The Distal-less (Dlx) homeobox genes of mammals are expressed in many tissues of the developing organism including the limbs, craniofacial skeleton and the forebrain. In the forebrain, Dlx1, Dlx2, Dlx5 and Dlx6 play a critical role in driving tangential migration of GABAergic progenitors from the ventral telencephalon to their final destinations, notably the neocortex and the striatum. These Dlx genes are organised into convergently transcribed clusters with short intergenic regions that contain notable cis regulators elements (CREs) that drive Dlx expression in unique subdomains of the developing ventral telencephalon. Previous studies have characterised Dlx regulation including but not limited to the direct activation of these CREs by effector proteins. However, to date very little work has been done to examine how the forebrain Dlx genes may be regulated at the level of the chromatin. To explore this, I used in silico and in vivo methods to examine some key histone modifications of the Dlx1/2 and Dlx5/6 bigene clusters in the developing forebrain; namely H3K27Ac, H3K4me3, H3K4me1 and H3K27me3. I found that within the Dlx expressing ganglionic eminences (GE), at midgestation, the Dlx loci are marked by bivalent chromatin which is enriched in both permissive H3K4me3 and repressive H3K27me3 marks. By performing ChIP-qPCR on the GE tissue of embryonic mice with targeted deletions of enhancer CREs, I found that these CREs play unique roles in shaping the chromatin. Removal of one of these CREs has widespread effects on the chromatin at both loci. Since these changes in chromatin signatures do not accompany significant changes in expression of histone modifying genes, we believe these CREs play yet-to-be determined roles in recruiting the modifying proteins to the loci, thereby establishing bivalent chromatin to fine-tune Dlx expression.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

Made available in DSpace on 2018-08-02T00:16:27Z (GMT). No. of bitstreams: 1 tese_7844_Dissertação Marina Marques Bonomo.pdf: 1383403 bytes, checksum: d97f7f2062882542783b7ec7d1a23233 (MD5) Previous issue date: 2014-02-14 / RESUMO A aplicação de lodo de esgoto na agricultura é uma prática crescente em muitos países devido a seus inúmeros benefícios para o solo e para as plantas, representando uma alternativa de disposição final prática e proveitosa para o resíduo. A utilização do lodo contribui no ciclo bioquímico dos nutrientes minerais e serve como fonte de matéria orgânica, micro e macro-nutrientes para o solo e para a cultura, promovendo um estímulo no crescimento da planta, principalmente de espécies de alta exigência nutricional. Porém, é de extrema importância que seja realizado o monitoramento dos efeitos do resíduo sobre os organismos expostos, considerando a presença de contaminantes e seu potencial tóxico para o ambiente. O objetivo deste trabalho foi avaliar as respostas biológicas da aplicação agrícola de doses crescentes de lodo de esgoto higienizado, por meio da integração destas com análises químicas, visando elucidar os benefícios e potenciais riscos da utilização deste resíduo. Para isso, foram realizadas avaliações morfológicas, anatômicas, fisiológicas e bioquímicas em Carica papaya, em adição a análises citogenéticas por meio do teste de Allium cepa. Os teores nutricionais observados nas análises químicas corroboram o potencial de utilização do lodo de esgoto higienizado como insumo agrícola, bem como os baixos níveis dos contaminantes analisados fazem com que o resíduo esteja dentro dos padrões exigidos pela legislação. Por outro lado, as análises dos efeitos citogenéticos observados em A. cepa e bioquímicos complementam os resultados observados na avaliação de crescimento de C. papaya para a confirmação de que a dosagem a ser aplicada no solo deve visar o suprimento das necessidades nutricionais da planta, porém sem causar danos e exigências metabólicas mais severas. Palavras-chave: mamão, Allium cepa, estresse oxidativo, crescimento vegetal, anatomia vegetal, resíduo sólido.

PC4/PCSK4 is the major Proprotein Convertase (PC) enzyme that plays a key role in mammalian fertilisation. It is detected in the acrosomal granules of round spermatids, acrosomal ridges of elongated spermatids and sperm plasma membrane overlying the acrosome with K-X-K/X-R as its preferred cleavage motif. Such motifs are present in male germ cell proteins ADAMs, proPACAP and proIGF-1/2 and these precursor proteins are processed most likely by PC4 during spermatogenesis, sperm maturation and sperm-egg interaction. For fertilization to occur, the mature sperm must penetrate the Zona Pelucida (ZP) and bind to the egg. Previously, PC4 null mouse sperm and wild type sperm treated with a specific PC4-inhibitor have shown to reduced abilities to penetrate the cumulus mass, bind to ZP and fertilize eggs. These findings suggest that sperm-PC4 plays an important role in fertilization and hence regulation of its activity is crucial for successful fertilization. But how PC4 activity is regulated in vivo is not yet clear. Recently, in epididymal fluid a serpin (serine protease inhibitor) called CRES has been described but the protease linked to this serpin in epididymis has not been identified. However in endocrine cells where CRES is also expressed, it inhibits PC2 enzyme. Thus based on localization and preliminary study, we propose that PC4 is the target enzyme for CRES in the reproductive tract. During sperm migration and storage in epididymis, sperm PC4 activity may be modulated by CRES so that premature sperm activation may not occur. Our data showed that CRES inhibits PC4 both in vitro (with IC50 in µM range) as well as ex vivo in human placenta trophoblast cell lines. Moreover CRES was found to be cleaved by PC4 suggesting a Serpin-Protease binding type of mechanism in the inhibition of protease activity. Taken together, we conclude that CRES regulates PC4 activity in reproductive tract crucial for mammalian fertilization.

Cystatin Related Epididymal Spermatogenic protein (CRES) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES is enigmatic and dependent on localization of its isoforms, which is the objective of this study. The initial approach was to investigate testicular and epididymal origins of these isoforms by immunohistochemistry and immunogold cytochemistry. To further pinpoint CRES localization we then selectively extracted and fractionated epididymal spermatozoa in order to find by immunoblotting which sperm fractions contained CRES isoforms. Immunohistochemical analysis of mouse spermatogenesis showed that CRES was expressed in the tail cytoplasm of elongating spermatids from step 9-16, with a pattern reminiscent of outer dense fibre (ODF) proteins. Ultrastructural immunocytochemistry revealed that the immunogold label was concentrated over growing ODFs and mitochondrial sheath in the testes which persisted in spermatozoa through the epididymis. Sequential extractions of isolated sperm tails with Triton X-100-dithiothreitol (DTT) to remove the mitochondrial sheath, whose extract contained an unrelated 66 kDa immunoreactive band, followed by either sodium dodecyl sulfate (SDS)-DTT or urea-DTT to solubilise accessory fibres of the tail revealed a 14 kDa immunoreactive band associated with the ODF. In addition, Western blots revealed glycosylated and non-glycosylated CRES isoforms in nonyl phenoxylpolyethoxylethanol (NP40) extracts of the caput, but not cauda, sperm. Immunohistochemical analysis of the caput and cauda epithelium showed that CRES is secreted by the Golgi apparatus of the ii initial segment, fills the proximal caput lumen, and disappears by mid caput. Western blots of caput and cauda tissue and luminal fluid revealed 14 and 19 kDa immunoreactive bands in caput tissues and luminal fluid, but not in the cauda. This study concludes that there are two origins of CRES, one arising in the testis and the other in the epididymis. Testicular CRES is ionically and covalently associated with the ODF while epididymal CRES is detergent soluble and is most likely associated temporarily with the surface of caput epididymal sperm. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2010-09-03 14:22:01.913