An Investigation of Enzyme/Prodrug Systems for Cancer Gene Therapy

Chemotherapy, radiotherapy and surgery, the current treatments for cancer, cure only fifty percent of patients. Gene therapy, where therapeutic genes are transferred directly to the tumour, offers a new form of cancer treatment. However, gene transfer techniques that are currently available can only achieve gene expression in a small proportion of the tumour cells, and this may hinder many gene therapy approaches. It may therefore be important to select genes for cancer therapy that offer the potential for a significant bystander effect, where cells not expressing the therapeutic gene are killed, as well as targeted cells. At present there appear to be two strategies for cancer gene therapy that achieve bystander killing, and hence require targeted expression in only a proportion of the cells. The first depends on stimulation of the immune system by, for example, expression of specific cytokines that increase proliferation of tumoricidal cells, while the second type of bystander effect is associated with enzyme / prodrug systems, which are the focus of the work described in this thesis. The mechanisms and efficacies of the Herpes Simplex virus thymidine kinase / ganciclovir (tk / GCV), human thymidine phosphorylase / 5'-deoxy-5-fluorouridine (tp / DFUR) and E.coli cytosine deaminase / 5-fluorocytosine (cd / FC) enzyme / prodrug systems have been examined. In culture, each enzyme / prodrug combination induced cell death in tumour cells expressing the suicide gene and, in mixed cultures of cells that do or do not express the gene, death occurred in both cell types, indicative of a bystander effect. With the tk / GCV system, in human and rodent cell lines, a correlation has been demonstrated between the bystander effect and gap junctional intercellular communication (GJIC). When cultures lack GJIC, no bystander effect was observed, but only a low level of communication was required to generate extensive bystander killing. Increased enzyme activity, on the other hand, improved the bystander effect, suggesting that high levels of tk expression will be important. Additionally, it has been shown that transfer of toxic metabolites from tk+ to tk- cells occurs within two hr of GCV application, before degenerative events were detected, indicating that apoptosis is the result, not the cause, of the tk / GCV bystander effect. Three of the cell lines used to investigate the tk / GCV system in culture were also used to examine the bystander effect in experimental tumours in nude mice. A correlation between the bystander effect in culture and in vivo was demonstrated in two of the cell lines but not the third, which suggests that other factors are important in vivo. In the tp / DFUR and cd / FC systems, bystander killing was due to transfer of toxic metabolites through the medium, and did not depend on GJIC or cell-cell contact. Bystander killing seen with these systems was weaker than that seen in the tk / GCV system. Combining the tk / GCV system with the tp / DFUR or cd / FC systems in culture was able to produce more extensive bystander killing than either system separately. The improvement was also observed using a cdtk fusion protein. The results in this thesis suggest that the choice of enzyme / prodrug therapy for cancer will be determined by the characteristics of each tumour. In addition they suggest the use of fusion proteins, to combine therapeutic proteins, warrants further investigation

Detecting control flow in Smarphones: Combining static and dynamic analyses

International audienceSecurity in embedded systems such as smartphones requires protection of confidential data and applications. Many of security mechanisms use dynamic taint analysis techniques for tracking information flow in software. But these techniques cannot detect control flows that use conditionals to implicitly transfer information from objects to other objects. In particular, malicious applications can bypass Android system and get privacy sensitive information through control flows. We propose an enhancement of dynamic taint analysis that propagates taint along control dependencies by using the static analysis in embedded system such as Google Android operating system. By using this new approach, it becomes possible to protect sensitive information and detect most types of software exploits without reporting too many false positives

Carriage of goods by sea - from Hague to Rotterdam: safer waters

The back bone of international trade has always been international transport. Without good transport networks, the movement of goods and services from one frontier to another would be an uphill task, and would greatly hinder development in international trade. The impact of such poor transport networks would reflect negatively on economies that rely on international trade for the growth of their nations. Nevertheless, perfect transport networks would be useless if the performance of the business of carriage was not regulated by a law developed to meet the standards established by time, and that would regulate the relationship of the parties under contracts of carriage, mainly the carrier, consignor and consignee, so as to ensure certainty and equality in the allocation of risks between the parties thereunder. This research focuses on the carriage of goods by sea. Like most other modes of transport, one of the major issues that arises in the business of carriage of goods by sea is the conflict between the carrier, consignor and consignee, with regards to the allocation of risk in the carriage. Over the years, early rules that were developed to regulate the relationship of the parties under contracts of carriage of goods by sea placed the carrier in a dominant position over the consignor. The carrier issued a standard bill of lading which exempted him from almost all liability for damage or loss of the goods in his care. The consignors and bona fide third parties, not satisfied with the terms of carriage contracts brought a lot of pressure to bear on their governments to enact legislation protecting their interests in the transaction. The United States of America were the first to pass such national law revising the position of the parties under contracts of carriage. In 1893 the United States of America passed the Harter Act. This Act aimed at imposing limits of liability on the carrier to which no derogation could be brought. However, this was a dangerous precedence which was going to hinder international trade rather than improve on it, as different nations developing local legislation on carriage meant conflict of laws. In order to avoid the extensive nationalisation of carriage laws, the international maritime community set to develop rules that would regulate carriage by sea. Over the years convention has succeeded convention such that today four international regimes (The Hague Rules, Hague-Visby Rules, Hamburg Rules and Rotterdam Rules), exist regulating carriage of goods by sea. This research takes an in-depth look at these regimes that were developed to regulate carriage by sea, and the author aims to identify a particular regime that meets the standards of modern day practice of carriage of goods, and advocate for the ratification of this regime, to the exclusion of all others so as to foster uniformity, certainty and equality in the business of carriage of goods by sea

Carriage of goods by sea - from Hague to Rotterdam: safer waters

The back bone of international trade has always been international transport. Without good transport networks, the movement of goods and services from one frontier to another would be an uphill task, and would greatly hinder development in international trade. The impact of such poor transport networks would reflect negatively on economies that rely on international trade for the growth of their nations. Nevertheless, perfect transport networks would be useless if the performance of the business of carriage was not regulated by a law developed to meet the standards established by time, and that would regulate the relationship of the parties under contracts of carriage, mainly the carrier, consignor and consignee, so as to ensure certainty and equality in the allocation of risks between the parties thereunder. This research focuses on the carriage of goods by sea. Like most other modes of transport, one of the major issues that arises in the business of carriage of goods by sea is the conflict between the carrier, consignor and consignee, with regards to the allocation of risk in the carriage. Over the years, early rules that were developed to regulate the relationship of the parties under contracts of carriage of goods by sea placed the carrier in a dominant position over the consignor. The carrier issued a standard bill of lading which exempted him from almost all liability for damage or loss of the goods in his care. The consignors and bona fide third parties, not satisfied with the terms of carriage contracts brought a lot of pressure to bear on their governments to enact legislation protecting their interests in the transaction. The United States of America were the first to pass such national law revising the position of the parties under contracts of carriage. In 1893 the United States of America passed the Harter Act. This Act aimed at imposing limits of liability on the carrier to which no derogation could be brought. However, this was a dangerous precedence which was going to hinder international trade rather than improve on it, as different nations developing local legislation on carriage meant conflict of laws. In order to avoid the extensive nationalisation of carriage laws, the international maritime community set to develop rules that would regulate carriage by sea. Over the years convention has succeeded convention such that today four international regimes (The Hague Rules, Hague-Visby Rules, Hamburg Rules and Rotterdam Rules), exist regulating carriage of goods by sea. This research takes an in-depth look at these regimes that were developed to regulate carriage by sea, and the author aims to identify a particular regime that meets the standards of modern day practice of carriage of goods, and advocate for the ratification of this regime, to the exclusion of all others so as to foster uniformity, certainty and equality in the business of carriage of goods by sea

Determining the burden of fungal infections in Zimbabwe.

Zimbabwe currently faces several healthcare challenges, most notably HIV and associated infections including tuberculosis (TB), malaria and recently outbreaks of cholera, typhoid fever and COVID-19. Fungal infections, which are also a major public health threat, receive considerably less attention. Consequently, there is dearth of data regarding the burden of fungal diseases in the country. We estimated the burden of fungal diseases in Zimbabwe based on published literature and 'at-risk' populations (HIV/AIDS patients, survivors of pulmonary TB, cancer, chronic obstructive pulmonary disease, asthma and patients receiving critical care) using previously described methods. Where there was no data for Zimbabwe, regional, or international data was used. Our study revealed that approximately 14.9% of Zimbabweans suffer from fungal infections annually, with 80% having tinea capitis. The annual incidence of cryptococcal meningitis and Pneumocystis jirovecii pneumonia in HIV/AIDS were estimated at 41/100,000 and 63/100,000, respectively. The estimated prevalence of recurrent vulvovaginal candidiasis (RVVC) was 2,739/100,000. The estimated burden of fungal diseases in Zimbabwe is high in comparison to other African countries, highlighting the urgent need for increased awareness and surveillance to improve diagnosis and management

Cellular automaton rules conserving the number of active sites

This paper shows how to determine all the unidimensional two-state cellular automaton rules of a given number of inputs which conserve the number of active sites. These rules have to satisfy a necessary and sufficient condition. If the active sites are viewed as cells occupied by identical particles, these cellular automaton rules represent evolution operators of systems of identical interacting particles whose total number is conserved. Some of these rules, which allow motion in both directions, mimic ensembles of one-dimensional pseudo-random walkers. Numerical evidence indicates that the corresponding stochastic processes might be non-Gaussian.Comment: 14 pages, 5 figure

Very static enforcement of dynamic policies

Security policies are naturally dynamic. Reflecting this, there has been a growing interest in studying information-flow properties which change during program execution, including concepts such as declassification, revocation, and role-change. A static verification of a dynamic information flow policy, from a semantic perspective, should only need to concern itself with two things: 1) the dependencies between data in a program, and 2) whether those dependencies are consistent with the intended flow policies as they change over time. In this paper we provide a formal ground for this intuition. We present a straightforward extension to the principal flow-sensitive type system introduced by Hunt and Sands (POPL’06, ESOP’11) to infer both end-to-end dependencies and dependencies at intermediate points in a program. This allows typings to be applied to verification of both static and dynamic policies. Our extension preserves the principal type system’s distinguishing feature, that type inference is independent of the policy to be enforced: a single, generic dependency analysis (typing) can be used to verify many different dynamic policies of a given program, thus achieving a clean separation between (1) and (2). We also make contributions to the foundations of dynamic information flow. Arguably, the most compelling semantic definitions for dynamic security conditions in the literature are phrased in the so-called knowledge-based style. We contribute a new definition of knowledge-based progress insensitive security for dynamic policies. We show that the new definition avoids anomalies of previous definitions and enjoys a simple and useful characterisation as a two-run style property

Polymorphisms in toll-like receptor genes and susceptibility to pulmonary aspergillosis

Toll-like receptors (TLRs) are important components of innate immunity. We investigated the association between polymorphisms in the TLR2, TLR4, and TLR9 genes and susceptibility to noninvasive forms of pulmonary aspergillosis. A significant association was observed between allele G on Asp299Gly (TLR4) and chronic cavitary pulmonary aspergillosis (odds ratio [OR], 3.46; P =.003). Susceptibility to allergic bronchopulmonary aspergillosis was associated with allele C on T-1237C (TLR9) (OR, 2.49; P =. 043). No particular polymorphism was associated with severe asthma with fungal sensitization. These findings reinforce the importance of innate immunity in the pathogenesis of different forms of aspergillosis.Fundação para a Ciência e Tecnologia, Portugal (POCI/SAU-ESP/61080/ 2004 and fellowship to A.C., contract SFRH/BD/11837/2003); CAPES (Brazilian government) (grant to A.P); and the Fungal Research Trust, United Kingdom

Generation of a human iPSC-derived cardiomyocyte/fibroblast engineered heart tissue model

Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types, such as cardiac fibroblasts, into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore, the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs, incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis