen Beitrag zur Entwicklung einer Immunprophylaxe darstellen. Ziel dieser Arbeit war es, sekretorische E. tenella-Proteine zu identifizieren und ausgewählte cDNA-Sequenzen in DNA-Immunisierungsstudien zu überprüfen. Um ein DNA-Immunisierungsprotokoll für Hühner zu erarbeiten, wurden die cDNAs von drei schon bekannten E. tenella-Antigenen alleine oder in Fusion zur cDNA des stabilisierenden enhanced green fluorescence protein (EGFP) in zwei unterschiedliche DNA-Immunisierungsvektoren (pCDNA3, pVR1012) kloniert. Die Überprüfung der Serokonversion zeigte, dass nach DNA-Immunisierung von Hühnern mit beiden Vektoren bzw. mit und ohne EGFP-Fusion vergleichbare Immunantworten induziert wurden. D.h. mit dem etablierten DNA-Immunisierungsprotokoll konnte eine Expression heterologer (Eimerien) Gene im Huhn erzielt werden. Um neue sekretorische E. tenella-Proteine zu identifizieren wurden bioinformatische und experimentelle Methoden eingesetzt. Über die bioinformatischen Analysen wurden aus E. tenella-expressed sequence tag (EST)-Datenbanken 54 putativ sekretierte E. tenella-Sequenzen extrahiert für die aufgrund ihrer beschriebenen Funktion und Lokalisierung eine Beteiligung an der Wirts-Parasit-Interaktion abgeleitet wurde. Mittels funktioneller Komplementierung in Hefen konnten aus einer E. tenella-Sporozoiten-cDNA-Bank 25 sekretorische Sequenzen identifiziert werden. Die cDNA-Sequenzen von 13 neu identifizierten sekretorischen Proteinen wurden in DNA-Immunisierungsstudien überprüft. Die Ergebnisse zeigten, dass beide Methoden geeignet sind, um sekretorische Proteine von E. tenella zu identifizieren. Aufgrund der Erkenntnisse dieser Arbeit, sollten jedoch weitere verbesserte Immunisierungsprotokolle erarbeitet werden, um die immunprotektive Wirkung der isolierten Kandidaten-Antigene von E. tenella in Hühnern zu überprüfen. Wie die vorliegende Arbeit bestätigt, eignet sich die Methode der DNA-Immunisierung vermutlich als Grundlage solcher Verfahren, wobei zukünftig die Induktion der essentiellen intestinalen Immunantwort im Vordergrund stehen sollte. / The protozoan parasite Eimeria tenella is highly pathogenic in chickens and causes caecal coccidosis that contribute to high economic losses in poultry farming. At the moment no vaccine based on a single antigen is available. The identification and characterization of proteins that are involved in host parasite interaction could particularly contribute to the development of immunoprophylactic control strategies. In order to establish a DNA immunisation protocol, cDNA of three already known E. tenella antigens were subcloned alone or in fusion to the stabilising fusion partner enhanced green flurescence protein (EGFP) into two different DNA immunisation vectors (pCDNA3, pVR1012). Following DNA immunisation of chickens using both vectors with or without EGFP fusion, respectively, the induction of comparable immune responses were shown by serum conversion. This implies it was possible to achieve heterologous (Eimeria) gene expression in chickens with the established immunisation protocol. In order to identify new E. tenella secretory proteins bioinformatic and experimental approaches were used. Following bioinformatic analysis 54 putatively secretory E. tenella sequences were identified for which roles in host parasite interaction were surmised, due to their localisation and function. The identification of secreted proteins from a E. tenella sporozoite cDNA library using a functional complementation assay in yeast resulted in 25 unique sequences. The cDNA of 13 newly identified secretory proteins were tested in DNA immunisation studies. The results showed that both methods are capable of identifying secretory proteins of E. tenella. Owing to the results presented here, new immunisation protocols should be established to test the immune protective capacity of candidate antigens of E. tenella in chickens. The present study confirmed the method of DNA immunisation as a basic tool for such new protocols. In future, however, DNA immunisation protocols should focus on the induction of essential intestinal immune responses.