Resumo: A glucoamilase é uma enzima hidrolítica que catalisa a liberação sucessiva de β-D-glicose a partir do amido e oligossacarídeos relacionados. Neste trabalho foram estudadas as glucoamilases de Aspergillus awamori expressas em levedura Saccharomyces cerevisiae. Foram utilizadas duas linhagens alteradas denominadas M1 e M2, e uma linhagem selvagem (WT), utilizada como parâmetros na comparação dos resultados. As enzimas foram produzidas em fermentação submersa, e amidos de diferentes origens vegetais foram utilizados como uma fonte extra de carbono na produção das enzimas. O melhor substrato para a produção da glucoamilase selvagem e da mutante M2 foi o amido de batata com 8,2 e 6,6 U/mL, respectivamente. Para a linhagem M1 foi o amido de mandioca com atividade enzimática de 5,9 U/mL. O amido de milho mostrou ser um substrato menos indicado para a produção destas enzimas. Para a purificação foi preparada uma coluna de afinidade com resina sepharoseTM 6B epóxi ativada ligada a acarbose, onde diferentes concentrações do ligante foram avaliadas. A coluna apresentou boa eficiência no processo de purificação conforme análise por eletroforese SDS-PAGE, com massas moleculares estimada em 100 kDa. A temperatura ótima de atividade das enzimas M1 e M2 foi 65 °C, enquanto que a selvagem teve sua atividade máxima em 60 °C. O pH ótimo de atuação das enzimas foi 4,5. As glucoamilases mutantes apresentaram maior termoestabilidade que a glucoamilase selvagem durante o processo de termoinativação, destacando principalmente a glucoamilase M2. A meia vida a 70 °C foi de 8,1 minutos para a enzima mutante M2, 4,1 minutos para a M1 e 3,0 minutos para a enzima selvagem. A energia de ativação para a desnaturação (Ead) foi de 252,9 e 262,8 KJ mol-1 para as enzimas M1 e M2 respectivamente, e de 234,3 KJ mol-1 para a selvagem. A maior energia dos mutantes indica maior resistência... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glucoamylase is a hydrolytic enzyme that catalyzes the consecutive liberation of β-D-glucose from starch and related oligosaccharides. In this work glucoamylases from Aspergillus awamori expressed in the yeast Saccharomyces cerevisiae were studied. Two mutant strains, denominated M1 and M2, were used and one wild strain (WS) was used as parameter to compare the results. The enzymes were produced in submerged fermentation and starches from different botanical origins were used as extra carbon source for enzyme production. The best substrate for the production of wild glucoamylase and of mutant M2 was potato starch with 8.2 and 6.6 U/mL, respectively. For strain M1 the best substrate was cassava starch with enzymatic activity of 5.9 U/mL. Corn starch revealed to be a less indicated starch for the production of these enzymes. For purification, an affinity column was prepared with activated SepharoseTM 6B epoxy linked to acarbose, and different concentrations of ligand were evaluated. The column exhibited good efficiency during the purification process according to SDS-PAGE analysis, with molecular masses estimated in 100 kDa. Optimum temperature for activities of M1 and M2 enzymes was 65°C, while the wild one exhibited maximum activity at 60°C. Optimum pH for enzyme action was 4.5. Mutant glucoamylases presented higher thermostability than wild glucoamylase during the thermoinactivation process, with M2 standing out. Half life at 70°C was of 8.1 minutes for mutant enzyme M2, 4.1 minutes for M1 and 3.0 minutes for wild enzyme. Activation energy for denaturation (Ead) was 252.9 and 262.8 KJ mol-1 for enzymes M1 and M2 respectively, and 234.3 KJ mol-1 for the wild one. The higher energy of the mutants indicates higher resistance of the protein structure, since more energy is required for the molecule to enter a transition and unfolding state. Thermodynamic parameter ΔG was higher... (Complete abstract click electronic access below) / Orientador: Roberto da Silva / Coorientador: Heloiza Ferreira Alves-Prado / Banca: Maria de Lourdes T. de M. Polizeli / Banca: Fernando Araripe Gonçalves Torres / Banca: Henrique Ferreira / Banca: Eleonora Cano Carmona / Doutor

O crescente número de bactérias resistentes é devido principalmente ao número limitado de modos de ação dos antibióticos, contra os quais bactérias criam mecanismo de resistência. Há, portanto, necessidade de terapias com espectro de ação mais amplo, atingindo diferentes alvos moleculares. A inativação fotodinâmica (IFD) pode ser uma dessas terapias, pois baseia-se na geração de espécies reativas que atacam diversas moléculas, e não um alvo específico. Fotossensibilizadores (FSs) absorvem luz em comprimento de onda específico e a energia absorvida pode ser transferida a um oxigênio molecular, gerando espécies reativas de oxigênio (EROs). Tais espécies são altamente citotóxicas e produzem reações de oxidação que levam à morte celular. Um dos alvos das EROs são fosfolipídios insaturados das membranas biológicas. O objetivo desta dissertação é investigar a interação dos FSs curcumina e azul de metileno com fosfolipídios e o efeito da foto-ativação desses FSs em um mimético de membrana bacteriana. Para tanto, foram usados filmes de Langmuir do extrato lipídico de Escherichia coli e dos lipídios sintéticos isolados DOPE, POPG e cardiolipina. As isotermas de pressão com o extrato de E coli indicam interação entre os FSs e os lipídios do filme, aumentando a área ocupada. A irradiação do filme na presença de curcumina aumenta sua estabilidade, o que sugere formação de hidroperóxidos de lipídio, mais hidrofílicos, pela ação do oxigênio singleto. Nos filmes dos lipídios isolados só a curcumina é incorporada, havendo aumento na área ocupada pelo filme, e redução no potencial de superfície. Nenhum efeito decorrente da irradiação desses filmes foi detectado. Um filme Langmuir-Blodgett (LB) de extrato de E. coli com curcumina foi submetido a quatro ciclos de fotoclareamento seguido de recuperação da fluorescência visualizados num microscópio confocal. A intensidade da fluorescência aumentou após o primeiro ciclo, indicativo de mudança conformacional para alocar maior quantidade de curcumina, o que corrobora a hipótese da formação de hidroperóxidos. / The growing number of resistant bacteria is mainly due to the limited number of modes of action of antibiotics, against which bacteria create resistance. There is, therefore, a need for therapies with broader action spectrum, reaching different molecular targets. Photodynamic inactivation (PDI) may be one of these therapies, because it is based on the generation of reactive species that attack several molecules, not a specific target. Photosensitizers (FSs) absorb light at a specific wavelength and the absorbed energy can be transferred to a molecular oxygen, generating reactive oxygen species (ROS). Such species are highly cytotoxic and produce oxidation reactions that lead to cell death. One of the targets of ROS are unsaturated phospholipids from biological membranes. The objective of this dissertation is to investigate the interaction of the FSs curcumin and methylene blue with phospholipids and the effect of photoactivation of these FSs on a bacterial membrane mimetic. For this purpose, Langmuir films of the lipid extract of Escherichia coli and the synthetic lipids DOPE, POPG and cardiolipin were used. The surface pressure isotherms with the E. coli extract indicate interaction between the FSs and the lipids of the film, increasing the occupied area. The irradiation of the film in the presence of curcumin increases its stability, which suggests the formation of more hydrophilic lipid hydroperoxides by the action of singlet oxygen. In the synthetic lipid films only curcumin is incorporated, with increase in the area occupied by the film, and reduction in surface potential. No effect from irradiation of these films was detected. A Langmuir-Blodgett (LB) film of E. coli extract with curcumin was submitted to four cycles of photobleaching followed by fluorescence recovery visualized in a confocal microscope. The intensity of the fluorescence increased after the first cycle, indicative of conformational change to allocate a larger amount of curcumin, which corroborates the hypothesis of hydroperoxide formation.

Ground water resources are heavily used for drinking water supply and often as a receptacle for waste water. One concern is the possible contamination of wetland areas by ground water receiving septic system infiltration. To investigate this, two tracer studies were performed using the bacteriophage PRD-1 by seeding septic systems adjacent to wetlands with the phage and monitoring migration towards wetland areas. Transport velocities were evaluated based on appearance of tracer in sampling wells at various distances from the injection point. Velocities were estimated to be 0.25 m/d and 0.4 m/d at the two sites. Some retardation with respect to the conservative tracer SF6 was observed, with a factor of about 1.5. Due to dry conditions, the water table was well below surface, so transport of the virus into surface water was not observed. Survival of public-health-related microorganisms in ground water is also a concern. The effects of temperature and total dissolved solids (TDS) on survival of 5 groups of indicator organisms were evaluated in controlled experiments. TDS did not have significant effects on inactivation of these microbes up to 1000 mg/l, but there was indication of reduced inactivation of enterococci at TDS concentrations of 3000 mg/l. Increased temperature consistently resulted in more rapid inactivation. Survival in aquifer and reservoir water samples was also evaluated, and significant effects due to water type, temperature, and pasteurization treatment were observed. Inactivation was more rapid in surface water sources, and pasteurization enhanced survival. For enterococci and DNA coliphage, pasteurization effects were more pronounced in surface water. DNA coliphage and perhaps fecal coliform appeared to be the more-conservative indicator organisms for aquifer injection monitoring. Lastly, it was observed that inactivation rates were considerably slower in pore water of saturated limestone than in the bulk water column of similar water sources and conditions, particularly for enterococci and fecal coliform.

<p>A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia. </p><p>In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases. </p><p>In summary, a suggested model of molecular pathogenesis of cervical carcinoma is as follows. Specific types of HPV infect one or more committed stem cells in the basal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin) or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CIN steps. Less efficient LOH events would lead to CIN steps where some other unknown factors require to be added to facilitate the formation of carcinoma. In the absence of LOH events no carcinoma develops from the HPV-infected stem cells.</p>

<p>Background. Platelets (thrombocytes) are the smallest cells in the blood. Platelet fulfils functions as formation of blood clots when bleeding. Low levels leads to bleeding while high levels increase the risk of thrombosis (obstruction of the circulatory flow system). Platelet transfusions may be required for patients with systemic bleeding and for patients at higher risk of bleeding because of coagulation defects, sepsis (presence of bacteria in the bloodstream), or platelet dysfunction related to medication or disease. A pathogen-reduction system for platelet components would be a useful method since it reduces the risk of bacterial, protozoa, viral and white blood cell contamination. The Intercept Blood System method (IBS) for platelets, destroys DNA and RNA and was validated against the routine method in order to reduce pathogen transmission risk during transfusion. The validation of IBS, the trombocyte count for100 buffy coat concentrates from 2007 were compared to values for 100 buffy coat concentrates from 2006 that had been treated with gamma-radiation. Akademiska sjukhuset in Uppsala has a requirement that 75% of the platelet concentrates contain at least 300*10 9 platelets per unit. IBS fulfilled to 94% compared to 98% for the routine method.</p><p>Thus, the IBS-method was well above the required value and is now used at</p><p>Akademiska sjukhuset in Uppsala.</p>

A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia. In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases. In summary, a suggested model of molecular pathogenesis of cervical carcinoma is as follows. Specific types of HPV infect one or more committed stem cells in the basal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin) or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CIN steps. Less efficient LOH events would lead to CIN steps where some other unknown factors require to be added to facilitate the formation of carcinoma. In the absence of LOH events no carcinoma develops from the HPV-infected stem cells.

Background. Platelets (thrombocytes) are the smallest cells in the blood. Platelet fulfils functions as formation of blood clots when bleeding. Low levels leads to bleeding while high levels increase the risk of thrombosis (obstruction of the circulatory flow system). Platelet transfusions may be required for patients with systemic bleeding and for patients at higher risk of bleeding because of coagulation defects, sepsis (presence of bacteria in the bloodstream), or platelet dysfunction related to medication or disease. A pathogen-reduction system for platelet components would be a useful method since it reduces the risk of bacterial, protozoa, viral and white blood cell contamination. The Intercept Blood System method (IBS) for platelets, destroys DNA and RNA and was validated against the routine method in order to reduce pathogen transmission risk during transfusion. The validation of IBS, the trombocyte count for100 buffy coat concentrates from 2007 were compared to values for 100 buffy coat concentrates from 2006 that had been treated with gamma-radiation. Akademiska sjukhuset in Uppsala has a requirement that 75% of the platelet concentrates contain at least 300*10 9 platelets per unit. IBS fulfilled to 94% compared to 98% for the routine method. Thus, the IBS-method was well above the required value and is now used at Akademiska sjukhuset in Uppsala.

Type-II Ribosome Inactivating Proteins from Abrus precatorius: Cytotoxicity and Mechanism of Cell Death A/B toxins produced by bacteria and plants are among the deadliest molecules known. The plant type-II ribosome inactivating proteins (RIPs) are prototype of A/B toxins. They are two subunit proteins with a toxic A subunit that harbors an RNA N-glycosidase activity and a lectin like B subunit which allows toxin entry into cells. The toxicity of A chain is due to its RNA-N-glycosidase activity which cleaves the bond between the ribose sugar and the adenine at position 4324 as demonstrated in rat liver ribosomes. The B- chain, a lectin, binds to the cell surface receptors terminating in galactose sugars and allows toxin entry into cells. The seeds of the subtropical climber Abrus precatorius contain two RIPs: the potent toxic lectin abrin and the relatively less toxic Abrus agglutinin. The toxic property of RIPs has widespread applications in the field of agriculture and medicine. The cells of our body commit suicide in response to genetic or environmental cues by the process, apoptosis or programmed cell death which results in the safe clearance of the dead cells without affecting the extra-cellular milieu. Apoptosis is essential for development, tissue homeostasis, and defense against pathogens. It involves the interplay of multiple pathways that are initiated and executed by a family of proteases termed caspases. Several plant type-I and type-II RIPs as well as bacterial toxins have been shown to induce apoptosis in cultured cell lines. Though many agents that inhibit macromolecular synthesis in cells induce DNA fragmentation and morphological changes associated with apoptosis, the link between protein synthesis inhibition by these toxins and apoptosis remains elusive. Though extensive studies have been carried out on several RIPs for e.g. ricin and shiga toxin, only few reports are available in literature on the mechanisms of toxicity exhibited by abrin, a type-II RIP, of South-East Asian origin. Earlier studies from the laboratory have focused on the sensitivity and mechanism of abrin induced cell death in Jurkat, a cell line of haematopoietic lineage and its variants. In the same direction, the objectives of my study were: (1) To delineate the structure-function relationship of Abrus agglutinin-I in comparison with abrin, (2) To establish monoclonal antibodies to the A subunit of abrin, analyzing their neutralizing effect on abrin toxicity in vitro and in vivo and (3) To delineate the pathway and determine the kinetics of apoptosis induced by abrin on cell lines of epithelial lineage. The thesis will be presented in three four chapters. The first chapter, ‘Introduction’, begins with a brief history of RIPs, followed by the description of their distribution and classification. The transport of toxins which is a unique property of this class of proteins is discussed in detail and supported with appropriate figures. Also, information pertaining to the structure of abrin and apoptosis induced by RIPs is written in brief. In the second chapter of the thesis the structural and functional studies of Abrus agglutinin-I (APA-I) as compared to abrin are discussed. Abrin and APA-I share a high degree of homology, however, previous reports by Liu et al., indicate that APA-I is many fold less toxic in cell free systems as compared to abrin. In our studies, APA-I was found to be less toxic on cultured cell lines. The IC50 value of protein synthesis inhibition by abrin was found to be 0.4 ng/ml for both Jurkat and MCF-7 cell lines. A 20-1000 fold difference was observed in the sensitivity of these cell lines to APA-I. The extent of apoptosis induced by APA-I in A3I9.2 a caspases-8 mutant Jurkat variant cell line was comparable to abrin indicating that the apoptosis induction by APA-I might not be through the extrinsic pathway. instead, our studies showed that APA-I induced apoptosis followed the mitochondrial pathway of cell death, in a caspase dependent manner similar to that of abrin. Unlike other agglutinins like wheat germ agglutinin, the agglutinating ability of the agglutinin-I had no role in the apoptosis induced. Protein synthesis inhibition appeared to be mandatory for the apoptosis induced by APA-I. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison to that of the reported structure of abrin. The substitution of Asn200 in abrin with Pro199 in agglutinin-I seems to be a major cause for the decreased toxicity. This perhaps is not a consequence of any kink formation by Pro residue in the helical segment, as reported by others earlier but due to fewer interactions that proline can possibly have with the bound substrate. Passive immuno-neutralization by administration of neutralizing antibodies is widely used as therapy against poisoning by various toxins. In case of type-II RIPs like ricin, antibodies to the toxic subunit were proven to have better protective efficacy than those to the lectin subunit. Neutralizing antibodies to abrin are not reported in literature. Therefore, a panel of monoclonal antibodies (mAbs) to the recombinant A chain of abrin was developed in our laboratory and characterized, which is presented in the third chapter of the thesis. Of these, D6F10 a high affinity antibody, exhibited neutralizing effect on abrin induced cytotoxicity on different cell lines tested. Antibodies may neutralize biological toxins in multiple ways; our studies suggested that mAb D6F10 interferes in the earliest event i.e. attachment of the toxin to the cell surface. Significantly, with the administration of mice with mAb D6F10 the prophylactic effect of the mAb could be demonstrated. In chapter 4, the sensitivity, kinetics of proteins synthesis inhibition and the mechanism of abrin induced cell death in cell lines of epithelial lineage is presented. Both sensitivity and kinetics of MCF-7/pv, Ovcar3, and T47D cells appeared comparable while, a variant culture of MCF-7 over-expressing caspases-3 was 50 times more sensitive to abrin. There was no significant difference in the binding of abrin between MCF-7/pv and MCF-7/C3+ cells. Previous studies in our laboratory indicated that abrin induced apoptosis is a caspases-3 dependent process. Also, in several systems it has been shown that caspases-3 is an indispensable molecule for apoptotic cell death. To test the absolute requirement of caspase-3, we examined abrin-induced apoptosis in a human breast cancer cell line MCF-7/pv reportedly deficient in caspases-3. Unlike other molecules like cisplatin, apoptosis induced by abrin in the MCF- 7/pv cells was found to be caspase -3 independent. However faster kinetics of apoptosis is observed, indicating that there is amplification of the apoptotic signals in the presence of caspases-3 resulting in an early onset of DNA fragmentation. The kinetics of protein synthesis inhibition and apoptosis follows similar kinetics in Jurkat cells while there is a time lapse between the two events in epithelial cells. Even with very high concentrations of abrin no detectable apoptosis was observed within 24 h in epithelial cells. The onset of fragmentation occurs after 24 h in the cell lines tested as opposed to Jurkat where it is observed as early as 6 h. Inhibition of caspases rescued the toxins from DNA fragmentation suggesting that the toxin does not cause direct nuclear damage in the cell line which does not involve the activation of caspases.

Access to sustainable sanitation systems is a determining factor in human health and economic development. However, more than a third of the world’s population lives without access to improved sanitation facilities. To meet the sanitation United Nations Millennium Development target, "halve, by 2015, the proportion of people without sustainable access to safe drinking water and basic sanitation", a wide range of non conventional sanitation technologies have been implemented in developing countries, including waterless systems. These systems function by diverting urine away from feces and collecting, storing, and dehydrating the fecal material in watertight dehydration vaults. From a public health perspective, adequate inactivation of fecal pathogens in a sanitation system is essential before any use or disposal of fecal material. In rural areas of El Salvador, the solar toilet is capable of inactivating fecal pathogens and reducing the prevalence of parasitic infections in its users when compared to other waterless systems. Nevertheless, not all solar toilets are able to inactivate completely Ascaris spp. ova after the recommended storage period. Un-ionized ammonia (NH3) has the potential to inactivate pathogens in solutions and sludge, including Ascaris spp. ova. This study hypothesized that adding ammonia to the solar toilet will improve the technology since pathogen inactivation with ammonia could be potentiated by the alkaline medium and high temperatures achieved inside the toilet vaults. To evaluate this approach, a series of experiments in solution and biosolid were performed in a laboratory environment using physical and chemical parameters similar to those achieved by the solar toilet. Eggs of the swine Ascaris species, Ascaris suum, were used as model in all experiments. In ammonia solution, the parasite ova were stored for a period of three days and; in biosolid, the parasite ova were stored for two months. Urea was used as the source of ammonia in biosolid. In addition to the experiments with ammonia, normal viability and morphological changes within the parasite ova during incubation in vitro at 28 C° were investigated and described to complement current literature published. Results from the experiments in ammonia solution indicated that addition of ammonia (1% and 2%) could improve the system since the critical parameters that significantly reduced A. suum ova viability to zero in three days could be achieved by the solar toilet: temperature of 35°C or higher and pH value of 9.3. Results from the experiments in biosolid further showed that inactivation of A. suum ova was faster in samples exposed to urea and to temperatures higher than 28°C. All samples exposed to urea achieved 100% inactivation after 14 days (28°C), 3 days (35°C) and 24 hours (40°C and 45°C). Survival analysis of the data showed that there was a significant difference (p value <.0001) between the inactivation achieved in the samples exposed to urea (1% and 2%) and the samples not exposed to urea. A logistic regression analysis estimated the effect of Urea (Treatment, OR: 25.9), Temperature (OR: 1.8), and Storage (OR: 1.17) on inactivation. Results from the experiment with A. suum ova in normal incubation solution showed that the ova went through clearly identified morphological changes at different speed of development. Two new additional stages of development were identified (Pre-larva 1 and Pre-larva 2) and no significant statistical difference was observed among the viability reported early in incubation and the one reported after three weeks of in vitro incubation, indicating that early stages of development may be use as an alternative to reduce the time to report viability. The results of this study suggest that inactivation of Ascaris spp ova by ammonia is possible in fecal material stored in the solar toilet or any other dry toilet, if the following physical and chemical conditions are met: a closed vault with a minimum temperature of 28°C; an initial pH of 8.3, minimum moisture of 27.5%, and addition of 1% urea to the biosolid. At 28°C longer storage time would be required for 100% inactivation while at higher temperatures less time of storage would be necessary. A community intervention is recommended to include field conditions and human behavior as other predictors for Ascaris spp. inactivation by ammonia.

Ctcf is a "master regulator" of the genome that plays a role in a variety of gene regulatory functions as well as in genome architecture. Evidence from studying the epigenetic process of X-chromosome inactivation suggests that, in certain cases, Ctcf might carry out its functions through interacting with RNA. Using mouse embryonic stem (ES) cells and a modified protocol for UV-crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq), Ctcf is found to interact with a multitude of transcripts genome-wide, both protein-coding mRNA (or noncoding transcripts therein) as well as many long-noncoding RNA (lncRNA). Examples of the latter include both well-characterized species from imprinted loci and previously unannotated transcripts from intergenic space. RNA binding targets of Ctcf are validated by a variety of biochemical methods, and Ctcf is found to interact with RNA through its C-terminal domain, distinct from its DNA-binding zinc-finger domain. Ctcf chromatin immunoprecipitation (ChIP)-seq done in parallel reveals distinct but correlated binding of Ctcf to DNA and RNA. In addition, allelic analysis of Ctcf ChIP pattern reveals significant differences between Ctcf binding to the presumptive inactive and active X chromosomes. Together, the current work reveals a further layer of complexity to Ctcf biology by implicating a role for Ctcf-RNA interactions in its recruitment to genomic binding sites.