Identification and characterization of new genes and pathways regulating spermatogonial cell fate

Spermatogenesis, i.e sperm production in the seminiferous tubules of the testis, is a complex process that takes over one month to complete. Life-long ability of sperm production ultimately lies in a small population of undifferentiated cells, called spermatogonial stem cells (SSCs). These cells give rise to differentiating spermatogonia, which are committed to mature into spermatozoa. SSCs represent a heterogeneous population of cells and many aspects of their basic biology are still unknown. Understanding the mechanisms behind the cell fate decision of these cells is important to gain more insights into the causes of infertility and testis cancer. In addition, an interesting new aspect is the use of testis-derived stem cells in regenerative medicine. Our data demonstrated that adult mouse testis houses a population of Nanog-expressing spermatogonia. Based on mRNA and protein analysis these cells are enriched in stage XII of the mouse seminiferous epithelial cycle. The cells derived from this stage have the highest capacity to give rise to ES cell-like cells which express Oct4 and Nanog. These cells are under tight non- GDNF regulation but their fate can be dictated by activating p21 signalling. Comparative studies suggested that these cells are regulated like ES cells. Taken together these data imply that pluripotent cells are present in the adult mammalian testis. CIP2A (cancerous inhibitor of PP2A) has been associated with tumour aggressiveness and poor prognosis. In the testis it is expressed by the descendants of stem cells, i.e. the spermatogonial progenitor cells. Our data suggest that CIP2A acts upstream of PLZF and is needed for quantitatively normal spermatogenesis. Classification of CIP2A as a cancer/testis gene makes it an attractive target for cancer therapy. Study on the CIP2A deficient mouse model demonstrates that systemic inhibition of CIP2A does not severely interfere with growth and development or tissue or organ function, except for the spermatogenic output. These data demonstrate that CIP2A is required for quantitatively normal spermatogenesis. Hedgehog (Hh) signalling is involved in the development and maintenance of many different tissues and organs. According to our data, Hh signalling is active at many different levels during rat spermatogenesis: in spermatogonia, spermatocytes and late elongating spermatids. Localization of Suppressor of Fused (SuFu), the negative regulator of the pathway, specifically in early elongating spermatids suggests that Hh signalling needs to be shut down in these cells. Introduction of Hh signalling inhibitor resulted in an increase in germ cell apoptosis. Follicle-stimulating hormone (FSH) and inhibition of receptor tyrosine kinases resulted in down-regulation of Hh signalling. These data show that Hh signalling is under endocrine and paracrine control and it promotes germ cell survival.

A Farewell to Flat Biology. Three-dimensional Cell Culture Models in Cancer Drug Target Identification and Validation

Cells of epithelial origin, e.g. from breast and prostate cancers, effectively differentiate into complex multicellular structures when cultured in three-dimensions (3D) instead of conventional two-dimensional (2D) adherent surfaces. The spectrum of different organotypic morphologies is highly dependent on the culture environment that can be either non-adherent or scaffold-based. When embedded in physiological extracellular matrices (ECMs), such as laminin-rich basement membrane extracts, normal epithelial cells differentiate into acinar spheroids reminiscent of glandular ductal structures. Transformed cancer cells, in contrast, typically fail to undergo acinar morphogenic patterns, forming poorly differentiated or invasive multicellular structures. The 3D cancer spheroids are widely accepted to better recapitulate various tumorigenic processes and drug responses. So far, however, 3D models have been employed predominantly in the Academia, whereas the pharmaceutical industry has yet to adopt a more widely and routine use. This is mainly due to poor characterisation of cell models, lack of standardised workflows and high throughput cell culture platforms, and the availability of proper readout and quantification tools. In this thesis, a complete workflow has been established entailing well-characterised 3D cell culture models for prostate cancer, a standardised 3D cell culture routine based on high-throughput-ready platform, automated image acquisition with concomitant morphometric image analysis, and data visualisation, in order to enable large-scale high-content screens. Our integrated suite of software and statistical analysis tools were optimised and validated using a comprehensive panel of prostate cancer cell lines and 3D models. The tools quantify multiple key cancer-relevant morphological features, ranging from cancer cell invasion through multicellular differentiation to growth, and detect dynamic changes both in morphology and function, such as cell death and apoptosis, in response to experimental perturbations including RNA interference and small molecule inhibitors. Our panel of cell lines included many non-transformed and most currently available classic prostate cancer cell lines, which were characterised for their morphogenetic properties in 3D laminin-rich ECM. The phenotypes and gene expression profiles were evaluated concerning their relevance for pre-clinical drug discovery, disease modelling and basic research. In addition, a spontaneous model for invasive transformation was discovered, displaying a highdegree of epithelial plasticity. This plasticity is mediated by an abundant bioactive serum lipid, lysophosphatidic acid (LPA), and its receptor LPAR1. The invasive transformation was caused by abrupt cytoskeletal rearrangement through impaired G protein alpha 12/13 and RhoA/ROCK, and mediated by upregulated adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A, and Rac/ PAK pathways. The spontaneous invasion model tangibly exemplifies the biological relevance of organotypic cell culture models. Overall, this thesis work underlines the power of novel morphometric screening tools in drug discovery.

Identification and characterization of CIP2A as a novel oncogenic inhibitor of PP2A

Inhibition of the tumor suppressor protein phosphatase 2A (PP2A) activity has been identified as one of the five key alterations required for human cell transformation. Regardless of this crucial role in human cancer development, the detailed mechanisms by which PP2A inhibition occurs in human cancers remain largely uncharacterized. PP2A regulates a plethora of cellular signaling cascades. One of the targets of PP2A is Myc oncoprotein, which is destabilized and degraded in response to PP2A-mediated dephosphorylation of Myc serine 62. In this study we identify Cancerous Inhibitor of PP2A (CIP2A) as a previously uncharacterized endogenous inhibitor of PP2A in human cancer cells. CIP2A inhibits PP2A activity leading to subsequent stabilization of the Myc protein. CIP2A promotes malignant growth of cancer cells in vitro and xenograft tumor formation in vivo and is overexpressed in cancer. Moreover, we explored the effect of CIP2A on global transcriptional profiles and validated a CIP2A-dependent transcriptional signature. Analysis of the CIP2A signature revealed both Myc-dependent and -independent functions for CIP2A. Importantly, we demonstrate that the CIP2A signature has clinical relevance in human breast cancer subtypes. Finally, we identify the genes potentially mediating the long-term growth suppression in CIP2A depleted cancer cells. Taken together, this work identifies CIP2A as a novel human oncoprotein and describes its function in cancer cells. These results may open novel possibilities for patient stratification and therapeutic intervention of cancer.

Avaliação de microcalcificações mamárias de acordo com as classificações do Breast Imaging Reporting and Data System (BI-RADS TM) e de Le Gal

OBJETIVO: avaliar a acurácia da mamografia para o diagnóstico de microcalcificações mamárias suspeitas, com as classificações do Breast Imaging Reporting and Data System (BI-RADS TM) e Le Gal em comparação com o resultado histopatológico utilizado como padrão-ouro. MÉTODOS: foram selecionados dos arquivos dos blocos cirúrgicos, 130 casos operados com mamografias contendo somente microcalcificações mamárias, inicialmente classificadas como suspeitas sem lesões detectáveis ao exame clínico. Estas foram reclassificadas por dois examinadores, utilizando as classificações de Le Gal e BI-RADS TM, obtendo-se diagnóstico de consenso. As biópsias foram revistas por dois patologistas e foi obtido diagnóstico de consenso. A leitura das mamografias e a revisão das lâminas foram feitas em duplo-cego. As análises estatísticas utilizadas neste estudo foram o teste do chi2, o modelo Fleiss quadrático para VPP e o programa Epi-Info 6.0. RESULTADOS: a correlação entre a análise histopatológica e mamográfica, usando BI-RADS TM e Le Gal, mostrou a mesma sensibilidade de 96,4%, especificidade de 55,9 e 30,3%, valor preditivo positivo (VPP) de 37,5% e 27,5% e acurácia de 64,6 e 44,6%, respectivamente. Quando discriminamos por categorias de BI-RADS TM, obtivemos VPPs: categoria 2, 0%; categoria 3, 1,8%; categoria 4, 31,6% e categoria 5, 60%. Os VPPs pela classificação de Le Gal foram: categoria 2, 3,1%; categoria 3, 18,1 %; categoria 4, 26,4%; categoria 5, 66,7% e não classificável, 5,2%. CONCLUSÕES: observou-se uma maior precisão com a classificação de BI-RADS TM, porém não se conseguiu reduzir a ambigüidade na avaliação das microcalcificações mamárias.

Identification and application of bile metabolites to assess the exposure of fish to pharmaceuticals in the environment

Thousands of tons of pharmaceuticals are consumed yearly worldwide. Due to the continuous and increasing consumption and their incomplete elimination in wastewater treatment plants (WWTP), pharmaceuticals and their metabolites can be detected in receiving waters, although at low concentrations (ng to low μg/L). As bioactive molecules the presence of pharmaceuticals in the aquatic environment must be considered potentially hazardous for the aquatic organisms. In this thesis, the biotransformation and excretion of pharmaceuticals in fish was studied. The main biotransformation pathways of three anti‐inflammatory drugs, diclofenac, naproxen and ibuprofen, in rainbow trout were glucuronidation and taurine conjugation of the parent compounds and their phase I metabolites. The same metabolites were present in fish bile in aquatic exposures as in fish dosed with intraperitoneal injection. Higher bioconcentration factor in bile (BCFbile) was found for ibuprofen when compared to diclofenac and naproxen. Laboratory exposure studies were followed by a study of uptake of pharmaceuticals in a wild fish population living in lake contaminated with WWTP effluents. Of the analyzed 17 pharmaceuticals and six phase I metabolites, only diclofenac, naproxen and ibuprofen was present in bream and roach bile. It was shown, that diclofenac, naproxen and ibuprofen excreted by the liver can be found in rainbow trout and in two native fish species living in the receiving waters. In the bream and roach bile, the concentrations of diclofenac, naproxen and ibuprofen were roughly 1000 times higher than those found in the lake water, while in the laboratory exposures, the bioconcentration of the compounds and their metabolites in rainbow trout bile were at the same level as in wild fish or an order of magnitude higher. Thus, the parent compounds and their metabolites in fish bile can be used as a reliable biomarker to monitor the exposure of fish to environmental pharmaceuticals present in water receiving discharges from WWTPs.

Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis

The present study evaluated the pheno- and genotypical antimicrobial resistance profile of coagulase-negative Staphylococcus (CNS) species isolated from dairy cows milk, specially concerning to oxacillin. Of 100 CNS isolates, the S. xylosus was the prevalent species, followed by S. cohnii, S. hominis, S. capitis and S. haemolyticus. Only 6% were phenotypically susceptible to the antimicrobial agents tested in disk diffusion assay. Penicillin and ampicillin resistance rates were significantly higher than others antimicrobials. Four isolates were positive to mecA gene (4%), all represented by the S. xylosus species. The blaZ gene was detected in 16% of the isolates (16/100). It was noticed that all mecA + were also positive to this gene and the presence of both genes was correlated to phenotypic beta-lactamic resistance. We conclude that CNS species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic and genotypic tests, which has implications for treatment and management decisions.

Regulation of self-renewal and detection of karyotypic changes of pluripotent human embryonic stem cells

Human embryonic stem cells are pluripotent cells capable of renewing themselves and differentiating to specialized cell types. Because of their unique regenerative potential, pluripotent cells offer new opportunities for disease modeling, development of regenerative therapies, and treating diseases. Before pluripotent cells can be used in any therapeutic applications, there are numerous challenges to overcome. For instance, the key regulators of pluripotency need to be clarified. In addition, long term culture of pluripotent cells is associated with the accumulation of karyotypic abnormalities, which is a concern regarding the safe use of the cells for therapeutic purposes. The goal of the work presented in this thesis was to identify new factors involved in the maintenance of pluripotency, and to further characterize molecular mechanisms of selected candidate genes. Furthermore, we aimed to set up a new method for analyzing genomic integrity of pluripotent cells. The experimental design applied in this study involved a wide range of molecular biology, genome-wide, and computational techniques to study the pluripotency of stem cells and the functions of the target genes. In collaboration with instrument and reagent company Perkin Elmer, KaryoliteTM BoBsTM was implemented for detecting karyotypic changes of pluripotent cells. Novel genes were identified that are highly and specifically expressed in hES cells. Of these genes, L1TD1 and POLR3G were chosen for further investigation. The results revealed that both of these factors are vital for the maintenance of pluripotency and self-renewal of the hESCs. KaryoliteTM BoBsTM was validated as a novel method to detect karyotypic abnormalities in pluripotent stem cells. The results presented in this thesis offer significant new information on the regulatory networks associated with pluripotency. The results will facilitate in understanding developmental and cancer biology, as well as creating stem cell based applications. KaryoliteTM BoBsTM provides rapid, high-throughput, and cost-efficient tool for screening of human pluripotent cell cultures.

Identification and antimicrobial resistance of members from the Enterobacteriaceae family isolated from canaries (Serinus canaria)

Abstract: The Enterobacteriaceae family contains potentially zoonotic bacteria, and their presence in canaries is often reported, though the current status of these in bird flocks is unknown. Therefore, this study aimed to identify the most common genera of enterobacteria from canaries (Serinus canaria) and their antimicrobial resistance profiles. From February to June of 2013, a total of 387 cloacal swab samples from eight domiciliary breeding locations of Fortaleza city, Brazil, were collected and 58 necropsies were performed in canaries, which belonged to the Laboratory of Ornithological Studies. The samples were submitted to microbiological procedure using buffered peptone water and MacConkey agar. Colonies were selected according to their morphological characteristics on selective agar and submitted for biochemical identification and antimicrobial susceptibility. A total of 61 isolates were obtained, of which 42 were from cloacal swabs and 19 from necropsies. The most isolated bacteria was Escherichia coli with twenty five strains, followed by fourteen Klebsiellaspp., twelve Enterobacterspp., seven Pantoea agglomerans, two Serratiaspp. and one Proteus mirabilis. The antimicrobial to which the strains presented most resistance was sulfonamides with 55.7%, followed by ampicillin with 54.1% and tetracycline with 39.3%. The total of multidrug-resistant bacteria (MDR) was 34 (55.7%). In conclusion, canaries harbor members of the Enterobacteriaceae family and common strains present a high antimicrobial resistance rate, with a high frequency of MDR bacteria.

Biomolecular screening for inhibitors of butyrylcholinesterase : identification and characterization using in vitro and in silico tools

Drug discovery is a continuous process where researchers are constantly trying to find new and better drugs for the treatment of various conditions. Alzheimer’s disease, a neurodegenerative disease mostly affecting the elderly, has a complex etiology with several possible drug targets. Some of these targets have been known for years while other new targets and theories have emerged more recently. Cholinesterase inhibitors are the major class of drugs currently used for the symptomatic treatment of Alzheimer’s disease. In the Alzheimer’s disease brain there is a deficit of acetylcholine and an impairment in signal transmission. Acetylcholinesterase has therefore been the main target as this is the main enzyme hydrolysing acetylcholine and ending neurotransmission. It is believed that by inhibiting acetylcholinesterase the cholinergic signalling can be enhanced and the cognitive symptoms that arise in Alzheimer’s disease can be improved. Butyrylcholinesterase, the second enzyme of the cholinesterase family, has more recently attracted interest among researchers. Its function is still not fully known, but it is believed to play a role in several diseases, one of them being Alzheimer’s disease. In this contribution the aim has primarily been to identify butyrylcholinesterase inhibitors to be used as drug molecules or molecular probes in the future. Both synthetic and natural compounds in diverse and targeted screening libraries have been used for this purpose. The active compounds have been further characterized regarding their potencies, cytotoxicity, and furthermore, in two of the publications, the inhibitors ability to also inhibit Aβ aggregation in an attempt to discover bifunctional compounds. Further, in silico methods were used to evaluate the binding position of the active compounds with the enzyme targets. Mostly to differentiate between the selectivity towards acetylcholinesterase and butyrylcholinesterase, but also to assess the structural features required for enzyme inhibition. We also evaluated the compounds, active and non-active, in chemical space using the web-based tool ChemGPS-NP to try and determine the relevant chemical space occupied by cholinesterase inhibitors. In this study, we have succeeded in finding potent butyrylcholinesterase inhibitors with a diverse set of structures, nine chemical classes in total. In addition, some of the compounds are bifunctional as they also inhibit Aβ aggregation. The data gathered from all publications regarding the chemical space occupied by butyrylcholinesterase inhibitors we believe will give an insight into the chemically active space occupied by this type of inhibitors and will hopefully facilitate future screening and result in an even deeper knowledge of butyrylcholinesterase inhibitors.

Effects of anthropogenic and natural hydrological changes on the behavior of the acidic metal discharge from acid sulfate soils in a river- and lake system in western Finland

Larsmo-Öjasjön i Österbotten skapades genom invallningar på 1960-talet pga. industrins behov av sötvatten. Sedan dess har vattenområdet drabbats av återkommande försurning och fiskdöd, och invallningen har ofta beskyllts för problemen. Avhandlingen undersöker syrabelastningen i området; bl.a. hur markanvändning, hydrologi och klimatförändringen påverkar belastningen. Konsekvenserna undersöks med fiskyngel som bioindikator, och olika miljömetoder testas och diskuteras. Ökad kunskap om försurningen hjälper oss att tillämpa effektiva miljömetoder och få förbättrad vattenkvalitet i framtiden. Den primära orsaken till den försämrade vattenkvaliteten under de senaste 40 åren är intensiv dikning av svavelrika sediment. Detta leder till oxidering av svavlet till svavelsyra och uppkomst av sura sulfatjordar. Syran löser upp mängder med toxiska metaller som spolas ut i vattendragen. Undersökningen visar att tiotusentals ton svavelsyra tillsammans med stora mängder metaller rinner till Larsmo-Öjasjön per år från sura sulfatjordar. Åarna bidrar med mest belastning, men den sammanlagda belastningen från de otaliga dikena och bäckarna är oväntat stor. Andra potentiella källor till försurningen, t.ex. muddringar och humussyror, beräknas vara obetydliga. Syra- och metallbelastningen varierar kraftigt med hydrologin, dvs. störst belastning sker under vår- och höstflöden. En eventuell klimatförändring kan ändra på avrinningsmönstret och orsaka mera belastning vintertid. Den årligt återkommande syra- och metallbelastningen kan ofta hindra lakens förökning, vilket kan ha större långtgående konsekvenser för fiskpopulationerna än de relativt sällsynta stora surchockerna med synlig fiskdöd. För att förebygga skador på vattendragen bör man undvika att dränera svavelrika sedimenten. På redan existerande sura sulfatjordar visade sig kontroll av grundvattennivån kunna möjliggöra en effektiverad markanvändning utan märkbart ökade miljökonsekvenser.

Floral biology and breeding system of three Ipomoea weeds

The floral biology of three weeds, Ipomoea cairica, I. grandifolia and I. nil (Convolvulaceae), was studied in Botucatu and Jaboticabal, São Paulo, in southeastern Brazil. The three species are melittophilous, with a varied set of floral visitors, but with some overlapping. Cluster analysis using Jacquard similarity index indicated a greater similarity among different plant species in the same locality than among the populations at different places, in relation to floral visitor sets. The promiscuous and opportunistic features of the flowers were shown, with such type of adaptation to pollination being advantageous to weeds since pollinator availability is unpredictable at ruderal environments.

Cyanobacterial occurrence and detection of microcystin by planar chromatography in surface water of Billings and Guarapiranga Reservoirs, SP, Brazil

Billings and Guarapiranga Reservoirs were deeply affected by environmental disturbances, which more evident consequence are the cyanobacterial blooms. Microcystins are the most common cyanotoxin in freshwaters and more than 70 types are known. Different methods for microcystins analysis in water can be used, among which ELISA and HPLC are the most frequently employed. However, less sophisticated and more economic methods can also be used. This is the case of planar chromatography (thin-layer chromatography) method previously used in cyanotoxins purification but gradually replaced by others. Posterior optimization of the microcystin chromatography conditions and because of its simplicity, rapidity, efficiency and low cost, this method is again considered an option for the analysis of microcystins and nodularins. Considering the importance of Billings and Guarapiranga Reservoirs for drinking water supplies and the few scientific data about cyanobacteria and cyanotoxins in these water bodies, the aims of this work are to analyze the biodiversity of cyanobacteria in the Billings and Guarapiranga Reservoirs and the detection of dissolved microcystins in the water. It was possible to identify 17 species of cyanobacteria, 9 of them being potentially toxic. In Billings Reservoir Microcystis aeruginosa (Kützing) Kützing and Cylindrospermopsis raciborskii (Woloszynska) Seenayya & Subba Raju are the most common species, while in Guarapiranga Reservoir only M. aeruginosa was considered as a common species. Microcystins were detected in all Billings Reservoir samples and in only one sample from Guarapiranga Reservoir.

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.