Evaluation of DNA enzymes targeted against the RNA component of human telomerase

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is currently no effective cure. Consequently, developing new therapies and elucidating effective targets is crucial for this fatal disease. In recent years, DNA enzymes, deoxyribonucleic acid molecules with enzymatic activity, have emerged. In the same manner as ribozymes, DNA enzymes are able to effect cleavage of RNA in a sequence-specific manner, and operate with catalytic efficiency. In this study, two DNA enzymes were designed to target the template region of human telomerase RNA (hTR), utilising the 10-23 and 8-17 catalytic motifs elucidated by Santoro and Joyce (1997). Telomerase is an RNA-dependent DNA polymerase, which stabilises telomere lengths by adding hexameric repeats (TTAGGG in humans) to chromosome termini, thus preventing the telomere shortening that usually occurs during mitotic cell division. Telomerase activity, whilst absent in normal somatic tissues, is present in almost 90% of all tumours. Thus, there is speculation that telomerase may be the much sought universal target for therapeutic intervention in cancer. In vitro cleavage assays showed both DNA enzymes to be catalytically competent. Unmodified phosphodiester (PO) backbone DNA enzymes were rapidly degraded in the presence of serum, with a half-life of 10 minutes. The common approach of introducing phosphorothioate (PS) linkages was used in an effort to overcome this instability. As a result of concurrent activity and stability studies on the DNA enzymes with various numbers of PS linkages, the DNA enzymes with a PO core and PS arms were chosen for use in further cell work. The cleavage activity of both was shown to be specific and affected by temperature, pH, MgCI2 concentration and enzyme concentration. Both DNA enzyme motifs reduced telomerase activity in cell lysates, as assessed by the telomerase repeat amplification protocol (TRAP) with an IC50 of 100nM. DNA enzymes being polyanionic molecules do not readily cross biological barriers. Cellular association of naked DNA enzyme was inefficient at less than 2%. Cellular delivery of the DNA enzymes was effectively improved using commercial cationic lipid formulations. However, the lipid-mediated delivery of DNA enzymes to U87-MG cells over a 4-hour period did not significantly inhibit cell proliferation compared to controls. This is possibly due to an expected lag period between the inhibition of telomere maintenance and cell death. Therefore, biodegradable polymer microspheres were investigated as a potential delivery option for prolonged and sustained delivery. In vitro release profiles showed that after an initial burst, sustained release of DNA enzymes was observed over 35 days. Finally, the efficacy and specificity of the DNA enzymes were demonstrated in a luciferase based reporter assay. Specific inhibition of luciferase expression was displayed at 10nM. Thus DNA enzymes have potential against endogenous cellular targets.

The design of novel antifolates against pneumocystis carinii

The pneumonia caused by Pneumocystis carinii is ultimately responsible for the death of many acquired immunodeficiency syndrome (AIDS) patients. Large doses of trimethoprim and pyrimethamine in combination with a sulphonamide and/or pentamidine suppress the infection but produce serious side-effects and seldom prevent recurrence after treatment withdrawal. However, the partial success of the aforementioned antifolates, and also trimetrexate used alone, does suggest dihydrofolate reductase (DHFR) as a target for the development of antipneumocystis agents. From the DHFR inhibitory activities of 3'-substituted pyrimethamine analogues it was suggested that the 3'-(3'',3''-dimethyltriazen-1''-yl) substituent may be responsible for the greater activity for the P.carinii over the mammalian enzyme. Crystallographic and molecular modeling studies revealed considerable geometrical and electronic differences between the triazene and the chemically related formamidine functions that may account for the differences in DHFR inhibitory profiles. Structural and electronic parameters calculated for a series of 3'-(3'',3''-disubstitutedtriazen-1''-yl) pyrimethamine analogues did not correlate with the DHFR inhibitory activities. However, the in vitro screening against P.carinii DHFR revealed that the 3''-hydroxyethyl-3''-benzyl analogue was the most active and selective. Models of the active sites of human and P.carinii DHFRs were constructed using DHFR sequence and structural homology data which had identified key residues involved in substrate and cofactor binding. Low energy conformations of the 3'',3''-dimethyl and 3''-hydroxyethyl-3''-benzyle analogues, determined from nuclear magnetic resonance studies and theoretical calculations, were docked by superimposing the diaminopyrimidine fragment onto a previously docked pyrimethamine analogue. Enzyme kinetic data supported the 3''-hydroxyethyl-3''-benzyl moiety being located in the NADPH binding groove. The 3''-benzyl substituent was able to locate to within 3 AA of a valine residue in the active site of P.carinii DHFR thereby producing a hydrophobic contact. The equivalent residue in human DHFR is threonine, more hydrophilic and less likely to be involved in such a contact. This difference may account for the greater inhibitory activity this analogue has for P.carinii DHFR and provide a basis for future drug design. From an in vivo model of PCP in immunosuppressed rats it was established that the 3"-hydroxyethyl-3"-benzyl analogue was able to reduce the.P.carinii burden more effectively with increasing doses, without causmg any visible signs of toxicity. However, equivalent doses were not as effective as pentamidine, a current treatment of choice for Pneumocystis carinii pneumonia.

Developing a conceptual framework for investigating the resilience of construction SMEs and their supply chains against extreme weather events

Projections into future increasingly suggest that the intensity and frequency of Extreme Weather Events (EWEs) will increase in the future. This has demanded the business organisations as well to be prepared to face the increasing risk of EWEs, in order to ensure their business continuity. However, current evidence base suggests that businesses, especially SMEs, are not adequately prepared to face the threat of such events. Ability to adequately prepare them has been hindered by the lack of in depth studies addressing this issue. The paper presents a doctoral study designed to investigate the resilience of SMEs operating in the construction sector; which is said to be a highly vulnerable sector for the impacts of EWEs, and their supply chains to EWEs. A conceptual framework developed to investigate this issue is presented and explained. It is argued that the resilience of construction SMEs and their supply chains against EWEs can be improved by a combination of reducing their vulnerability, enhancing coping capacity and implementing coping mechanisms. Importance of undertaking a broader view to include the whole supply chain in making business decisions with regard to EWEs by SMEs is also highlighted.

Herman Chinery-Hesse:the Bill Gates of Ghana?

Herman Chinery-Hesse considers his plans for a new venture, a virtual mall that would enable African producers to sell their products worldwide through a new international payment system based on mobile phones and pre-paid scratch cards. In 2010, his operating company, Black Star Lines (BSL) Ghana Ltd is considering plans to launch shopAfrica53.com, and associated payment and distribution services in Ghana and the UK. This case teaches new approaches to poverty reduction through the realisation of entrepreneurial opportunities at the Bottom of the Pyramid (BoP) and is suitable for courses on social enterprise, entrepreneurship in general, and development studies seeking to incorporate more private sector approaches. It can also be adapted for courses such as international strategy or technology business.

Europol and the EU’s fight against organized crime: exploring the potential of experimentalist governance

While issues relating to the development, legitimacy and accountability of the European Police Office, Europol, have been intensively discussed in political and academic circles, the actual impact of Europol on policy-making in the European Union has yet to receive scholarly attention. By investigating the evolution and the role of Europol's organized crime reports, this article elaborates on whether Europol has been able to exert an influence beyond its narrowly defined mandate. Theoretically informed by the assumptions of experimentalist governance, the article argues that the different legal systems and policing traditions of EU member states have made it difficult for the EU to agree on a common understanding on how to fight against organized crime. This lack of consensus, which has translated into a set of vague and broadly formulated framework goals and guidelines, has enabled Europol to position its Organized Crime Threat Assessments as the point of reference in the respective EU policy-making area. Europol's interest in improving its institutional standing thereby converged with the interest of different member states to use Europol as a socialization platform to broadcast their ideas and to ‘Europeanize’ their national counter-organized crime policy.

Who speaks up for Inés Fonseca? Representing violence against vulnerable subjects and the ethics of care in fictional narrative about Alzheimer’s disease:Ahora tocad música de baile (2004) by Andrés Barba

This paper studies the Spanish fictional novel by Andrés Barba, Ahora tocad música de baile (2004), one of the first cultural texts dealing entirely with Alzheimer’s disease (AD) to appear in Spain. It argues that the significance of Barba’s fictional novel rests on two important issues: the ethics of representation of violence against vulnerable subjects and the ethics of care. The paper analyses how these two issues allow Barba to create a story in which the verbal and physical abuse to which the person living with Alzheimer’s disease is subjected places the reader, on the one hand, as voyeur/witness of the abuse; and, on the other, as interpreter, and ultimately judge, of the fine line that separates euthanasia, assisted suicide, and murder. The open ending of the novel defers all ethical and moral judgment to the reader. It examines how the novel offers a monolithic perspective about AD, in which care is presented as a burden. In fact, this study shows that the novel’s multi-layered structure and polyphonic nature places the emphasis on stigmas, stereotypes and negative metaphors around AD, as found in contemporary social discourses.

Developing methods for testing the resistance of white cabbage against Thrips tabaci

Since the damage of the onion thrips (Thrips tabaci Lindemann) first occurred on white cabbage in Hungary several observations have been carried out, both in Hungary and abroad, to assess varietal resistance. The use of a new evaluation method for field screening is described and the result of the monitoring of 64 varieties is reported. The most susceptible varieties were ‘Bejo 1860’, ‘SG 3164’, ‘Quisto’, ‘Green Gem’ and ‘Ramada’. On the other hand, ‘Golden Cross’, ‘Balashi’, ‘Riana’, ‘Autumn Queen’, ‘Leopard’, Ama-Daneza’ and ‘Galaxy’ suffered the least damage under natural infestation. Methods for testing the patterns of resistance are also described and evaluated. In case of plants at the few leaf growth stage significant negative correlation was found between egg mortality and the egg laying preference of adults. The results of the other antibiotic and antixenotic tests were greatly affected by differences in the physiological age and condition of the varieties.

Swimming against the tide: A study of a youth enrichment program seeking to empower inner-city Black youth

Serious inequities in our K-12 public education system, particularly in regard to the quality of education in predominately Black inner-city schools, are well-documented in the literature (Freeman, 1998; Ross, 1998). Moreover, there is general agreement that the most effective means of ameliorating that situation is through well-thought-out after-school programs and partnership initiatives (Beck, 1993; Gardner et al., 2001). ^ The purpose of this qualitative study was to examine the programmatic interventions of a youth enrichment program for inner-city Black youth currently in place at the Overtown Youth Center (OYC) in Miami, Florida, in order to: (a) discern those factors that support its claim that it is making a difference in students’ lives, (b) explore how any such factors are implemented, and (c) determine whether its interventions have served to equalize the playing field for these youth. ^ Two primary methods of data collection were used for this study. The first was participant observation conducted over the course of two years through a partnership initiative established and led by this author. The second was through in-depth interviews of the Center’s founder, staff, and students. Secondary methods used were the recording of informal conversations and the analysis of written documents. ^ Analysis of the data yielded four features of the Center that are indispensible to the students’ growth. The center provides the youth with (a) physical and psychological safety, (b) supportive relationships, (c) exposure to cultural and educational opportunities, and (d) assistance in building self-esteem. ^ The most significant finding of the study was that OYC has been successful at making a difference in students’ lives and at increasing their aspirations to attend college. By addressing the full spectrum of their needs, the Center has given them many of the necessary tools with which to compete and thereby helped equalize their opportunities to succeed in school and in life. ^ The study also noted a number of challenges for the Center to examine. The main issues that need to be addressed more seriously are staff turnover, staff indifference, nepotism, inconsistent student attendance, and insufficient focus on racial issues and African-American-centered education. Meeting those challenges would engender even greater positive outcomes.^

From Bilingualism to Interlinguistics: The Case Against the Deficit Model of Language Acquisition

The article argues against an ahistorical deficit model of Spanish/English bilingualism in educational practice based on interlinguistic research. The bidirectional facilitative effects of Hispanic bilingualism allow Spanish-speaking minorities to exploit their language background while learning academic English and integrating their language and culture into the American mainstream.

The Rise of Native Voices Against Natural Resource Extraction in Latin America [Student's Paper Series]

Latin America, a region rich in both energy resources and native heritage, faces a rising politico-social confrontation that has been growing for over two decades. While resources like oil and gas are exploited to enhance the state’s economic growth, indigenous groups feel threatened because the operations related to this exploitation are infringing on their homelands. Furthermore, they believe that the potential resource wealth found in these environmentally-sensitive regions is provoking an “intrusion” in their ancestral territory of either government agencies or corporations allowed by governmental decree. Indigenous groups, which have achieved greater political voice over the past decade, are protesting against government violations. These protests have reached the media and received international attention, leading the discourse on topics such as civil and human rights violations. When this happens, the State finds itself “between a rock and a hard place”: In a debate between indigenous groups’ rights and economic sustainability.

When group members go against the grain : an ironic interactive effect of group identification and normative content on healthy eating

Acknowledgements The research leading to these results has been funded by a Tandem Grant from the European Health Psychology Society/CREATE, awarded to the first and second author. The funder had no involvement in the study design, collection or analysis of data, writing the report, or the decision to submit this manuscript for publication.

Women against the war in Vietnam and in support of Vietnamese women

General note: Title and date provided by Bettye Lane.

Governmentality gone wild: How the separation of sex workers from 'communities' contributes to violence against sex workers

Sex workers are members of our communities, whether they are local or national communities. In law, mainstream media representations, and research sex workers are positioned as outside of or in opposition to communities. Even within marginalized communities sex workers are excluded when appeals to respectability politics are made. In this thesis I analyze three analytic sites from three areas of social life. The first chapter performs a textual analysis of The Bedford Decision (2013) and the resulting Protection of Communities and Exploited Persons Act (2014) as an examination of law. The second chapter is an analysis of filmic discourse on community, sex workers, and violence in the mainstream film London Road (2015) as an examination of mainstream media. The third chapter draws upon empirical research, i.e. in-depth interviews with three current and former sex workers in Ottawa, Canada and analyzes the transcripts using interpretative phenomenological analysis (IPA) to center how sex workers’ understanding of their work, community, and the laws and policies that are supposed govern and protect them. In my preface and conclusion I discuss some of the ethical dilemmas I encountered while conducting this research. My findings suggest that sex workers are being positioned and understood as outside of communities in ways that contribute to violence against sex workers. The implications of this research suggest that people who speak in the name of communities—communities in the sense of local neighborhood communities, activist communities, and national communities—need to recognize that sex workers are part of their communities and be accountable to ensuring they are treated as members. Researchers who conduct research on sex work and sex workers need to be accountable to their participants and the impacts their research may have on laws and policies. Sex workers are an over-researched population yet their voices are largely misappropriated or silenced in popular research and policy debates.

Characterisation of algicidal bacterial exometabolites against the lipid-accumulating diatom Skeletonema sp.

Microalgae are of increasing interest due to their occurrence in the environment as harmful algal blooms and as a source of biomass for the production of fine and bulk chemicals. A method for the low cost disruption of algal biomass for environmental remediation or bioprocessing is desirable. Naturally-occurring algal lytic agents from bacteria could provide a cost-effective and environmentally desirable solution. A screen for algal lytic agents against a range of marine microalgae has identified two strains of algicidal bacteria isolated from the coastal region of the Western English Channel. Both strains (designated EC-1 and EC-2) showed significant algicidal activity against Skeletonema sp. and were identified as members of Alteromonas sp. and Maribacter sp. respectively. Characterisation of the two bioactivities revealed that they are small extracellular metabolites displaying thermal and acid stability. Purification of the EC-1 activity to homogeneity and initial structural analysis has identified it as a putative peptide with a mass of 1266. amu.

Characterisation of algicidal bacterial exometabolites against the lipid-accumulating diatom Skeletonema sp.

Microalgae are of increasing interest due to their occurrence in the environment as harmful algal blooms and as a source of biomass for the production of fine and bulk chemicals. A method for the low cost disruption of algal biomass for environmental remediation or bioprocessing is desirable. Naturally-occurring algal lytic agents from bacteria could provide a cost-effective and environmentally desirable solution. A screen for algal lytic agents against a range of marine microalgae has identified two strains of algicidal bacteria isolated from the coastal region of the Western English Channel. Both strains (designated EC-1 and EC-2) showed significant algicidal activity against Skeletonema sp. and were identified as members of Alteromonas sp. and Maribacter sp. respectively. Characterisation of the two bioactivities revealed that they are small extracellular metabolites displaying thermal and acid stability. Purification of the EC-1 activity to homogeneity and initial structural analysis has identified it as a putative peptide with a mass of 1266. amu.