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.

Generation and Characterization of Knockout Mice and Analysis of Patient Material Demonstrates a Role for Tissue Inhibitor of Metalloproteinases 4 (Timp4) in the Cardiovascular System and Tumor Metastasis

This thesis focuses on tissue inhibitor of metalloproteinases 4 (TIMP4) which is the newest member of a small gene and protein family of four closely related endogenous inhibitors of extracellular matrix (ECM) degrading enzymes. Existing data on TIMP4 suggested that it exhibits a more restricted expression pattern than the other TIMPs with high expression levels in heart, brain, ovary and skeletal muscle. These observations and the fact that the ECM is of special importance to provide the cardiovascular system with structural strength combined with elasticity and distensibility, prompted the present molecular biologic investigation on TIMP4. In the first part of the study the murine Timp4 gene was cloned and characterized in detail. The structure of murine Timp4 genomic locus resembles that in other species and of the other Timps. The highest Timp4 expression was detected in heart, ovary and brain. As the expression pattern of Timp4 gives only limited information about its role in physiology and pathology, Timp4 knockout mice were generated next. The analysis of Timp4 knockout mice revealed that Timp4 deficiency has no obvious effect on the development, growth or fertility of mice. Therefore, Timp4 deficient mice were challenged using available cardiovascular models, i.e. experimental cardiac pressure overload and myocardial infarction. In the former model, Timp4 deficiency was found to be compensated by Timp2 overexpression, whereas in the myocardial infarct model, Timp4 deficiency resulted in increased mortality due to increased susceptibility for cardiac rupture. In the wound healing model, Timp4 deficiency was shown to result in transient retardation of re-epithelialization of cutaneous wounds. Melanoma tumor growth was similar in Timp4 deficient and control mice. Despite of this, lung metastasis of melanoma cells was significantly increased in Timp4 null mice. In an attempt to translate the current findings to patient material, TIMP4 expression was studied in human specimens representing different inflammatory cardiovascular pathologies, i.e. giant cell arteritis, atherosclerotic coronary arteries and heart allografts exhibiting signs of chronic rejection. The results showed that cardiovascular expression of TIMP4 is elevated particularly in areas exhibiting inflammation. The results of the present studies suggest that TIMP4 has a special role in the regulation of tissue repair processes in the heart, and also in healing wounds and metastases. Furthermore, evidence is provided suggesting the usefulness of TIMP4 as a novel systemic marker for vascular inflammation.

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.

Design and characterization of large area position sensitive radiation detectors

Planar, large area, position sensitive silicon detectors are widely utilized in high energy physics research and in medical, computed tomography (CT). This thesis describes author's research work relating to development of such detector components. The key motivation and objective for the research work has been the development of novel, position sensitive detectors improving the performance of the instruments they are intended for. Silicon strip detectors are the key components of barrel-shaped tracking instruments which are typically the innermost structures of high energy physics experimental stations. Particle colliders such as the former LEP collider or present LHC produce particle collisions and the silicon strip detector based trackers locate the trajectories of particles emanating from such collisions. Medical CT has become a regular part of everyday medical care in all developed countries. CT scanning enables x-ray imaging of all parts of the human body with an outstanding structural resolution and contrast. Brain, chest and abdomen slice images with a resolution of 0.5 mm are possible and latest CT machines are able to image whole human heart between heart beats. The two application areas are presented shortly and the radiation detection properties of planar silicon detectors are discussed. Fabrication methods and preamplifier electronics of the planar detectors are presented. Designs of the developed, large area silicon detectors are presented and measurement results of the key operating parameters are discussed. Static and dynamic performance of the developed silicon strip detectors are shown to be very satisfactory for experimental physics applications. Results relating to the developed, novel CT detector chips are found to be very promising for further development and all key performance goals are met.

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis

Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG) for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs) anti-C. fetus subsp. venerealis of the IgM (1) and IgG (14) classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs): two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

Detection and characterization of fibropapilloma associated herpesvirus of marine turtles in Rio Grande do Sul, Brazil

Fibropapillomatosis (FP) is a benign tumoral disease that affects sea turtles, hampering movement, sight and feeding, ultimately leading to death. In Brazil, the disease was described for the first time in 1986. Research suggests the involvement of a herpesvirus in association with environmental and genetic factors as causal agents of FP. The objective of the present study was to detect and characterize this herpesvirus in sea turtles living in the coast of state Rio Grande do Sul (RS), Brazil. From October 2008 to July 2010, 14 turtles were observed between the beaches of Torres and Tavares, of which 11 were green turtles (Chelonia mydas) and 3 were loggerhead turtles (Caretta caretta). All turtles were young and mean curved carapace length was 37.71±7.82cm, and varied from 31 to 55cm. Only one green turtle presented a 1cm, papillary, pigmented fibropapilloma. Skin and fibropapilloma samples were analyzed by conventional and real time PCR assays to detect and quantify herpesvirus. All skin samples were negative, though the fibropapilloma specimen was positive in both tests. Viral load was 9,917.04 copies of viral genome per milligram of tissue. The DNA fragment amplified from the fibropapilloma sample was sequenced and allocated in the Atlantic phylogeographic group. This study reports the first molecular characterization of herpesvirus associated with fibropapilloma in turtles from the coast of RS.

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

First isolation and characterization of Leptospira interrogans serogroup Australis from swine in Brazil

The purpose of this study was to report the first recovery and characterization of Leptospira interrogans (serogroup Australis) from urine of swine in Brazil. The isolate was studied by serogrouping, MLVA, PGFE, and partial sequencing of rrs and secY. It was serogrouped as serogroup Australis, probably serovar Bratislava (titre 1,600), and sequenced as Leptospira interrogans. The MLVA and PGFE profiles also suggested the isolate as serovar Bratislava, since they were indistinguishable from reference strains Balico and Jez Bratislava. This is the first Leptospira interrogans serogroup Australis isolate, probably serovar Bratislava, obtained in Brazil.

Occurrence and characterization of Campylobacter spp.isolates in dogs, cats and children

To improve the understanding of implications of Campylobacterspp. infections in pets and children of different environments were analysed 160 faecal samples from children and 120 from pets (103 dogs and 17 cats). Campylobacter spp. were detected in 6.87% of the children and in 18.3% of the dogs and cats. From 33 stool samples positive for Campylobacter spp., 57.6% were identified as C. jejuni, and 33.4% were identified as C. coli. More than 50% of the isolates in pets were resistant to ceftiofur, sulphazotrim, norfloxacin and tetracycline. In humans, most of the isolates were resistant to amoxicillin, cefazolin, ceftiofur, erythromycin and norfloxacin. From 19 isolates of C. jejuni, 11 isolates from children and 5 from dogs contained two to four of the virulence genes flaA, pldA, cadF or ciaB.We found an association between the presence of virulence genes and diarrhoea. Furthermore, an association was observed between the presence of Campylobacter spp. and diarrhoea in dewormed pets with blood picture suggestive of bacterial infection, and the therapeutic use of antibiotics was associated with more positive detection of Campylobacterspp. in the faeces of pets. Our data indicate that virulent strains of Campylobacter spp. can be risk factor to diarrhoea in animals, and that high resistance to antimicrobial agents is common in pets.

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.

Purification and characterisation of a lectin from the red marine alga Pterocladiella capillacea (S.G. Gmel.) Santel. & Hommers.

A lectin present in the marine red alga Pterocladiella capillacea was purified and characterised by extraction of soluble proteins (crude extract) in 20 mM Tris-HCl buffer, pH 7.5. Among the analysed erythrocytes (human blood group A, B and O and the animals ox, goat, chicken and rabbit) the lectin agglutinated specifically rabbit erythrocytes. The hemagglutinating activity assay showed that the lectin was not dependent on divalent cations and was shown to be inhibited by the glycoproteins avidin and mucin. The purification procedure was conduced by precipitation of the crude extract with 80% saturation ammonium sulfate (F0/80) followed by affinity chromatography on guar-gum column. The lectin of P. capillacea was purified 14.5 fold and had a recovery of 27.4% of the original total specific activity present in the crude extract. The absence of carbohydrate suggested that the lectin is not a glycoprotein. The molecular mass of P. capillacea lectin, determined by gel filtration, was 5.8 kDa. SDS-PAGE in the presence of ß-mercaptoethanol gave one band, indicating that the native lectin is a monomeric protein. The activation energy of denaturation process (D G') was calculated to be 106.87 kJ . mol-1 at 70 ºC.

Synthesis and Characterization of Cellulose fiber-silica nanocomposites

Cellulose fiber-silica nanocomposites with novel mechanical, chemical and thermal properties have potential to be widely applied in different area. Monodispered silica nanoparticles play an important role in enhancing hybrids properties of hardness, strength, thermal stability etc. On the other hand, cellulose is one of the world’s most abundant and renewable polymers and possesses several unique properties required in many areas and biomedicine. The aim of this master thesis is to study if silica particles from reaction of sodium silicate and sulphuric acid can be adsorbed onto cellulose fiber surfaces via in situ growth. First, nanosilica particles were synthesized. Effect of pH and silica contents were tested. In theoretical part, introduction of silica, methods of preparation of nanosilica from sodium silicate, effect factors and additives were discussed. Then, cellulose fiber-silica nanocomposites were synthesis via route from sodium silicate and route silicic acid. In the experiment of route from sodium silicate, the effects of types of sodium silicate, pH and target ratio of silica to fiber were investigated. From another aspect, the effects of types of sodium silicate, fiber concentration in mixture solution and target ratio of silica to fiber were tested in the experiment of route from silicic acid. Samples were investigated via zeta potential measurement, particle size distribution, ash content measurement and Scanning Electron Microscopy (SEM). The Results of the experiment of preparing silica sol were that the particle size of silica sol was smaller prepared in pH 11.7 than that prepared in pH 9.3. Then in the experiment of synthesis of cellulose fiber-silica nanocomposites, it was concluded that the zeta potential of all the samples were around -16 mV and the highest ash content of all the samples was only 1.4%. The results of SEM images showed only a few of silica particles could be observed on the fiber surface, which corresponded to the value of ash content measurement.

Isolation and characterization of a reserve protein from the seeds of Opuntia ficus-indica (Cactaceae)

We describe here the isolation and characterization of a major albumin from the seeds of Opuntia ficus-indica (Cactaceae). This protein has a molecular mass of 6.5 kDa and was isolated by a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of this protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The N-terminal amino acid sequence of this protein is Asp-Pro-Tyr-Trp-Glu-Gln-Arg.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.