Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.

Rhetoric and Representation : Exploring the Cultural Meaning of the Natural Sciences in Contemporary Popular Science Writing and Literature

Kirjallisuuden- ja kulttuurintutkimus on viimeisten kolmen vuosikymmenen aikana tullut yhä enenevässä määrin tietoiseksi tieteen ja taiteen suhteen monimutkaisesta luonteesta. Nykyään näiden kahden kulttuurin tutkimus muodostaa oman kenttänsä, jolla niiden suhdetta tarkastellaan ennen kaikkea dynaamisena vuorovaikutuksena, joka heijastaa kulttuurimme kieltä, arvoja ja ideologisia sisältöjä. Toisin kuin aiemmat näkemykset, jotka pitävät tiedettä ja taidetta toisilleen enemmän tai vähemmän vastakkaisina pyrkimyksinä, nykytutkimus lähtee oletuksesta, jonka mukaan ne ovat kulttuurillisesti rakentuneita diskursseja, jotka kohtaavat usein samankaltaisia todellisuuden mallintamiseen liittyviä ongelmia, vaikka niiden käyttämät metodit eroavatkin toisistaan. Väitöskirjani keskittyy yllä mainitun suhteen osa-alueista popularisoidun tietokirjallisuuden (muun muassa Paul Davies, James Gleick ja Richard Dawkins) käyttämän kielen ja luonnontieteistä ideoita ammentavan kaunokirjallisuuden (muun muassa Jeanette Winterson, Tom Stoppard ja Richard Powers) hyödyntämien keinojen tarkasteluun nojautuen yli 30 teoksen kattavaa aineistoa koskevaan tyylin ja teemojen tekstianalyysiin. Populaarin tietokirjallisuuden osalta tarkoituksenani on osoittaa, että sen käyttämä kieli rakentuu huomattavassa määrin sellaisille rakenteille, jotka tarjoavat mahdollisuuden esittää todellisuutta koskevia argumentteja mahdollisimman vakuuttavalla tavalla. Tässä tehtävässä monilla klassisen retoriikan määrittelemillä kuvioilla on tärkeä rooli, koska ne auttavat liittämään sanotun sisällön ja muodon tiukasti toisiinsa: retoristen kuvioiden käyttö ei näin ollen edusta pelkkää tyylikeinoa, vaan se myös usein kiteyttää argumenttien taustalla olevat tieteenfilosofiset olettamukset ja auttaa vakiinnuttamaan argumentoinnin logiikan. Koska monet aikaisemmin ilmestyneistä tutkimuksista ovat keskittyneet pelkästään metaforan rooliin tieteellisissä argumenteissa, tämä väitöskirja pyrkii laajentamaan tutkimuskenttää analysoimalla myös toisenlaisten kuvioiden käyttöä. Osoitan myös, että retoristen kuvioiden käyttö muodostaa yhtymäkohdan tieteellisiä ideoita hyödyntävään kaunokirjallisuuteen. Siinä missä popularisoitu tiede käyttää retoriikkaa vahvistaakseen sekä argumentatiivisia että kaunokirjallisia ominaisuuksiaan, kuvaa tällainen sanataide tiedettä tavoilla, jotka usein heijastelevat tietokirjallisuuden kielellisiä rakenteita. Toisaalta on myös mahdollista nähdä, miten kaunokirjallisuuden keinot heijastuvat popularisoidun tieteen kerrontatapoihin ja kieleen todistaen kahden kulttuurin dynaamisesta vuorovaikutuksesta. Nykyaikaisen populaaritieteen retoristen elementtien ja kaunokirjallisuuden keinojen vertailu näyttää lisäksi, kuinka tiede ja taide osallistuvat keskusteluun kulttuurimme tiettyjen peruskäsitteiden kuten identiteetin, tiedon ja ajan merkityksestä. Tällä tavoin on mahdollista nähdä, että molemmat ovat perustavanlaatuisia osia merkityksenantoprosessissa, jonka kautta niin tieteelliset ideat kuin ihmiselämän suuret kysymyksetkin saavat kulttuurillisesti rakentuneen merkityksensä.

Relocating the American Dream : The America of the 1960s as Portrayed by the New Journalists Norman Mailer, Hunter S. Thompson, and Tom Wolfe

The concept of the American Dream was subject to a strong re-evaluation process in the 1960s, as counterculture became a prominent force in American society. A massive generation of young people, moved by the Vietnam War, the hippie movement, and psychedelic experimentation, created substantial social turbulence in their efforts to break out of conventional patterns and to create a new kind of society. This thesis outlines and analyses the concept of the American Dream in popular imagination through three works of new journalism. My primary data consists of Tom Wolfe’s The Electric Kool-Aid Acid Test (1967), Hunter S. Thompson’s Fear and Loathing in Las Vegas: A Savage Journey to the Heart of the American Dream (1971), and Norman Mailer’s Armies of the Night: History as a Novel, the Novel as History (1968). In defining the American Dream, I discuss the history of the concept as well as its manifestations in popular culture. Because of its elusive and amorphous nature, the concept of the American Dream can only be examined in cultural texts that portray the values, sentiments, and customs of a certain era. I have divided the analytical section of my thesis into three parts. In the first part I examine how the authors discuss the American society of their time in relation to ideology, capitalism, and the media. In the second part I focus on the Vietnam War and the controversy it creates in relation to the notions of freedom and patriotism. In the third part I discuss how the authors portray the countercultural visions of a better America that challenged the traditional interpretations of the American Dream. I also discuss the dark side of the new dream: the problems and disillusions that came with the effort to change the world. This thesis is an effort to trace the relocation of the American Dream in the context of the 1960s counterculture and new journalism. It hopes to provide a valuable addition to the cultural history of the sixties and to the effort of conceptualizing the American Dream.

Mediated music makers : Constructing author images in popular music

The subject of the thesis is the mediated construction of author images in popular music. In the study, the construction of images is treated as a process in which artists, the media and the members of the audience participate. The notions of presented, mediated and compiled author images are used in explaining the mediation process and the various authorial roles of the agents involved. In order to explore the issue more closely, I analyse the author images of a group of popular music artists representing the genres of rock, pop and electronic dance music. The analysed material consists mostly of written media texts through which the artists authorial roles and creative responsibilities are discussed. Theoretically speaking, the starting points for the examination lie in cultural studies and discourse analysis. Even though author images may be conceived as intertextual constructions, the artist is usually presented as a recognizable figure whose purpose is to give the music its public face. This study does not, then, deal with musical authors as such, but rather with their public images and mediated constructions. Because of the author-based functioning of popular music culture and the idea of the artist s individual creative power, the collective and social processes involved in the making of popular music are often superseded by the belief in a single, originating authorship. In addition to the collective practices of music making, the roles of the media and the marketing machinery complicate attempts to clarify the sharing of authorial contributions. As the case studies demonstrate, the differences between the examined author images are connected with a number of themes ranging from issues of auteurism and stardom to the use of masked imagery and the blending of authorial voices. Also the emergence of new music technologies has affected not only the ways in which music is made, but also how the artist s authorial status and artistic identity is understood. In the study at hand, the author images of auteurs, stars, DJs and sampling artists are discussed alongside such varied topics as collective authorship, evaluative hierarchies, visual promotion and generic conventions. Taken altogether, the examined case studies shed light on the functioning of popular music culture and the ways in which musical authorship is (re)defined.

Den brændende Fakkel : Om dobbeltgrebet i Knud Lyne Rahbeks liv og revolutionsroman

Professor Knud Lyne Rahbek was a novelist, playwright, poet, magazine editor, journalist, socialite person, host of the Bakkehus , historian, theatre manager, translator, publisher etc., but his versatility either side of 1800 is better known than read and more despised than understood. In terms of methodology, the thesis is based on biographical, historical and philological research, while at the same time making use of formalistic and close reading methods. This study begins and ends with 7th of February 1800, when Kamma and Knud Lyne Rahbek join the exiled P.A. Heiberg at the inn near Frederiksberg Castle. What falls between is an interpretation of Rahbek s works in the service of democracy, human rights and freedom of the press as a pragmatic navigation between activities - both subversive and legitimate. Posterity mistook this range as mere spinelessness, and Rahbek was relegated to the literary and historical margins as an anachronism and as a jack of all trades, who did not know what he really wanted and therefore flitted about in so many fields just to be present. But Rahbek s problem was not one of standpoint, but rather how to find a balance between totalizing attitudes and confrontations between rebellious idealism and deep-rooted absolutism, without foregoing his belief in enlightenment, humanism and tolerance. In this way, and also through his personal conduct, which at that time was seen as jovial bonhommie, he made his contribution to the development of modern democratic Denmark in the full awareness of a popular, peaceful and down-to-earth community. Rahbek s principal work about the event of the French Revolution, which provides the focus for the above, is Camill og Constance. Et Revolutions Skilderie (1799). For today s reader, the novel about the revolution is an obvious example of a historical novel, as it does not only provide fictionalized information about past events placing them in a generally accepted perspective of historical development, but also gives the characters qualities, which, in Rahbek s words, allows the real events to influence the fictional characters. From this point of view, the novel of the revolution has shifted the benchmark for the first real historical novel on the European literary scene back by fifteen years. Lacking the aura so easily foisted on fearless iconoclasts or tragic losers, Rahbek s contribution may seem modest in spite of its enormous volume; but only when it is not evaluated in its full context, which is the development of Denmark towards an international democratic society.

Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

Novel conditional mouse models for targeted kidney research : Emphasis on the analysis of the biological function of nephrin

Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.

Identification of two novel human neuronal ceroid lipofuscinosis genes

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.

Identification of the Meckel syndrome gene (MKS1) exposes a novel ciliopathy

Meckel syndrome (MKS, MIM 249000) is an autosomal recessive developmental disorder causing death in utero or shortly after birth. The hallmarks of the disease are cystic kidney dysplasia and fibrotic changes of the liver, occipital encephalocele with or without hydrocephalus and polydactyly. Other anomalies frequently seen in the patients are incomplete development of the male genitalia, club feet and cleft lip or palate. The clinical picture has been well characterized in the literature while the molecular pathology underlying the disease has remained unclear until now. In this study we identified the first MKS gene by utilizing the disease haplotypes in Finnish MKS families linked to the MKS1 locus on chromosome 17q23 (MKS1) locus. Subsequently, the genetic heterogeneity of MKS was established in the Finnish families. Mutations in at least four different genes can cause MKS. These genes have been mapped to the chromosomes 17q23 (MKS1), 11q13 (MKS2), 8q22 (MKS3) and 9q33 (MKS4). Two of these genes have been identified so far: The MKS1 gene (this work) and the MKS3 gene. The identified MKS1 gene was initially a novel human gene which is conserved among species. We found three different MKS mutations, one of them being the Finnish founder mutation. The information available from MKS1 orthologs in other species convinced us that the MKS1 gene is required for normal ciliogenesis. Defects of the cilial system in other human diseases and model organisms actually cause phenotypic features similar to those seen in MKS patients. The MKS3 (TMEM67) gene encodes a transmembrane protein and the gene maps to the syntenic Wpk locus in the rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. The available information from these two genes suggest that MKS1 would encode a structural component of the centriole required for normal ciliary functions, and MKS3 would be a transmembrane component most likely required for normal ciliary sensory signaling. The MKS4 locus was localized to chromosme 9q32-33 in this study by using an inbred Finnish family with two affected and two healthy children. This fourth locus contains TRIM32 gene, which is associated to another well characterized human ciliopathy, Bardet Biedl syndrome (BBS). Future studies should identify the MKS4 gene on chromosome 9q and confirm if there are more than two genes causing MKS Finnish families. The research on critical signaling pathways in organogenesis have shown that both Wnt and Hedgehog pathways are dependent on functional cilia. The MKS gene products will serve as excellent model molecules for more detailed studies of the functional role of cilia in organogenesis in more detail.

Oncolytic Adenoviruses for Gynecologic Cancer

Gene therapy is a promising novel approach for treating cancers resistant to or escaping currently available modalities. Treatment approaches are based on taking advantage of molecular differences between normal and tumor cells. Various strategies are currently in clinical development with adenoviruses as the most popular vehicle. Recent developments include improving targeting strategies for gene delivery to tumor cells with tumor specific promoters or infectivity enhancement. A rapidly developing field is as well replication competent agents, which allow improved tumor penetration and local amplification of the anti-tumor effect. Adenoviral cancer gene therapy approaches lack cross-resistance with other treatment options and therefore synergistic effects are possible. This study focused on development of adenoviral vectors suitable for treatment of various gynecologic cancer types, describing the development of the field from non-replicating adenoviral vectors to multiple-modified conditional replicating viruses. Transcriptional targeting of gynecologic cancer cells by the use of the promoter of vascular endothelial growth factor receptor type 1 (flt-1) was evaluated. Flt-1 is not expressed in the liver and thus an ideal promoter for transcriptional targeting of adenoviruses. Our studies implied that the flt-1 promoter is active in teratocarcinomas.and therefore a good candidate for development of oncolytic adenoviruses for treatment of this often problematic disease with then poor outcome. A tropism modified conditionally replicating adenovirus (CRAd), Ad5-Δ24RGD, was studied in gynecologic cancers. Ad5-Δ24RGD is an adenovirus selectively replication competent in cells defective in the p16/Rb pathway, including many or most tumor cells. The fiber of Ad5-Δ24RGD contains an integrin binding arginine-glycine-aspartic acid motif (RGD-4C), allowing coxackie-adenovirus receptor independent infection of cancer cells. This approach is attractive because expression levels of CAR are highly variable and often low on primary gynecological cancer cells. Oncolysis could be shown for a wide variety of ovarian and cervical cancer cell lines as well as primary ovarian cancer cell spheroids, a novel system developed for in vitro analysis of CRAds on primary tumor substrates. Biodistribution was evaluated and preclinical safety data was obtained by demonstrating lack of replication in human peripheral blood mononuclear cells. The efficicacy of Ad5-Δ24RGD was shown in different orthotopic murine models including a highly aggressive intraperitoneal model of disseminated ovarian cancer cells, where Ad5-Δ24RGD resulted in complete eradication of intraperitoneal disease in half of the mice. To further improve the selectivity and specificity of CRAds, triple-targeted oncolytic adenoviruses were cloned, featuring the cyclo-oxygenase-2 (cox-2) promoter, E1A transcomplementation and serotype chimerism. Those viruses were evaluated on ovarian cancer cells for specificity and oncolytic potency with regard to two different cox2 versions and three different variants of E1A (wild type, delta24 and delta2delta24). Ad5/3cox2Ld24 emerged as the best combination due to enhanced selectivity without potency lost in vitro or in an aggressive intraperitoneal orthotopic ovarian tumor model. In summary, the preclinical therapeutic efficacy of the CRAds tested in this study, taken together with promising biodistribution and safety data, suggest that these CRAds are interesting candidates for translation into clinical trials for gynecologic cancer.