Thesis (PhD)--Macquarie University. Division of Society, Culture, Media & Philosophy, Department of Media. / Bibliography: leaves 391-427. / Preamble or redesigning Poland: 1989-2004 -- Introducing Polish cinema -- Of (post) transitional Polishness and film -- Of the Polish film industry, culture and criticism 1989-2004: a story in three acts -- Of nostalgia and arcadias in heritage films -- Of the People's Republic and its memory -- Of the transitional ethos -- Of (post) transitional comic relief -- Conclusion. / This thesis examines patterns of (post) transitional developments in Polish cinema between 1989 and 2004. It proposes a three-stage approach to the analysis of transitional continuities in films about the mythical Polish past, The Polish People's Republic (PRL), the contemporary reality and contemporary Polish comedies. / Mode of access: World Wide Web. / iv, 427 leaves

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L’implantation retardée ou diapause embryonnaire décrit l'arrêt ou le retardement pendant l'embryogenèse. Chez le vison, la diapause est corrélée avec une sécrétion pituitaire insuffisante de la prolactine, ayant pour résultat la différentiation incomplète du corpus luteum et réduction de la progestérone. Des études antérieures suggèrent que le blastocyste de vison en diapause demeure dans un état de quiescence ou se développe lentement. Pour élucider ceci, la réplication de l'ADN a été étudiée. Les résultats démontrent synthèse de l’ADN et prolifération cellulaire dans les embryons au stade de morula, avant la diapause et dans les blastocystes après la réactivation. La réplication de l'ADN a été également détectée dans des blastocystes en diapause et en diapause prolongée. L'implantation est considérée comme une interaction bidirectionnelle entre le blastocyste et l'utérus. Il a été montré que les prostaglandines sont importantes pour la vascularisation de l’utérus au moment de l’implantation et peuvent réactiver l'utérus des visons après la diapause. La concentration protéinique et la localisation de la phospholipase citosolique A2 (CPLA2) et de la cyclooxygenase 2 (COX2) ont été étudiées dans l'utérus de vison. L’expression de la CPLA2 et COX2 étaient sur-régulées au moment de l'implantation. Il est connu que la prolactine active les corpus luteum des visons. L'idée de un lien entre la prolactine et la voie de signalisation des prostaglandines a été testée en mesurant les récepteurs de prolactine. Les résultats montrent une augmentation de l’expression des récepteurs de prolactine à l'implantation suggérant que la prolactine pourrait activer la voie de prostaglandine à l'utérus par son propre récepteur. La conclusion, les embryons pendant la diapause ne sont pas arrêtées complètement et les protéines liées à la voie de prostaglandine sont implique dans la réactivation de l'utérus. / Delayed implantation or diapause describes arrest or retardation during embryogenesis. In mink, diapause is related to insufficient pituitary prolactin secretion, resulting in incomplete differentiation of the corpus luteum with reduced progesterone concentration. The mink blastocyst at diapause was believed to be totally quiescent or expanding at a low rate. To explore this, DNA replication was studied. Results showed synthesis of DNA, and thus cell proliferation at the morulae stage before diapause and at the blastocyst following activation. DNA replication was detected not only at diapause but also at extended diapause. Furthermore, implantation is considered as a two-way interaction between the blastocyst and the uterus. It has been shown that prostaglandins are important for vascularization of the uterus and products of the prostaglandin pathway could reactivate the mink uterus following diapause. Protein concentration and localization was studied for cytosolic phospholipase A2 (CPLA2) and cyclooxygenase 2 (COX2) in mink uterus. Expression of CPLA2 and COX2 was up regulated at implantation. It is know that prolactin is the factor that activates the mink corpus luteum. The idea of a link between prolactin and prostaglandin pathway was investigated by quantifying the prolactin receptors in the uterus. Results showed an increase of prolactin receptors at implantation suggesting that prolactin could activate the prostaglandin pathway at the uterus through its own receptor. In conclusion, embryos during diapause are not completely arrested, and proteins related to the prostaglandin pathway are implicated in reactivation of the uterus.

Cytomegalovirus (CMV) is a significant cause of mortality and morbidity in immunocompromised patients and an important cause of birth defects if acquired in utero. The licensed CMV antivirals, ganciclovir, cidofovir and foscarnet, all target the viral DNA polymerase. For each drug prolonged use is associated with significant toxicities and development of drug resistance. None are approved for use during pregnancy. Therefore, development of new anti-CMV drugs that target different pathways would be beneficial. All herpesviruses encode an alkaline nuclease. That genetic disruption of the CMV alkaline nuclease, UL98, reduces CMV replication by 1,000-fold suggests that UL98 may be a useful target for development of novel anti-CMV drugs. Moreover, using herpes simplex virus type 1 Hsiang and Ho found that the anthraquinone emodin inhibits activity of the viral alkaline nuclease, blocks viral replication in cell culture, and reduces viral pathogeneses in a mouse model (Brit. J. of Pharm., 2008). Earlier studies also showed that anthraquinone derivatives including emodin have anti-CMV activity (Barnard et al., Antiviral Research 1992 & 1995), although the mechanism of CMV inhibition has not been further studied. We therefore sought to confirm the anti-CMV activities of emodin and related anthraquinone derivatives, to characterize their mechanisms of action, and to determine specifically if they act through inhibition of UL98. Using a luciferase-based CMV yield reduction assay emodin inhibited CMV replication (IC50 = 4.9 μM); however, that the TD50 for cytotoxicity (determined using an luciferase-based cell viability assay) was only 2-fold higher suggested that emodin may act non-specifically. Two additional anthraquinone derivatives (acid blue 40 and alizarin violet R) inhibited CMV only at high concentrations (IC50 = 238; 265 μM) that were also cytotoxic. Atanyl blue PRL, however, exhibited anti-CMV activity (IC50 = 6.3 μM) with low cytotoxicity (TD50 = 216 μM). Thus, characterization of atanyl blue PRL (impact on gene expression, GFP expression, viral spread, infectivity, time of addition studies, and inhibition of UL98 nuclease activity) should be informative. Atanyl blue PRL appears to block immediate-early gene expression and reduce early and late gene expression. Atanyl blue PRL also blocked GFP expression, reduced viral spread, and also lowered the infectivity of CMV. Finally, atanyl blue PRL inhibits UL98 alkaline nuclease activity at an IC50 of 5.7 μM. This suggests that atanyl blue PRL may inhibit CMV through inhibition of UL98. Thus, atanyl blue PRL represents a novel class of anti-herpesvirals and provides a lead structure for structure based drug discovery.

L’implantation retardée ou diapause embryonnaire décrit l'arrêt ou le retardement pendant l'embryogenèse. Chez le vison, la diapause est corrélée avec une sécrétion pituitaire insuffisante de la prolactine, ayant pour résultat la différentiation incomplète du corpus luteum et réduction de la progestérone. Des études antérieures suggèrent que le blastocyste de vison en diapause demeure dans un état de quiescence ou se développe lentement. Pour élucider ceci, la réplication de l'ADN a été étudiée. Les résultats démontrent synthèse de l’ADN et prolifération cellulaire dans les embryons au stade de morula, avant la diapause et dans les blastocystes après la réactivation. La réplication de l'ADN a été également détectée dans des blastocystes en diapause et en diapause prolongée. L'implantation est considérée comme une interaction bidirectionnelle entre le blastocyste et l'utérus. Il a été montré que les prostaglandines sont importantes pour la vascularisation de l’utérus au moment de l’implantation et peuvent réactiver l'utérus des visons après la diapause. La concentration protéinique et la localisation de la phospholipase citosolique A2 (CPLA2) et de la cyclooxygenase 2 (COX2) ont été étudiées dans l'utérus de vison. L’expression de la CPLA2 et COX2 étaient sur-régulées au moment de l'implantation. Il est connu que la prolactine active les corpus luteum des visons. L'idée de un lien entre la prolactine et la voie de signalisation des prostaglandines a été testée en mesurant les récepteurs de prolactine. Les résultats montrent une augmentation de l’expression des récepteurs de prolactine à l'implantation suggérant que la prolactine pourrait activer la voie de prostaglandine à l'utérus par son propre récepteur. La conclusion, les embryons pendant la diapause ne sont pas arrêtées complètement et les protéines liées à la voie de prostaglandine sont implique dans la réactivation de l'utérus. / Delayed implantation or diapause describes arrest or retardation during embryogenesis. In mink, diapause is related to insufficient pituitary prolactin secretion, resulting in incomplete differentiation of the corpus luteum with reduced progesterone concentration. The mink blastocyst at diapause was believed to be totally quiescent or expanding at a low rate. To explore this, DNA replication was studied. Results showed synthesis of DNA, and thus cell proliferation at the morulae stage before diapause and at the blastocyst following activation. DNA replication was detected not only at diapause but also at extended diapause. Furthermore, implantation is considered as a two-way interaction between the blastocyst and the uterus. It has been shown that prostaglandins are important for vascularization of the uterus and products of the prostaglandin pathway could reactivate the mink uterus following diapause. Protein concentration and localization was studied for cytosolic phospholipase A2 (CPLA2) and cyclooxygenase 2 (COX2) in mink uterus. Expression of CPLA2 and COX2 was up regulated at implantation. It is know that prolactin is the factor that activates the mink corpus luteum. The idea of a link between prolactin and prostaglandin pathway was investigated by quantifying the prolactin receptors in the uterus. Results showed an increase of prolactin receptors at implantation suggesting that prolactin could activate the prostaglandin pathway at the uterus through its own receptor. In conclusion, embryos during diapause are not completely arrested, and proteins related to the prostaglandin pathway are implicated in reactivation of the uterus.

A busca pelo repertório genético por trás de características comportamentais e reprodutivas de espécies de primatas tem desafiado nosso grupo de pesquisa. A premissa principal nesse tipo de estudo baseia-se na hipótese de que um traço fenotípico (seja ele fisiológico, comportamental, etc) compartilhado por um grupo taxonômico inteiro deve ser determinado por um repertório genético comum a esses táxons. Considerando a complexidade de muitas dessas características, temos ampliado nossos estudos para vários genes da rede OXT – AVP – PRL, usando abordagens in silico, in vitro, e in vivo. Na presente Tese, o conjunto gênico de estudo foi selecionado por uma metodologia baseada na ontologia biológica, com critério de seleção para características como comportamento materno, amamentação e aspectos reprodutivos, tais como comportamento de acasalamento e de corte. Essa seleção resultou em 12 genes candidatos para o estudo: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR e TRH. Exploramos aqui esse conjunto gênico da rede OXT – AVP – PRL através da mineração de dados, buscando por seus ortólogos nas várias espécies de primatas e de outros mamíferos, bem como através de novos dados de sequências da região codificadora dos genes PRLR e PRLH, em um conjunto de espécies de primatas do Novo Mundo (NWM, conforme sigla em inglês). Adicionalmente, sequências das regiões codificadoras de PRLR foram também obtidos em espécies de marsupiais. Com o objetivo de elucidar os padrões evolutivos dos genes de interesse, utilizamos análises como os testes NsSites e Branch Sites do pacote PAML, assim como vários testes populacionais clássicos, para diferentes conjuntos amostrais. Além disso, foi feita a predição da estrutura secundária das proteínas-alvo, utilizando metodologia específica do programa PSIPRED, bem como o PONDER-FIT, para a caracterização de aminoácidos intrinsecamente desordenados. Nossos resultados das análises in silico sugerem que os genes da família de receptores da vasopressina (AVPR1A, AVPR1B, e AVPR2) apresentam um padrão compatível com seleção positiva em mamíferos placentários. Alguns dos sítios com sinal de seleção apresentam motivos lineares (SLiMS) preditos no receptor AVPR2, que podem ter facilitado a emergência de novidades adaptativas, conforme foi sugerido para a espécie do rato-canguru Dipodomys ordii, que habita regiões áridas. As análises dos dados originais da região codificadora do gene PRLR em 17 espécies de NWM revelaram vários sítios presentes na forma longa do receptor com alta probabilidade de estarem sob seleção positiva, sendo que alguns deles (posições 507, 532 e 572) estão associados com o parto gemelar, uma característica das espécies de Callitrichidae. Adicionalmente verificamos no ramo dos Siimiformes um motivo linear de interação que reconhece domínios SH3 (Src Homology 3). Os domínios SH3 e os seus locais de ligação foram descritos para centenas de proteínas; eles fornecem à célula um meio particularmente conveniente e adaptável de interação específica proteína-proteína, que pode ser de importância funcional. Esse trabalho como um todo contribuiu para o conhecimento do repertório genético relacionado à complexa rede de mecanismos neuroendócrinos associados à emergência de traços adaptativos, tanto fisiológicos quanto comportamentais em diferentes clados de mamíferos. / The search for the genetic repertoire behind behavioral and reproductive features of Primate species has challenged our research group. The principal premise in this kind of study is based on the hypothesis that a phenotypic trait (either physiological, behavioral, etc.) shared by an entire taxonomic group should be determined by a genetic repertoire common to these taxa. Considering the complexity of many of these features, we have expanded our studies for several genes of the OXT - AVP - PRL network, using in silico, in vitro, and in vivo approaches. In the present Thesis, the set of genes for the study was selected by a methodology based on biological ontology, with features like maternal behavior, breastfeeding, and reproductive aspects, such as mating and courtship behavior. This selection resulted in 12 candidate genes for the study: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR, and TRH. We explored here this gene set of the OXT – AVP – PRL network through data mining, searching for their orthologues in many Primate species and other mammals, as well as through new sequence data from the PRLR and PRLH coding region in a set of New World Monkey (NWM) species. Additionally, sequences from the PRLR coding regions were also obtained in marsupial species. To elucidate evolutionary patterns of the genes of interest, we used the NsSites and Branch Sites tests from PAML package, as well as several classic population tests, for different sample sets. In addition, we predicted the secondary structure of target proteins, using a specific methodology of the PSIPRED program, as well as PONDER-FIT for prediction of intrinsically disordered amino acids. Our in silico results suggest that the genes of the vasopressin receptor family (AVPR1A, AVPR1B, and AVPR2) present a pattern compatible with positive selection in placental mammals. Some of the sites with selection signals have linear motifs (SLiMS) predicted in the AVPR2 receptor, which may have facilitated the emergence of adaptive novelties, as was suggested for the kangaroo rat Dipodomys ordii, which inhabits arid regions. Analyses of the original PRLR coding region data on 17 NWM species revealed several sites present in the long form of the receptor with a high probability of being under positive selection, some of them (positions 507, 532 and 572) being associated with twin births, a characteristic of Callitrichidae species. Additionally, we verified in the Siimiformes branch a linear interaction motif that recognizes SH3 domains (Src Homology 3). The SH3 domains and their ligands were described for hundreds of proteins; they provide a particularly convenient and adaptable medium of specific protein-protein interaction to the cell, which can be of functional importance. This work as a whole contributed to the knowledge of the genetic repertoire connected to the complex network of neuroendocrine mechanisms associated to the emergence of physiological and behavioral adaptive traits in different mammalian clades.

A busca pelo repertório genético por trás de características comportamentais e reprodutivas de espécies de primatas tem desafiado nosso grupo de pesquisa. A premissa principal nesse tipo de estudo baseia-se na hipótese de que um traço fenotípico (seja ele fisiológico, comportamental, etc) compartilhado por um grupo taxonômico inteiro deve ser determinado por um repertório genético comum a esses táxons. Considerando a complexidade de muitas dessas características, temos ampliado nossos estudos para vários genes da rede OXT – AVP – PRL, usando abordagens in silico, in vitro, e in vivo. Na presente Tese, o conjunto gênico de estudo foi selecionado por uma metodologia baseada na ontologia biológica, com critério de seleção para características como comportamento materno, amamentação e aspectos reprodutivos, tais como comportamento de acasalamento e de corte. Essa seleção resultou em 12 genes candidatos para o estudo: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR e TRH. Exploramos aqui esse conjunto gênico da rede OXT – AVP – PRL através da mineração de dados, buscando por seus ortólogos nas várias espécies de primatas e de outros mamíferos, bem como através de novos dados de sequências da região codificadora dos genes PRLR e PRLH, em um conjunto de espécies de primatas do Novo Mundo (NWM, conforme sigla em inglês). Adicionalmente, sequências das regiões codificadoras de PRLR foram também obtidos em espécies de marsupiais. Com o objetivo de elucidar os padrões evolutivos dos genes de interesse, utilizamos análises como os testes NsSites e Branch Sites do pacote PAML, assim como vários testes populacionais clássicos, para diferentes conjuntos amostrais. Além disso, foi feita a predição da estrutura secundária das proteínas-alvo, utilizando metodologia específica do programa PSIPRED, bem como o PONDER-FIT, para a caracterização de aminoácidos intrinsecamente desordenados. Nossos resultados das análises in silico sugerem que os genes da família de receptores da vasopressina (AVPR1A, AVPR1B, e AVPR2) apresentam um padrão compatível com seleção positiva em mamíferos placentários. Alguns dos sítios com sinal de seleção apresentam motivos lineares (SLiMS) preditos no receptor AVPR2, que podem ter facilitado a emergência de novidades adaptativas, conforme foi sugerido para a espécie do rato-canguru Dipodomys ordii, que habita regiões áridas. As análises dos dados originais da região codificadora do gene PRLR em 17 espécies de NWM revelaram vários sítios presentes na forma longa do receptor com alta probabilidade de estarem sob seleção positiva, sendo que alguns deles (posições 507, 532 e 572) estão associados com o parto gemelar, uma característica das espécies de Callitrichidae. Adicionalmente verificamos no ramo dos Siimiformes um motivo linear de interação que reconhece domínios SH3 (Src Homology 3). Os domínios SH3 e os seus locais de ligação foram descritos para centenas de proteínas; eles fornecem à célula um meio particularmente conveniente e adaptável de interação específica proteína-proteína, que pode ser de importância funcional. Esse trabalho como um todo contribuiu para o conhecimento do repertório genético relacionado à complexa rede de mecanismos neuroendócrinos associados à emergência de traços adaptativos, tanto fisiológicos quanto comportamentais em diferentes clados de mamíferos. / The search for the genetic repertoire behind behavioral and reproductive features of Primate species has challenged our research group. The principal premise in this kind of study is based on the hypothesis that a phenotypic trait (either physiological, behavioral, etc.) shared by an entire taxonomic group should be determined by a genetic repertoire common to these taxa. Considering the complexity of many of these features, we have expanded our studies for several genes of the OXT - AVP - PRL network, using in silico, in vitro, and in vivo approaches. In the present Thesis, the set of genes for the study was selected by a methodology based on biological ontology, with features like maternal behavior, breastfeeding, and reproductive aspects, such as mating and courtship behavior. This selection resulted in 12 candidate genes for the study: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR, and TRH. We explored here this gene set of the OXT – AVP – PRL network through data mining, searching for their orthologues in many Primate species and other mammals, as well as through new sequence data from the PRLR and PRLH coding region in a set of New World Monkey (NWM) species. Additionally, sequences from the PRLR coding regions were also obtained in marsupial species. To elucidate evolutionary patterns of the genes of interest, we used the NsSites and Branch Sites tests from PAML package, as well as several classic population tests, for different sample sets. In addition, we predicted the secondary structure of target proteins, using a specific methodology of the PSIPRED program, as well as PONDER-FIT for prediction of intrinsically disordered amino acids. Our in silico results suggest that the genes of the vasopressin receptor family (AVPR1A, AVPR1B, and AVPR2) present a pattern compatible with positive selection in placental mammals. Some of the sites with selection signals have linear motifs (SLiMS) predicted in the AVPR2 receptor, which may have facilitated the emergence of adaptive novelties, as was suggested for the kangaroo rat Dipodomys ordii, which inhabits arid regions. Analyses of the original PRLR coding region data on 17 NWM species revealed several sites present in the long form of the receptor with a high probability of being under positive selection, some of them (positions 507, 532 and 572) being associated with twin births, a characteristic of Callitrichidae species. Additionally, we verified in the Siimiformes branch a linear interaction motif that recognizes SH3 domains (Src Homology 3). The SH3 domains and their ligands were described for hundreds of proteins; they provide a particularly convenient and adaptable medium of specific protein-protein interaction to the cell, which can be of functional importance. This work as a whole contributed to the knowledge of the genetic repertoire connected to the complex network of neuroendocrine mechanisms associated to the emergence of physiological and behavioral adaptive traits in different mammalian clades.

A busca pelo repertório genético por trás de características comportamentais e reprodutivas de espécies de primatas tem desafiado nosso grupo de pesquisa. A premissa principal nesse tipo de estudo baseia-se na hipótese de que um traço fenotípico (seja ele fisiológico, comportamental, etc) compartilhado por um grupo taxonômico inteiro deve ser determinado por um repertório genético comum a esses táxons. Considerando a complexidade de muitas dessas características, temos ampliado nossos estudos para vários genes da rede OXT – AVP – PRL, usando abordagens in silico, in vitro, e in vivo. Na presente Tese, o conjunto gênico de estudo foi selecionado por uma metodologia baseada na ontologia biológica, com critério de seleção para características como comportamento materno, amamentação e aspectos reprodutivos, tais como comportamento de acasalamento e de corte. Essa seleção resultou em 12 genes candidatos para o estudo: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR e TRH. Exploramos aqui esse conjunto gênico da rede OXT – AVP – PRL através da mineração de dados, buscando por seus ortólogos nas várias espécies de primatas e de outros mamíferos, bem como através de novos dados de sequências da região codificadora dos genes PRLR e PRLH, em um conjunto de espécies de primatas do Novo Mundo (NWM, conforme sigla em inglês). Adicionalmente, sequências das regiões codificadoras de PRLR foram também obtidos em espécies de marsupiais. Com o objetivo de elucidar os padrões evolutivos dos genes de interesse, utilizamos análises como os testes NsSites e Branch Sites do pacote PAML, assim como vários testes populacionais clássicos, para diferentes conjuntos amostrais. Além disso, foi feita a predição da estrutura secundária das proteínas-alvo, utilizando metodologia específica do programa PSIPRED, bem como o PONDER-FIT, para a caracterização de aminoácidos intrinsecamente desordenados. Nossos resultados das análises in silico sugerem que os genes da família de receptores da vasopressina (AVPR1A, AVPR1B, e AVPR2) apresentam um padrão compatível com seleção positiva em mamíferos placentários. Alguns dos sítios com sinal de seleção apresentam motivos lineares (SLiMS) preditos no receptor AVPR2, que podem ter facilitado a emergência de novidades adaptativas, conforme foi sugerido para a espécie do rato-canguru Dipodomys ordii, que habita regiões áridas. As análises dos dados originais da região codificadora do gene PRLR em 17 espécies de NWM revelaram vários sítios presentes na forma longa do receptor com alta probabilidade de estarem sob seleção positiva, sendo que alguns deles (posições 507, 532 e 572) estão associados com o parto gemelar, uma característica das espécies de Callitrichidae. Adicionalmente verificamos no ramo dos Siimiformes um motivo linear de interação que reconhece domínios SH3 (Src Homology 3). Os domínios SH3 e os seus locais de ligação foram descritos para centenas de proteínas; eles fornecem à célula um meio particularmente conveniente e adaptável de interação específica proteína-proteína, que pode ser de importância funcional. Esse trabalho como um todo contribuiu para o conhecimento do repertório genético relacionado à complexa rede de mecanismos neuroendócrinos associados à emergência de traços adaptativos, tanto fisiológicos quanto comportamentais em diferentes clados de mamíferos. / The search for the genetic repertoire behind behavioral and reproductive features of Primate species has challenged our research group. The principal premise in this kind of study is based on the hypothesis that a phenotypic trait (either physiological, behavioral, etc.) shared by an entire taxonomic group should be determined by a genetic repertoire common to these taxa. Considering the complexity of many of these features, we have expanded our studies for several genes of the OXT - AVP - PRL network, using in silico, in vitro, and in vivo approaches. In the present Thesis, the set of genes for the study was selected by a methodology based on biological ontology, with features like maternal behavior, breastfeeding, and reproductive aspects, such as mating and courtship behavior. This selection resulted in 12 candidate genes for the study: AVP, AVPR1A, AVPR1B, ESR1, FOS, HCRT, OXT, OXTR, PRL, PRLH, PRLR, and TRH. We explored here this gene set of the OXT – AVP – PRL network through data mining, searching for their orthologues in many Primate species and other mammals, as well as through new sequence data from the PRLR and PRLH coding region in a set of New World Monkey (NWM) species. Additionally, sequences from the PRLR coding regions were also obtained in marsupial species. To elucidate evolutionary patterns of the genes of interest, we used the NsSites and Branch Sites tests from PAML package, as well as several classic population tests, for different sample sets. In addition, we predicted the secondary structure of target proteins, using a specific methodology of the PSIPRED program, as well as PONDER-FIT for prediction of intrinsically disordered amino acids. Our in silico results suggest that the genes of the vasopressin receptor family (AVPR1A, AVPR1B, and AVPR2) present a pattern compatible with positive selection in placental mammals. Some of the sites with selection signals have linear motifs (SLiMS) predicted in the AVPR2 receptor, which may have facilitated the emergence of adaptive novelties, as was suggested for the kangaroo rat Dipodomys ordii, which inhabits arid regions. Analyses of the original PRLR coding region data on 17 NWM species revealed several sites present in the long form of the receptor with a high probability of being under positive selection, some of them (positions 507, 532 and 572) being associated with twin births, a characteristic of Callitrichidae species. Additionally, we verified in the Siimiformes branch a linear interaction motif that recognizes SH3 domains (Src Homology 3). The SH3 domains and their ligands were described for hundreds of proteins; they provide a particularly convenient and adaptable medium of specific protein-protein interaction to the cell, which can be of functional importance. This work as a whole contributed to the knowledge of the genetic repertoire connected to the complex network of neuroendocrine mechanisms associated to the emergence of physiological and behavioral adaptive traits in different mammalian clades.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.

Indiana University-Purdue University Indianapolis (IUPUI) / The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.