Rozprawy doktorskie na temat „CRC”

Запропоновано новий спосіб швидкого декодування кодів CRC (Cyclic Redundancy Code) в каналах з паралельним надходженням вхідних даних. Використання теорії паралельних ЛПС (лінійних послідовнісних схем) та математичного представлення симетрії часу дозволяє вдвічі прискорити CRC-контроль. Такий спосіб контролю може бути використано в системах зберігання та архівації даних.
Предложен новый способ быстрого декодирования кодов CRC (Cyclic Redundancy Code) в каналах с параллельным поступлением входных данных. Использование теории параллельных ЛПС (линейнных последовательностных схем) и математического представления симметрии времени позволяет в два раза ускорить CRC-контроль. Такой способ контроля может быть использовать в системах сохранения и архивирования данных.
A new method for fast decoding of CRC (Cyclic Redundancy Code) in channels with parallel input data is proposed. The use of the theory of linear finite state machine (LFSM) and the mathematical representation of time symmetry makes it possible to double the speed of CRC control. Such a control method can be used in data storage and archiving systems.

Tese de Doutoramento em Ciências Veterinárias, especialidade de Ciências Biológicas e Biomédicas
Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related death in the Western world. Dll4/Notch signaling has been shown to regulate tumor angiogenesis and cancer stem cell maintenance in CRC, but how it affects the intestinal precancerous lesions that lead to CRC initiation is not known. Therefore we evaluated the role of Dll4/Notch pathway during intestinal tumorigenesis. For that we used two well-established mouse models of CRC, the ApcMin/+ autochthonous transgenic model and the azoxymethane plus dextran sodium sulphate chemically induced model of chronic colitis associated-cancer (CAC). First we analyzed the protein expression pattern of Dll4 and other Notch pathway members in these settings relatively to that in the normal gut. Then we evaluated the effect of endothelial-specific or ubiquitous Dll4 deregulation and performed a therapeutic trial with the Dll4 inhibitor Dll4-Fc. This protein was administered alone, and in combination with the epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitor erlotinib to assess if the anti-Dll4 therapy mediated vascular defects impaired the delivery of other anti-cancer drugs to the tumors. We observed that the Notch pathway is activated in the two studied models of CRC. The normal protein expression pattern of Notch pathway members in the gut is altered in chronic colitis and in ApcMin/+ and colitis-driven intestinal tumors. Dll4 is the most upregulated ligand in the intestinal adenomas in both models of CRC and is present in both tumor epithelium and stroma. Both Dll4 blockade (endothelial-specific and ubiquitously) and activation (endothelial-specific) have an inhibitory effect on intestinal tumor initiation and growth by promoting a noncompetent vasculature or decreasing the vessel density, respectively. Besides its angiogenic related effects, Dll4/Notch pathway promotes excessive inflammation in CAC, sustains the tumor stem cell pool and tumor proliferation synergistically with Wnt signaling, and inhibits differentiation mainly of the secretory cells. In addition, the effectiveness of erlotinib is not affected by Dll4-Fc, where these therapies additively inhibit intestinal tumorigenesis.
RESUMO - Avaliação in vivo da função da sinalização intercelular Dll4/Notch no desenvolvimento de tumores intestinais - O cancro colo-rectal (CCR) é o terceiro tipo de cancro mais comum e é uma das principais causas de morte no Mundo Ocidental. O CCR geralmente desenvolve-se esporadicamente, mas também pode ser hereditário como na síndrome polipose adenomatosa familiar (PAF). A PAF está associada à ativação da via de sinalização Wnt através de mutações no gene supressor tumoral Adenomatous Polyposis Coli (APC), que também ocorre frequentemente nos CCR esporádicos. O desenvolvimento do CCR também pode estar associado a inflamação crónica, nomeadamente à doença de Crohn (CD) e à colite ulcerativa (UC). Infelizmente o desenvolvimento de novas estratégias terapêuticas ou preventivas que tenham como alvo vias de sinalização críticas no desenvolvimento do CCR continua a ser extremamente necessário. Uma dessas estratégias tem como alvo a angiogénese tumoral. O primeiro agente anti-angiogénico a ser aprovado foi o Bevacizumab, que é usado em combinação com outros fármacos no tratamento do CCR metastático. No entanto, a utilização deste fármaco leva ao aparecimento de efeitos secundários e resistência tumoral e em tumores não metastáticos não se tem mostrado eficaz. Assim, continua a ser necessário desenvolver melhores estratégias terapêuticas anti-angiogénicas...
Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA) e União Europeia: Fundo Social Europeu

Die rollenbasierte Modellierung ist ein aktueller Forschungszweig, welcher Verfahren für die Analyse und die Lehre benötigt. Zu diesem Zweck präsentiert die Arbeit eine Erweiterung des klassischen, objektorientierten CRC-Karten-Verfahrens um rollenbasierte Konzepte. Diese basiert auf grundlegenden Eigenschaften rollenbasierter Elemente, wie Rollen, Objekte und Kontexte, welche modular in das CRC-Karten- Verfahren eingebunden werden. Weiterhin soll anhand einer empirische Studie ermittelt werden, wie gut das rollenerweiterte R-CRC-Karten-Verfahren für die Aufgaben in Analyse und Lehre geeignet ist. Das R-CRC-Karten-Verfahren soll letztendlich eine effiziente Möglichkeit bieten, Problemstellungen rollenbasiert zu analysieren und rollenbasierte Konzepte in der Lehre zu vermitteln.

With the rapid growth of Internet-of-Things (IoT), billions of devices are expected to be interconnected to provide various services appealing to users. Many devices will get an access to valuable information which is likely to increase the number of malicious attacks on these devices in the future. Therefore, security is considered as one of the most critical challenges in the development of IoT. In order to secure resource-constrained devices such as sensors or radio frequency identification (RFID) tags which form the backbone of IoT, lightweight cryptographic algorithms are required. This thesis focuses on the problem of message authentication. To authenticate a message means to verify that the message: (1) comes from the right sender (i.e. its authenticity), and (2) has not been modified (i.e. its integrity). It is challenging to use traditional message authentication methods in resource-constrained devices because typically they can allocate only a few hundred gates for implementing security due to their limited computing, storage and energy resources. To address these needs, a new message authentication algorithm based on a Cryptographic Cyclic Redundancy Check (C-CRC) was developed by KTH in collaboration with Ericsson. In this thesis, we implemented C-CRC and compared it with KECCAK Message Authentication Code (KMAC) standardized by the National Institute of Standards and Technology (NIST) in 2016. First, MATLAB and Verilog versions were developed for both algorithms. The comparison of these two versions allowed us to verify the correctness of algorithms functionality. After that, the Verilog descriptions were simulated in ModelSim and synthesized using Synopsys design compiler. Finally, placement and routing was performed using Cadence SoC Encounter. The evaluation results show that C-CRC outperforms KMAC in terms of area, power, throughput per area, and energy per bit. However, C-CRC is worse than KMAC in terms of latency. We have also investigated several different options of implementing C-CRC, including producing more than one bit of output per clock cycle. We found that such a technique improves throughput of C-CRC with the minimal penalty in area and power consumption

This paper is an exploration into the potential of a new collaborative tool called the DiamondTouch. An application was developed to support CRC Card Sessions and tested with different users. As part of this research, experiment participants created software designs, both using and not using the software, where metrics were gathered about their performance. The results are examined both in a qualitatively and quantitatively manner for insight into usability of the system. With these results, an evaluation is made on the hardware and the software. Suggestions are made about the future of this application and can provide guidance for developing other collaborative applications on the DiamondTouch.

Colorectal cancer (GRG) is the 3rd common cancer, and second leading cause of cancer associated mortality. Oncogenic mutations in the BRAF gene results in an altered protein structure, leading to constitutively activated MAPK signalling. Beneficial treatment strategies for this poor prognostic subgroup of GRG patients have yet to be identified. Hence this objective of this study was to identify novel treatment strategies in BRAF mutant GRG models. Our data has shown that MEK inhibition results in acute expression of pSTAT3, regulated through the c-METI JAK signalling axis. Taking a combined approach of JAKlMEK, or c-MET/MEK inhibition we have shown that these combinations results in significant increased apoptosis in BRAFMT GRG, and have potential as novel combinations. Furthermore, we are the first to show that MEK inhibition results in increased expression of the caspase 8 inhibitor c-FLlP, as a mechanism of resistance to apoptosis induction. Using a gene silencing and small-molecule inhibitor approach, we have identified that combined c-FLlP/MEK inhibition is a novel treatment strategy that may provide benefit for this subgroup of GRG patients.

Tourism has come to be recognised as a major contributor to national economies. In a knowledge-based economy (that emphasises the benefits of industry/government and academic research), a strong research base must underpin management of a tourist destination if it is to realise its full potential. The establishment of collaborative networks between industry, academia, and government in the strategic planning and management of cities and towns is becoming increasingly popular. However, the way in which the processes underlying these settings facilitate or inhibit eventual outcomes is poorly understood. If knowledge is to drive innovation and economic growth optimally, it is important not just to develop an understanding of the processes underlying the creation, diffusion and utilisation of knowledge in cooperative research settings, but also the relationships among them. Accordingly, the aim of this investigation is to examine the relationships among knowledge creation, diffusion and utilisation occurring in the Cooperative Research Centres (CRC) Program, specifically, the Gold Coast Visioning Project, with a view to identifying the most efficient means for formulating and disseminating research designed for industry and/or government application. Knowledge is defined as information that is imbued with meaning or relevance. However, this definition says little of the ways that individuals, groups and organisations acquire knowledge. While cognitive psychologists have produced several theories suggesting the structure and mechanisms of individual cognitive processes underlying the acquisition and use of knowledge, social scientists have sought to describe and explain the process by investigating the influence of social factors. Recent contributions to group learning have examined group composition, group size, familiarity among group members, and communication processes in an attempt to understand the ways in which groups acquire knowledge. Research shows that knowledge utilisation in organisations results from the interdependent influences of organisational processes and the control opportunities and control problems that arise through organisational structure. These frameworks provide accounts of how knowledge is utilised within an organisation, but not of how organisations learn. Recent research suggests that organisations learn through knowledge networks where organisational focus moves from the consideration and protection of boundaries to the management of (and care for) relationships. Therefore, organisations contain static (rules, norms and procedures) and dynamic (social relationships) elements that mutually influence the degree to which organisations learn. A synthesis of the available literature resulted in the development of a series of models that served not only to inform, but also be informed by the analysis of this investigation. A single case study, namely the Gold Coast Visioning Project, was used to examine the ways in which knowledge was created, disseminated and utilised in a CRC setting. This ethnographic investigation considered the process of knowledge creation through to utilisation at individual, group, organisational, and inter-organisational levels, while simultaneously examining the interrelated influences of social, cognitive, affective and communication factors. Throughout the project, data were collected through stakeholder interviews, various documents and participant observation of stakeholder meetings and workshops. Data were analysed using a grounded theory approach and methods of thick description. The results show that researchers and industry stakeholders bring different frames of reference, different expectations, and different knowledge bases to the exercise. This inhibited communication, and gave the appearance of dissension when, in fact, what was being sought was a common frame for understanding and communication. Additionally, the gap between industry and researcher worldviews generated the sense that industry was resisting or failing to understand what the research was seeking to achieve. Consequently, in order to manage the relationship, research plans and findings were communicated to industry in a teacher-to-student fashion, which fostered single-loop learning, and reduced industry stakeholders' sense of ownership in the process and findings. During the project, industry stakeholders frequently sought to have research come pre-packaged with "meaning", but researchers lacked the contextual knowledge necessary to specify the relevance of their research. The results also show that research findings need to be integrated and diffused to industry over time, and specific applications need to be formulated (and reformulated) in response to particular and changing needs of industry. As a result of this investigation, a model of 'best practice' has been developed with detailed recommendations for the design, implementation, and reporting of CRC-sponsored research to optimise its utility for end-users of such research. From a theoretical perspective, the findings of this study challenge the ways that current theories account for the ways in which knowledge is acquired and utilised since the results show that knowledge is constructed both socially and emotionally. Any investigation that seeks to understand how knowledge is acquired and utilised must consider social and affective influences. To ignore the role of emotion and values in the process of knowledge acquisition is to ignore a key component of an individual's reasoning capacity.

As Pseudomonas aeruginosa adapts to the Cystic Fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lung environments, mucoid strains often appear as a result of alginate overproduction. Such mucoid conversion is associated with the establishment of a chronic pulmonary infection. Alginate confers resistance to phagocytosis and has other pathogenic properties. The regulation of alginate production is complex and involves an alternate sigma factor, anti-sigmas and several DNA-binding transcriptional regulators. Here we examined the possibility that the catabolic repression control (Crc) protein repressor may affect alginate gene expression. A putative Crc binding site was observed adjacent to the ribosome binding site of algD, the first gene in the 12-gene alginate biosynthetic operon. We hypothesized that Crc binding here would act as a repressor of algD expression. Taking a genetic approach, Gateway technology was used to construct crc::GmR (nonpolar) mutants of P. aeruginosa strain PAO1 and its mucoid (mucA) mutant derivative, PDO300. The crc mutation had the expected phenotypes with respect to pyocyanin production, biofilm formation and diauxic growth. When a PalgD-lacZ (translational) fusion was tested, the crc mutant showed increased algD expression as predicted for a mRNA-binding repressor. Another Ptrc-algD-lacZ (translational) construct, but missing the upstream promoter (PalgD) elements, also showed increased activity in crc mutants as predicted if Crc was acting directly as a repressor of algD transcriptional / translational expression. However, this was not consistent with the production of alginate. The crc mutant of mucoid PDO300 showed lower levels of alginate production than its parent strain. Under conditions were the algD operon was induced by ammonium metavanadate in the growth medium to produce alginate, the crc mutation again resulted in a lower level of alginate production than wild-type, which was again inconsistent with the algD-lacZ expression data. This suggests that crc mutation, which has global effects on carbon flow in the cell, could be affecting metabolic pathways that feed precursors into the alginate biosynthetic pathway. Future directions for this research are discussed.

Tourism has come to be recognised as a major contributor to national economies. In a knowledge-based economy (that emphasises the benefits of industry/government and academic research), a strong research base must underpin management of a tourist destination if it is to realise its full potential. The establishment of collaborative networks between industry, academia, and government in the strategic planning and management of cities and towns is becoming increasingly popular. However, the way in which the processes underlying these settings facilitate or inhibit eventual outcomes is poorly understood. If knowledge is to drive innovation and economic growth optimally, it is important not just to develop an understanding of the processes underlying the creation, diffusion and utilisation of knowledge in cooperative research settings, but also the relationships among them. Accordingly, the aim of this investigation is to examine the relationships among knowledge creation, diffusion and utilisation occurring in the Cooperative Research Centres (CRC) Program, specifically, the Gold Coast Visioning Project, with a view to identifying the most efficient means for formulating and disseminating research designed for industry and/or government application. Knowledge is defined as information that is imbued with meaning or relevance. However, this definition says little of the ways that individuals, groups and organisations acquire knowledge. While cognitive psychologists have produced several theories suggesting the structure and mechanisms of individual cognitive processes underlying the acquisition and use of knowledge, social scientists have sought to describe and explain the process by investigating the influence of social factors. Recent contributions to group learning have examined group composition, group size, familiarity among group members, and communication processes in an attempt to understand the ways in which groups acquire knowledge. Research shows that knowledge utilisation in organisations results from the interdependent influences of organisational processes and the control opportunities and control problems that arise through organisational structure. These frameworks provide accounts of how knowledge is utilised within an organisation, but not of how organisations learn. Recent research suggests that organisations learn through knowledge networks where organisational focus moves from the consideration and protection of boundaries to the management of (and care for) relationships. Therefore, organisations contain static (rules, norms and procedures) and dynamic (social relationships) elements that mutually influence the degree to which organisations learn. A synthesis of the available literature resulted in the development of a series of models that served not only to inform, but also be informed by the analysis of this investigation. A single case study, namely the Gold Coast Visioning Project, was used to examine the ways in which knowledge was created, disseminated and utilised in a CRC setting. This ethnographic investigation considered the process of knowledge creation through to utilisation at individual, group, organisational, and inter-organisational levels, while simultaneously examining the interrelated influences of social, cognitive, affective and communication factors. Throughout the project, data were collected through stakeholder interviews, various documents and participant observation of stakeholder meetings and workshops. Data were analysed using a grounded theory approach and methods of thick description. The results show that researchers and industry stakeholders bring different frames of reference, different expectations, and different knowledge bases to the exercise. This inhibited communication, and gave the appearance of dissension when, in fact, what was being sought was a common frame for understanding and communication. Additionally, the gap between industry and researcher worldviews generated the sense that industry was resisting or failing to understand what the research was seeking to achieve. Consequently, in order to manage the relationship, research plans and findings were communicated to industry in a teacher-to-student fashion, which fostered single-loop learning, and reduced industry stakeholders' sense of ownership in the process and findings. During the project, industry stakeholders frequently sought to have research come pre-packaged with "meaning", but researchers lacked the contextual knowledge necessary to specify the relevance of their research. The results also show that research findings need to be integrated and diffused to industry over time, and specific applications need to be formulated (and reformulated) in response to particular and changing needs of industry. As a result of this investigation, a model of 'best practice' has been developed with detailed recommendations for the design, implementation, and reporting of CRC-sponsored research to optimise its utility for end-users of such research. From a theoretical perspective, the findings of this study challenge the ways that current theories account for the ways in which knowledge is acquired and utilised since the results show that knowledge is constructed both socially and emotionally. Any investigation that seeks to understand how knowledge is acquired and utilised must consider social and affective influences. To ignore the role of emotion and values in the process of knowledge acquisition is to ignore a key component of an individual's reasoning capacity.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Marketing and Management
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Le diabète est une dérégulation systémique chronique caractérisée par des perturbations métaboliques permanentes à l’origine de nombreuses complications, y compris le cancer. Le diabète augmente le risque du cancer colorectal (CRC) de 1,2 à 1,5 fois. Cependant, les voies moléculaires et cellulaires en jeu ne sont pas assez élucidées. Nos résultats témoignent d’une dérégulation de la voie AMPK/mTORC1 dans le diabète et le CRC avec une surexpression de la NADPH oxydase Nox4, augmentant ainsi la production de ROS. Ceci provoque un stress oxydatif qui s’élève en cas de diabète et contribue à la progression du CRC. De plus, nos résultats montrent que ce stress induit une altération de la voie de signalisation AMPK/mTORC1, aboutissant à une agressivité accrue du comportement des cellules cancéreuses du côlon et de la formation de polypes. Notre projet permet, d’une part, d’identifier de nouveaux mécanismes moléculaires impliqués dans la progression du CRC induite par le diabète et d’autre part, de développer des stratégies thérapeutiques efficaces pour inverser la progression du CRC chez les patients diabétiques
Diabetes is a chronic systemic malfunction characterized by persistent metabolic disturbances that culminate in a high rate of complications to which cancer was recently annexed. In fact, diabetes inflates colorectal cancer (CRC) risk by 1.2-1.5 folds. However, the cellular and molecular pathways involved are not well understood. Our results show that AMPK/mTORC1 pathway is deregulated in both diabetes and CRC. This was paralleled by an elevation in the expression of the NADPH oxidase Nox4 leading to an increase in ROS production. Furthermore, our results show that oxidative stress, secondary to alteration in the level and activity of Nox4 is augmented in diabetes and contributes to the progression of CRC. The resulting oxidative stress further led to an alteration in the signaling of the AMPK/mTORC1 pathways culminating in an exacerbated aggressiveness in cancer cell behavior and colon polyp formation. Our project allows the identification of novel molecular mechanisms involved in diabetes-induced CRC progression and development of effective therapeutic strategies to reverse the progression of CRC in diabetic patients

Colorectal cancer (CRC) is the second most common cancer among males and third among females across the world. In Australia it is the second most prevalent and the second leading cause of cancer-related mortality with the number of CRC incidences doubling over the last decade. While there has been a reduction of the incidence-adjusted mortality of CRC, a significant number of CRC detections are made at either the intermediate or later stages of the disease progression despite the roll out of a population based screening program for individuals aged 50 and over. Data shows that ‘one size fits all’ nature of the program despite recommendations from the NHMRC to screen according to the familial risk of an individual and inappropriate colonoscopy referrals, may have led to over screening those at average risk while potentially under-screening and missing those at an increased risk. Furthermore this program may have missed individuals under the age of 50 that have a high familial lifetime risk of developing CRC and require earlier CRC screening with a colonoscopy. It was hypothesised that implementing an online familial risk tool that notified both patients (aged 25-74) and their GPs of their familial CRC lifetime risk would increase the uptake of risk-appropriate screening among the study population relative to controls that receive usual care, during the 12 month study period. In doing so, this thesis used a complex intervention aimed at improving the rate of risk-appropriate screening for colorectal cancer (CRC) among an Australian General Practice population. This intervention utilised an online evidenced -based familial history algorithm, that stratified participants into three Australian National Health and Medical Research Council (NHMRC) recommended relative risk groups for screening CRC. These categories are based on a strong body of evidence showing that familial phenotype as measured by family history is a significant and non-modifiable risk factor for an increased lifetime risk of CRC. The algorithm used in the online tool was adapted from the NHMRC guidelines but were also updated by utilising the most recent evidence-base in addition to consulting with a group of experts. This algorithm was then programmed into an online website called “Which test is best?”. This website notified participants of their familial risk in addition to faxing or emailing this information to their consulting General Practitioner (GP). The website was piloted among members of a cancer consumer group (n=26), before being amended to improve clarity and the website interface. It was then implemented in a clustered RCT to evaluate its effect on risk-appropriate CRC screening. The intervention was implemented at both the cluster (GP practice) (Intervention n=27, Control n=28) and participant (eligible patients aged 25-74 with no personal history of CRC and/or inflammatory bowel diseases) level (Intervention, n=836, Control n=726). Those in the intervention arm were given access to the online website with risk tool and their family history information. In addition to their familial risk category with NHMRC recommended screening guidelines were forwarded to their consulting GP, while the control group had usual care. Both groups were followed up 12 months later to obtain their self-reported CRC screening information using the online survey. Thereafter, the control group was immediately given access to the online website with risk tool so that their family history information could be recorded and the level of risk-appropriate screening could be calculated for both groups. The results from this study showed ,that there was no significant difference in risk-appropriate screening rates amongst participants allocated to the intervention group compared to the control group as there was no main effect of allocation when included as a predictor within a binomial logistic regression when modelled to the GEE. However, participants allocated to the intervention group that were designated as belonging to the potentially high-risk category did engage in significantly higher levels of risk-appropriate screening when compared to the control group at 12 month follow-up. This was observed by a significant interaction effect of both family history and allocation in predicting risk-appropriate screening the final GEE model. Specifically, potentially high-risk individuals that were allocated to the intervention group had higher odds (about five times) of engaging in risk-appropriate screening when compared to those at population level risk that were assigned to either control or intervention, when controlling for other variables. This suggests that the online familial risk tool was effective in changing the behaviour of participants from the intervention group that were categorised as having a family history consistent with a potentially high risk (defined as having lifetime relative risk three times or greater of the general population) of developing CRC in their lifetimes. GPs from participating clusters were followed up by a survey (n=66) to assess their attitudes, knowledge and perceived barriers on utilising family history to risk-appropriately screen their average risk patients. Three important findings emerged from this survey. Firstly it shows that the majority of GPs in this study regard family history as the most important factor in screening their asymptomatic patients for CRC. It also shows that these GPs in principle strongly support the NHMRC guidelines, continuing education and peer-reviewed evidence as the most important knowledge factors in evaluating their CRC screening recommendations for asymptomatic patients, while being somewhat less influenced by government policy and their patients’ personal perceptions about the efficacy of a particular CRC screening test. However GPs appear very sensitive to their patients’ fears and anxiety over CRC screening, assessing this factor as the most important barrier to screening for CRC followed by their subjective lack of experience with CRC screening and time constraints imposed during the consultation. Findings also showed a substantial level of dissonance between what GPs believe to be appropriate CRC screening for their asymptotic patients and what they may be likely to recommend with 77% GPs self-reporting that they still refer up to 10 average risk asymptomatic patients to a colonoscopy during a typical month. Taken together the findings from this thesis show that that an intervention which aims to include both the patient and GP improves the uptake CRC risk-appropriate screening for individuals with potentially high-risk. It shows that a tailored risk tool, that supports GP triage may be sufficient to improve uptake of CRC screening modalities across all risk groups but may not be sufficient to encourage risk-appropriate screening of those from average and moderate risk. This is mainly due to persistent over-screening in the average-risk group within our study sample. Future studies may need to examine and differentiate between under screeners and over-screeners in order to target and tailor interventions to those groups separately.

The performance of turbo codes can often be improved by improving the weight spectra of such codes. Methods of producing the weight spectra of turbo codes have been investigated and many improvements were made to refine the techniques. A much faster method of weight spectrum evaluation has been developed that allows calculation of weight spectra within a few minutes on a typical desktop PC. Simulation results show that new high performance turbo codes are produced by the optimisation methods presented. The two further important areas of concern are the code itself and the decoding. Improvements of the code are accomplished through optimisation of the interleaver and choice of constituent coders. Optimisation of interleaves can also be accomplished automatically using the algorithms described in this work. The addition of a CRC as an outer code proved to offer a vast improvement on the overall code performance. This was achieved without any code rate loss as the turbo code is punctured to make way for the CRC remainder. The results show a gain of 0.4dB compared to the non-CRC (1014,676) turbo code. Another improvement to the decoding performance was achieved through a combination of MAP decoding and Ordered Reliability decoding. The simulations show a performance of just 0.2dB from the Shannon limit. The same code without ordered reliability decoding has a performance curve which is 0.6dB from the Shannon limit. In situations where the MAP decoder fails to converge ordered reliability decoding succeeds in producing a codeword much closer to the received vector, often the correct codeword. The ordered reliability decoding adds to the computational complexity but lends itself to FPGA implementation.

Colorectal cancer (CRC) progresses from precursor lesions or polyps via two main pathways – the traditional and serrated pathway. The traditional pathway is the conventional type, while the serrated neoplastic pathway has been recently identified and is not well characterised. The aim of this study was to evaluate molecular factors associated with the serrated pathway and study its progression from a serrated polyp to colorectal cancer using patient samples and mouse models. The first part of the study was to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such as MLH1, p16 and IGFBP7 in patient samples. The mouse models were then utilized to assess the contribution of BRAF V600E mutation along with the cell cycle regulators and tumour suppressor genes, p16Ink4a and p19Arf in the initiation and progression of CRC. The CIMP study was a prospective study in which the real-time based MethyLight assay was employed to evaluate CIMP and methylation status of p16, IGFBP7 and MLH1 in 154 serrated polyps and 63 adenomas. Samples were subjected to bisulfite modification and methylation levels were assessed using the Weisenberger et al panel of methylation markers (IGF2, SOCS1, NEUROG1, RUNX3 and CACNA1G) with ALU as the reference gene.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Science, Environment, Engineering and Technology
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Considéré comme un cancer de l'âge mûr, l'incidence du cancer colorectal ne cesse d'augmenter avec l'allongement de la vie. Dans la majorité des cas, le cancer colique est associé à une mutation du gène suppresseur de tumeur Apc, contrôlant l’activation de la signalisation Wnt/β-caténine. Afin, d'identifier de nouveaux acteurs de la tumorigenèse colique, notre laboratoire a développé des modèles murins de mutation du gène Apc qui ont pour avantage de mimer la pathologie humaine (Colnot et al, 2004 ; Andreu et al, 2005). La création de ces modèles a permis à l’équipe de démontrer i) qu’une activation physiologique de cette voie contrôle la prolifération des cellules souches et des progéniteurs ainsi que leur différenciation et ii) qu’une activation aigüe est suffisante pour déclencher l’initiation tumorale intestinale (Andreu et al, 2005; Andreu et al, 2008). Les travaux antérieurs à mon arrivée ont permis d’identifier différents évènements moléculaires et cellulaires induits en cascade suite à l’activation pathologique de la voie Wnt/β-caténine. Parmi ceux-ci, une induction transcriptionnelle de gènes impliqués dans l'autophagie a été mise en évidence. Ce processus d'auto-cannibalisme cellulaire est associé à de nombreuses pathologies telles que les maladies neurodégénératives ou infectieuses. Cependant, le rôle de l'autophagie dans le cancer reste ambivalent et son implication dans le cancer colique demeure inconnue.Dans ce contexte, mon travail de doctorat a consisté à répondre aux questions suivantes :L’induction transcriptionnelle de gène Atg s’accompagne-t-elle d’une activation du processus d’autophagie à tous les stades de la progression tumorale intestinale murine et humaine?Des études de transcriptomique à haut débit nous ont permis d’identifier une induction transcriptionnelle de gènes clés du processus d’autophagie tels qu’Atg7. Cependant, l’activation fonctionnelle de l’autophagie n’est pas toujours associée à une augmentation de la transcription des acteurs de ce processus. Nous nous sommes donc intéressés aux marqueurs couramment décris dans la littérature et permettant d’établir l’état d’activation du flux autophagique. Ainsi, nous avons étudié le niveau d’expression de ces marqueurs par des expériences de western-blot et d’immuno-marquages sur des échantillons tumoraux humains et murins à différents stades de la progression du CRC. L’inhibition de l’autophagie impacte-t-elle la carcinogénèse colorectale?Dans ce contexte, nous avons généré un modèle murin de délétion conditionnelle et simultanée d'un allèle du gène Apc et des deux allèles du gène Atg7 (gène clé de l'autophagie) spécifiquement dans les cellules épithéliales intestinales. Afin de suivre l'apparition et l'évolution des tumeurs au cours du temps, nous avons mis au point une nouvelle méthode non-invasive de reconstruction tridimensionnelle de côlons de souris, issue d'imagerie échographique à haute résolution. Dans le but de caractériser l’impact de l’inhibition de l’autophagie, les modifications propres à la cellule déficiente en autophagie ont été explorées, notamment le statut énergétique ainsi que les changements dans l’environnement immunitaire et microbien de l’épithélium intestinal. Finalement, l’inhibition génétique de l’autophagie dans notre modèle murin, prédisposé au développement de tumeurs intestinales, nous a permis de caractériser l’implication de l’autophagie dans la carcinogénèse colique, ainsi que les mécanismes moléculaires et cellulaires liant l’auto-cannibalisme cellulaire à la pathologie tumorale
Colorectal cancer is one of the major causes of cancer-related deaths. We took advantage of Apc mutant mice that mimic the adenomatous polyps that affect humans with an inactivated Apc gene, to gain insight into the critical events that affect the development of colorectal cancer. We show that autophagy, a catabolic pathway involved in the degradation of intracellular proteins and organelles, is activated in intestinal murine and human cancer and its inhibition has a crucial role in controlling tumorigenesis. We report that the in vivo conditional deletion of the essential autophagy gene Atg7 in intestinal epithelial cells inhibits the formation of pre-cancerous lesions resulting from Apc loss by enhancing immunosurveillance. The antibody-mediated depletion of CD8+ T cells demonstrated a critical role for CD8+ T cells in antitumoral responses resulting from the inhibition of autophagy. We used a broad-spectrum antibiotics treatment to show that the expansion of IFN-producing CD8+ T cells following the deletion of Atg7 is dependent on the intestinal microbiota and is associated with Paneth and goblet cell defects. In addition, the inhibition of autophagy affected tumor cell growth and restrained cancer growth for extended time periods. We demonstrate that the inhibition of autophagy in Apc tumor cells results in a stress response accompanied by metabolic defects, characterized by AMPK activation and p53 cell cycle arrest. This study suggests that autophagy inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer

Colorectal cancer (CRC), which includes cancer of the large bowel and rectum is the third most common cancer in men and the second in women and there is a poorer survival rate in less developed regions of the world such as West Africa mainly due to the ‘out of reach’ costs of chemotherapy. Evidence suggests that aspirin, a non-steroidal anti-inflammatory drug (NSAID) has the potential to decrease incidence of, or mortality from, a number of cancers including CRC through several mechanisms of action. However, this evidence is dampened by aspirin’s gastrointestinal (GI) toxicity, which have been found to be mostly age-dependent. The search for potential aspirin-related compounds with the same or better cytotoxic effects against cancer cells accompanied by a safer toxicity profile has been ongoing over the years and led to us to synthesise a number of novel aspirin analogues. One of the mechanisms of action suggested for the anticancer property of aspirin is the COX-dependent pathway. In this thesis SW480 cell line, a CRC cell line that is COX-2 negative and mismatch repair (MMR) proficient was used to study the possible COX-independent mechanism of action for aspirin, its analogues and diflunisal at 0.5 mM. Diflunisal was included in this study because it is also a salicylate with reports of having cytotoxic effects. OE33 and FLO1 oesophageal cancer cells were also employed in the epidermal growth factor receptor (EGFR) and synergy experiments to show effects were not just specific to SW480 cells alone. These aspirin analogues were synthesised, identified using nuclear magnetic resonance (NMR) and infra-red (IR) spectroscopy, and tested for purity using thin layer chromatography (TLC) and melting point. The findings of this study suggest that these compounds breakdown into salicylates and perturb epidermal growth factor (EGF) internalization with PN517 (fumaryldiaspirin) and PN590 (ortho-thioaspirin) also driving EGF co-localization with early-endosome antigen-1 (EEA1). The perturbation of the internalization of EGF by aspirin and PN517 was also observed by a time-lapse assay using live confocal imaging. These compounds also had specific effects on different tyrosine phosphorylation sites of the EGFR, with none but PN590 inhibiting 4 phosphorylation at Y1068, and all but PN502 (ortho-aspirin), PN548 (meta-aspirin) and PN549 (para-aspirin) inhibiting phosphorylation at Y1045 and Y1173. Given that the EGF internalization assay involved the cells being treated with compounds for 2 h, cells were also treated for this same time period and probed with pEGFR 1045, which resulted in the compounds having no significant effect on phosphorylation at that site which is responsible for the ubiquitination of the EGFR. Most of these compounds were apoptotic with some showing a combination of apoptosis and necrosis. Aspirin and its isomers drove apoptotic cell death in SW480 cells via the BCL2-BAX pathway while the thioaspirins appear to follow the p21 pathway by decreasing the expression of the protein. In addition, it was shown that PN502 (aspirin), PN517 and PN590 had synergistic effects when used in combination with oxaliplatin at ED50, ED75 and ED90 in SW480 CRC cells. The cytotoxicity of these compounds individually or in combination was determined using MTT assay followed by the use of the CompuSyn and CalcuSyn software to calculate combination index (CI), which indicated whether a drug combination was synergistic, antagonistic or additive. PN517 and PN524 were synergistic when used in combination with cisplatin in OE33 oesophageal cancer cells. Effect of these compounds on the EGFR indicates a delay or disruption of the signalling pathway involved in the proliferation of cancer cells, thus, translating into protection against tumour formation or progression while the synergistic effects of these compounds when used in combination with platinum compounds can provide patients with less toxic chemotherapeutic regimen especially in patients with CRC tumours that harbour mutant TP53 gene and normally resistant to oxaliplatin. It is therefore proposed that the perturbation of EGF internalization is a novel mechanism of action for aspirin and its analogues in cancer therapy. These positive findings shed light on the understanding of the possible mechanism of action for aspirins and gives hope for a more affordable, less toxic therapy for the prevention, treatment and management of cancer.

Background: Conventional chemotherapeutics target bulk tumour cells and generally leave cancer stem cell (CSC) populations unaffected. Recent literature characterized the presence and the role of CSC in several types of solid tumors, including colorectal cancer. Colorectal CSCs (CCSCs) display enhanced WNT/β-catenin pathway activity, sustaining self-renewal and tumor-initiating capacity. Thus, CCSCs are crucial for tumour recurrence and metastasis. As one of the main contributors to sustained self-renewal activity in CCSCs, enhanced formation of β-catenin/CBP complex is fostering transactivation of canonical WNT target genes such as c-myc. However, maintenance of healthy intestinal stem cells also dependents on the canonical WNT pathway. Thus, selective targeting CCSCs while sparring normal intestinal cells is still a significant challenge. Interestingly, Sam68 is a key mediator of the interaction between β-catenin and CBP. It has been reported as a “druggable” target to selectively disrupt β-catenin/CBP in CSCs. Indeed, CWP232228 successfully targets CSCs in AML by facilitating Sam68/CBP complex formation, and consequently lowering the abundance of β-catenin/CBP complexes. CWP232228 was clinically tested on multiple human cancers. Unfortunately, such clinical trials were halted due to unknown causes, and limited information was released on clinical safety and benefits. Consequently, developing more potent pharmacological modulators of Sam68/CBP complex formation is still highly relevant to eradicate CCSCs. Here we describe the discovery and characterization of a new CWP analog, known as YB-0158, which displays enhanced potency and neoplastic selectivity against CCSC. Methods and Results: Following the confirmation that ICG/CWP class of compounds bind to Sam68 in CSCs, I used in silico docking methods to screen for CWP analogs having high predicted affinity for Sam68 Cterminal proline-rich domain. Using high content imaging techniques, I confirmed our top candidate (YB-0158) as more potent vs. CWP parent molecule to compromise cell growth, to induce loss of pluripotency, and to increase Sam68 nuclear localization in a surrogate model of human CSCs. YB-0158 also displayed enhanced selective toxicity in colorectal cancer models vs. normal intestinal epithelium progenitor cells. Moreover, I confirmed that YB-0158 exert negative impact on cancer cell growth by inducing apoptosis and reducing proliferation. Lentiviral-based knockdowns explicitly displayed decrease in drug effectivity in the absence of Sam68, reinforcing the essential role of Sam68 mediating ICG-001/CWP response in CSCs. I demonstrated that Sam68 expression is enriched in tumor-initiating cell fractions derived from primary colorectal tumor tissues vs. bulk heterogeneous tumor organoids. Therefore, YB-0158 showed striking efficacy at supressing tumor-initiation activity in a patient-based serial organoid formation assay. Finally, YB-0158 eradicated CSCs activity in vivo as demonstrated by a syngeneic mouse-to-mouse serial transplantation assay. Conclusion: Overall, YB-0158 is a novel analog of CWP232228 with superior potency to target CCSCs activity through facilitation of Sam68 nuclear localization, thus reducing the interaction frequency between CBP and β-catenin.

Colorectal Cancer (CRC) is one of the most common forms of cancer worldwide (National Cancer Institute [NCI], 2007); its prevalence is also reflected in the Jordanian population (Jordanian Ministry of Health & Jordan Cancer Registry [MOH & JCR], 2008). It appears that CRC diagnosis and treatment modalities have a negative impact on patients’ physical, social, and emotional well-being and their quality of life (QOL). Alarmingly, up to 35% of CRC patients have clinically significant levels of psychological distress. Accordingly, better understanding of QOL and its psychosocial predictors will assist health professionals, especially oncology nurses, to recognize the effects of CRC and its treatment modalities on patients and to plan appropriate interventions to ameliorate these effects. This study was conducted in two phases using mixed methods in a sequentional-explanatory design to: (1) explore the relationships between hope, coping, psychological distress (depression and anxiety), age, gender, marital status, income, time since diagnosis and QOL among Jordanian CRC patients; (2) identify to what extent hope, coping, psychological distress (depression and anxiety), age, gender, marital status, income and time since diagnosis predicts QOL among Jordanian CRC patients; and (3) describe Jordanian CRC patients’ experiences and perceptions about QOL during their treatment period.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Nursing and Midwifery
Griffith Health
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The primary outcome of this thesis is found in three contributions. First, we developed an automatic converter from the cube representation of incompletely specified multiple-output Boolean function, given in Espresso format, to VHDL. The converter is designed specifically for updating functions of Feedback Shift Register (FSRs) in the Galois configuration, namely it reads in the description of a combinatorial function and adds register stages to the appropriate positions. The converter can handle both, Linear and Non-Linear feedback Shift Registers. The second contribution is modifying the automatic converter to design a tool which gives the user the opportunity to see the hardware characteristics of its circuit quickly from the espresso format of its design. The third contribution is applying the resulting converter to evaluate the results of the CRC generation algorithm presented in [6]. We computed the hardware characteristics such as area, timing and power dissipation on most popular CRCs in ASIC and FPGA technologies. Furthermore, we introduced a simple interface used to provide the user a good estimation of the power diagram during the executing time which is similar to probing the current of the circuit.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
This dissertation aims to analyze the Program of Continuing Education in accountancy, implemented by Resolution 945/02 passed by the Federal Council of Accountants, based on the analyses of data collected from 2003 to 2005 by independent auditors working in the state of Sergipe. Although it has also considered quantitative aspects, this research is primarily qualitative, having adopted a historical-critical focus, according to a dialectical approach; it endeavors to understand facts globally and not in isolation, deprived of a context. Semi-structured interviews were made with the auditors and representatives of the Regional Council of Accountants of Sergipe State. With the results, it was possible to delineate the profile of the members of this profession as well as his/her views on the Program. We found out their dissatisfaction with both the events realized in Sergipe and the possibility that the Program might be extended to the remaining accountancy sectors. It lacks structure and competence alike for its development in the State. The key point is the meager number of courses being offered by training agents, which are highly valued in the fulfillment of professional norms.
Esta dissertação teve como objetivo analisar o Programa de Educação Continuada na profissão contábil, com sua implantação a partir da Resolução nº 945/02 do Conselho Federal de Contabilidade, tomando como base uma análise dos resultados obtidos entre 2003 e 2005, pelos auditores independentes que atuam em Sergipe. A pesquisa é predominantemente qualitativa, apesar de contemplar aspectos quantitativos, e utilizou um enfoque histórico-crítico, dentro da abordagem dialética; buscando compreender os fatos de maneira articulada entre si, e não isoladamente do contexto. Foram realizadas entrevistas semi-estruturadas com os auditores e com representantes do Conselho Regional de Contabilidade de Sergipe. Através dos resultados obtidos, foi possível delinear o perfil desse profissional e a sua percepção em relação ao Programa. Verificamos que há uma insatisfação quanto aos eventos realizados em Sergipe e a possibilidade de expansão do Programa para os demais segmentos de contabilistas, falta estrutura e preparo para seu desenvolvimento em Sergipe. O determinante está na insuficiência de cursos promovidos pelas capacitadoras, que valem pontos para o cumprimento da norma profissional.

The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a pyrR- strain compared to the isogenic pyrR+ strain. Thus, pyrR is negatively regulated while the other pyr genes (except pyrC) are positively activated by PyrR. That no regulation was seen for pyrC is in keeping with the recent discovery of a second functional pyrC that is not regulated in P. aeruginosa. Gel filtration chromatography shows the PyrR protein exists in a dynamic equilibrium, and it is proposed that PyrR functions as a monomer in activating pyrD, pyrE and pyrF and as a dimeric repressor for pyrR by binding to the inverted repeat. A related study discovered that the catabolite repression control (Crc) protein was indirectly involved in pyr gene regulation, and shown to negatively regulate expression of PyrR at the posttranscriptional level.

According to the Swedish curriculum every child has the right to feel safe in school. Also according to CRC (committee on the rights of the child) the child has the right to be respected and provided for his or hers basic needs. The teachers must also develop equality for the children and they should also counteract insults. But even though we have these laws and rules some children feel unsafe at school. Every day 60 000 children are being bullied at schools in Sweden. This is a huge problem that needs to be addressed. The aim of this thesis is to investigate teachers in different schools and their strategies to handle bullying. The method is qualitative interviews of teachers. The result indicates that the teachers do see things differently from each other, and that can lead to complications later on. The complications do not only regard the student in that way that they are being treated differently in different schools. It can also affect the teachers in that way that they does not see the harm in what some kids do that other would call bullying.

Introduction: Van Gogh-Like 2 (VANGL2) is a scaffolding planar cell polarity protein involved in non-canonical Wnt signalling. It has been shown to have crucial roles in regulating epithelial development and homeostasis. Moreover, VANGL2 has been implicated in human cancers, with increased expression and copy number amplification seen in several cancer contexts. Many related components within this pathway have also been linked to cancer development, with VANGL2 expression known to regulate factors involved in cell migration and extracellular matrix (ECM) remodelling in cell lines. These cellular processes tend to be erroneously activated in cancer. VANGL2 is known to inhibit the classical driver pathway of colorectal cancer (CRC), canonical, or β- catenin dependant, Wnt signalling, in CRC cell lines. The aim of this thesis is to determine the expression of VANGL2 in CRC, and to investigate how VANGL2 may act to regulate intestinal homeostasis and disease. Methods: Transcriptional verification of VANGL2 expression in the mouse intestine was carried out by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and transcripts localised within the murine colon using RNA-In Situ Hybridisation (RNAISH). Expression and localisation of the VANGL2 protein and related non-canonical Wnt signalling components was confirmed using immuno-histochemistry (IHC). Furthermore, using a combination of human Tissue Micro-Array (TMA), transcriptional data and genomic data, we determined an association between VANGL2 on tumour grade and disease-free survival. To functionally validate the effects of VANGL2 on colorectal biology, we used a model in which VANGL2 is selectively deleted from the colonic epithelium using Villin-CreERT Vangl2flox mouse lines. Using a combination of molecular biology methods, we identified the ECM as differentially regulated following VANGL2 modulation. To test the role of VANGL2 in colorectal cancer, we used a murine colorectal cancer model in which adenomatous polyposis coli (APC) is deleted from colonic epithelium resulting in the formation of cancer concurrently with deletion of Vangl2. We evaluated survival of these mice as well as tumour number and size. Tumour tissue was analysed using IHC, qRT-PCR and 3-Dimensional organoid culture. Results: Within this thesis I have illustrated that the murine colonic epithelium expresses Vangl2, and other components known to interact with VANGL2 including Vangl1, Wnt5A, and Protein Tyrosine Kinase 7 (Ptk7). I have also shown that VANGL2 is expressed within the human colonic epithelium. I go on to show that 9.2% of human CRC possesses VANGL2 transcriptional alterations which correlates with a worsened disease-free survival (DFS) rate among patients. Using IHC, I also show that higher grade CRC is associated with increased VANGL2 expression. In our murine cancer model, mice with single or dual-copy loss of VANGL2 were found to have a reduced number of colonic tumours, while maintaining similar tumour size. Investigations to identify how VANGL2 may have control of tumour initiation were carried out focussing on the ECM. I found that, contrary to what I have discovered in the healthy murine colon, tumours from VANGL2-deficient mice had increased transcription of the ECM markers Secreted protein acidic and rich in cysteine (Sparc) and Decorin (Dcn), as well as increased expression of the ECM regulators Matrix Metallopeptidase 9 (Mmp9) and Tissue Inhibitor of Metalloproteinases 1 (Timp1). Changes in the ECM was also seen at the protein level, with increases in staining for the ECM components Col1 (Collagen, type I), and Laminin in VANGL2-deficient tissue. The ECM modulator Connective Tissue Growth Factor (Ctgf), is implicated in multiple cancers including CRC and is increased within VANGL2-deficient tumours at both the transcript and protein level, implicating Ctgf in increasing the ECM of these tumours.

Colorectal cancer (CRC) is the second most common cancer among males and third among females across the world. In Australia it is the second most prevalent and the second leading cause of cancer-related mortality with the number of CRC incidences doubling over the last decade. While there has been a reduction of the incidence-adjusted mortality of CRC, a significant number of CRC detections are made at either the intermediate or later stages of the disease progression despite the roll out of a population based screening program for individuals aged 50 and over. Data shows that ‘one size fits all’ nature of the program despite recommendations from the NHMRC to screen according to the familial risk of an individual and inappropriate colonoscopy referrals, may have led to over screening those at average risk while potentially under-screening and missing those at an increased risk. Furthermore this program may have missed individuals under the age of 50 that have a high familial lifetime risk of developing CRC and require earlier CRC screening with a colonoscopy. It was hypothesised that implementing an online familial risk tool that notified both patients (aged 25-74) and their GPs of their familial CRC lifetime risk would increase the uptake of risk-appropriate screening among the study population relative to controls that receive usual care, during the 12 month study period. In doing so, this thesis used a complex intervention aimed at improving the rate of risk-appropriate screening for colorectal cancer (CRC) among an Australian General Practice population. This intervention utilised an online evidenced -based familial history algorithm, that stratified participants into three Australian National Health and Medical Research Council (NHMRC) recommended relative risk groups for screening CRC. These categories are based on a strong body of evidence showing that familial phenotype as measured by family history is a significant and non-modifiable risk factor for an increased lifetime risk of CRC. The algorithm used in the online tool was adapted from the NHMRC guidelines but were also updated by utilising the most recent evidence-base in addition to consulting with a group of experts. This algorithm was then programmed into an online website called “Which test is best?”. This website notified participants of their familial risk in addition to faxing or emailing this information to their consulting General Practitioner (GP). The website was piloted among members of a cancer consumer group (n=26), before being amended to improve clarity and the website interface. It was then implemented in a clustered RCT to evaluate its effect on risk-appropriate CRC screening. The intervention was implemented at both the cluster (GP practice) (Intervention n=27, Control n=28) and participant (eligible patients aged 25-74 with no personal history of CRC and/or inflammatory bowel diseases) level (Intervention, n=836, Control n=726). Those in the intervention arm were given access to the online website with risk tool and their family history information. In addition to their familial risk category with NHMRC recommended screening guidelines were forwarded to their consulting GP, while the control group had usual care. Both groups were followed up 12 months later to obtain their self-reported CRC screening information using the online survey. Thereafter, the control group was immediately given access to the online website with risk tool so that their family history information could be recorded and the level of risk-appropriate screening could be calculated for both groups. The results from this study showed ,that there was no significant difference in risk-appropriate screening rates amongst participants allocated to the intervention group compared to the control group as there was no main effect of allocation when included as a predictor within a binomial logistic regression when modelled to the GEE. However, participants allocated to the intervention group that were designated as belonging to the potentially high-risk category did engage in significantly higher levels of risk-appropriate screening when compared to the control group at 12 month follow-up. This was observed by a significant interaction effect of both family history and allocation in predicting risk-appropriate screening the final GEE model. Specifically, potentially high-risk individuals that were allocated to the intervention group had higher odds (about five times) of engaging in risk-appropriate screening when compared to those at population level risk that were assigned to either control or intervention, when controlling for other variables. This suggests that the online familial risk tool was effective in changing the behaviour of participants from the intervention group that were categorised as having a family history consistent with a potentially high risk (defined as having lifetime relative risk three times or greater of the general population) of developing CRC in their lifetimes. GPs from participating clusters were followed up by a survey (n=66) to assess their attitudes, knowledge and perceived barriers on utilising family history to risk-appropriately screen their average risk patients. Three important findings emerged from this survey. Firstly it shows that the majority of GPs in this study regard family history as the most important factor in screening their asymptomatic patients for CRC. It also shows that these GPs in principle strongly support the NHMRC guidelines, continuing education and peer-reviewed evidence as the most important knowledge factors in evaluating their CRC screening recommendations for asymptomatic patients, while being somewhat less influenced by government policy and their patients’ personal perceptions about the efficacy of a particular CRC screening test. However GPs appear very sensitive to their patients’ fears and anxiety over CRC screening, assessing this factor as the most important barrier to screening for CRC followed by their subjective lack of experience with CRC screening and time constraints imposed during the consultation. Findings also showed a substantial level of dissonance between what GPs believe to be appropriate CRC screening for their asymptotic patients and what they may be likely to recommend with 77% GPs self-reporting that they still refer up to 10 average risk asymptomatic patients to a colonoscopy during a typical month. Taken together the findings from this thesis show that that an intervention which aims to include both the patient and GP improves the uptake CRC risk-appropriate screening for individuals with potentially high-risk. It shows that a tailored risk tool, that supports GP triage may be sufficient to improve uptake of CRC screening modalities across all risk groups but may not be sufficient to encourage risk-appropriate screening of those from average and moderate risk. This is mainly due to persistent over-screening in the average-risk group within our study sample. Future studies may need to examine and differentiate between under screeners and over-screeners in order to target and tailor interventions to those groups separately.

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O surgimento de novas metodologias ágeis para apoiar o desenvolvimento de sistemas, como a Extreme Programming (XP), vem causando impacto nas empresas de desenvolvimento de software, especialmente por sua flexibilidade nas mudanças de requisitos no decorrer do projeto. Assim, um melhor entendimento e representação estrutural dos requisitos tornam-se fundamental. Logo, esta pesquisa aplica o conceito das técnicas de refatoração de código dentro da Engenharia de Requisitos, que é focado na metodologia XP, por meios das User Stories. O trabalho aplica um conjunto de padrões e regras que permite aos requisitos expressos em cartões CRC serem refatorados através de pré e pós-condições, sendo que esses requisitos são descritos por mapas conceituais (MC) em formato OWL. Por sua vez, os MCs são convertidos em diagramas de classes da UML por meio da UML-MC que formaliza esta transformação. Dessa forma, o ambiente UStory-Refactory automatiza parcialmente o processo de refatoração e permite que os requisitos refatorados
The emergence of new agile methodologies to support systems development, as the Extreme Programming (XP), has been causing impact on software development companies, specially for its flexibility in the requirements changes during the project. Thus, a better understanding and structural representation of the requirements become basic. Then, this research applies the concept of the code refactoring techniques, inside of the Requirements Engineering, which is focused at XP methodology, through the User Stories. The work applies a set of standards and rules that allows the requirements expressed in CRC cards to be refactored through pre and post-conditions, and the requirements are described for conceptual maps (CMaps) in OWL format. In their turn, the CMaps are converted into UML classes diagrams by the UML-MC that formalizes this transformation. This way, the UStory-Refactory environment partially automatizes the refactoring process and allows the refactored requirements to be exported in OWL format, promoting

Pure rotational spectroscopy of the CrC (X-3 Sigma(-)) and CrCCH ((X) over tilde (6)Sigma(+)) radicals has been conducted using millimeter/sub-millimeter direct absorption methods in the frequency range 225-585 GHz. These species were created in an AC discharge of Cr(CO)(6) and either methane or acetylene, diluted in argon. Spectra of the CrCCD were also recorded for the first time using deuterated acetylene as the carbon precursor. Seven rotational transitions of CrC were measured, each consisting of three widely spaced, fine structure components, arising from spin-spin and spin-rotation interactions. Eleven rotational transitions were recorded for CrCCH and five for CrCCD; each transition in these cases was composed of a distinct fine structure sextet. These measurements confirm the respective (3)Sigma(-) and (6)Sigma(+) ground electronic states of these radicals, as indicated from optical studies. The data were analyzed using a Hund's case (b) Hamiltonian, and rotational, spin-spin, and spin-rotation constants have been accurately determined for all three species. The spectroscopic parameters for CrC were significantly revised from previous optical work, while those for CrCCH are in excellent agreement; completely new constants were established for CrCCD. The chromium-carbon bond length for CrC was calculated to be 1.631 angstrom, while that in CrCCH was found to be r(Cr-C) = 1.993 angstrom - significantly longer. This result suggests that a single Cr-C bond is present in CrCCH, preserving the acetylenic structure of the ligand, while a triple bond exists in CrC. Analysis of the spin constants suggests that CrC has a nearby excited (1)Sigma(+) state lying similar to 16 900 cm(-1) higher in energy, and CrCCH has a (6)Pi excited state with E similar to 4800 cm(-1). Published by AIP Publishing.

Colorectal cancer (CRC) is not a homogenous disease. Recent molecular classification of established tumours based on gene expression and (epi)genetic mutation burden, has revealed considerable disease heterogeneity. The relative importance of the epithelial and stromal tissue compartments varies between different tumour subtypes and this contributes to the observed clinical and molecular heterogeneity of CRC. The AIM of this study was to explore the role of the stroma in different precancerous pathologies (polyps) and in CRC. Stromal gene expression varies considerably between the different polyp subtypes (SSA and TVA) with a comparatively greater number of differentially expressed genes in serrated lesion stroma, suggesting the hypothesis that SSA lesions, usually initiated by BRAF mutations and methylation, require the recruitment of pro-tumorigenic stroma to enable lesion progression. In contrast, TVA are initiated by epithelial mutations that disrupt Wnt signaling (such as APC) and this is sufficient to drive tumourigenesis, irrespective of stromal influences. Moreover, transwell tissue culture 3D techniques and animal models revealed that fibroblasts support the cross species growth of mouse epithelial organoids and abrogate the normal media requirement for Noggin and EGF. Interestingly, mouse epithelium grown in this co culture system develop as spheroids rather than the branching organoids seen with media morphogen supplementation, indicating a phenotype modulating effect of the fibroblasts. Preliminary results revealed that fibroblasts have effects on cell proliferation and cell cycle regulation by upregulation of genes involved with cell cycle progression, DNA synthesis/repair, protein translation, vesicles mediated transport and lipid metabolism. Primary stromal cell cultures isolated from adenoma and colon cancer (CMS2 and CMS4) might in part represent the corresponding cancer microenvironment, thus providing a useful complement to the current cellular biochemistry and therapeutic research in CRC.
Il cancro del colon-retto (CRC) non è una malattia omogenea. Una recente classificazione molecolare sul CRC basata sull’ espressione genica e mutazioni (epi)genetiche, ha rivelato una notevole eterogeneità nella malattia. L' importanza dei compartimenti tissutali, epitelialiali e stromali, varia tra i diversi sottotipi di CRC e questo contribuisce all’ eterogeneità clinica e molecolare osservata nel CRC. Lo scopo di questo studio è stato quello di esplorare il ruolo dello stroma in diverse forme pretumorali (polipi) e nel CRC. Dalle analisi eseguite, e’ emerso che il profilo genetico dello stroma nelle due condizioni pre cancerogene di CRC oggetto di studio (TVA e SSA), differisce in maniera significativa, suggerendo l’ipotesi che i polipi di tipo SSA, in genere promossi da mutazioni nel gene BRAF, richiedono l'assunzione di stroma pro-cancerogeno per consentire la progressione della lesione. Al contrario, in lesioni di tipo TVA, le mutazioni epiteliali che interrompono la via di segnalazione Wnt (es. APC), siano sufficienti per promuovere la tumorigenesi a prescindere dall’ influenza dello stroma. Inoltre, con l’ausilio di tecniche di coltura 3D, inserti e modelli animali, e’ emerso che i fibroblasti sostengono la crescita degli organoidi murine senza l’impiego dei fattori di crescita Noggin e EGF. In particolare, i fibroblasti causano lo sviluppo di sferoidi piuttosto che di organoidi, indicando un effetto modulante sul fenotipo. I risultati preliminary, hanno rivelato che i fibroblasti hanno effetti sulla proliferazione cellulare e la regolazione del ciclo cellulare mediante la regolazione in maniera positiva dei geni coinvolti nella progressione del ciclo cellulare, nella sintesi e/o riparazione del DNA, nella traduzione di proteine, nel trasporto mediato da vescicole e nel metabolismo dei lipidi. I fibroblasti isolati da adenoma e CRC (CMS2 e CMS4) potrebbero in parte rappresentare il microambiente tumorale, fornendo così un utile complemento biochimico e cellulare sulla ricerca terapeutica nel tumore del colon.

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Since the Industrial Revolution, individuals search for professions which grant them social status. The first current of thought of the Theory of Professions claims that professions were created to meet the society's needs. However, subsequent theories (which have been adopted for this paper), presume that the main objective of any profession is to attain social status. In order to reach this goal, the society must develop a positive representation of the profession and its professionals. The social representations are non-scientific explanations of reality, making common and familiar objects accessible to a whole group through historical insight, full of cognitive approaches and interpretative distortions of individuals about objects. The literature analyzed here shows that the stereotypes created for the accountancy professional are predominantly negative, making the task of recruiting good students more difficult in this field. Professional class agencies are the main responsible for the profession, and their target is the attainment of professional status. Faced with this situation, this paper aims to identify and analyze the stereotypes in the rhetoric of the professional class agency for Brazilian accountants from 1962 to 2014. Through an exploratory and qualitative research, it was possible making a prior selection of stereotypes by means of the analysis of literature extracted from the 285 informative newsletters published by the CRC/SP. The data has been analyzed from the perspective of a historical-economical recapitulation, in parallel with the main accountancy milestones in Brazil. The obtained results confirm what Jeacle (2008) states: there is a relationship between positive and negative stereotypes, since both had the same behavior in many decades and, whenever the negative stereotypes were confirmed, the positive ones were confirmed as well. It was not possible establishing the same relationship with the neutral stereotypes group. The results also indicate that the accountancy professionals adapt themselves to the external changes by changing the signs of representation exposed to the society. It was also possible to observe that, in times of international crisis and threat to the accountancy professional outlook, the CRC/SP kept sharing signs that would depict this professional as competent and trustworthy.
Desde a revolu????o industrial os indiv??duos buscam profiss??es que mais lhe trar??o status social. A primeira corrente da Teoria das Profiss??es defende que as profiss??es surgiram para atender necessidades da sociedade, entretanto, as teorias posteriores, e adotadas por esse trabalho, assumem que o principal objetivo de qualquer profiss??o ?? o status social. A fim de atender a esse objetivo ?? necess??rio que a sociedade crie uma representa????o positiva sobre a profiss??o e seus profissionais. As representa????es sociais s??o explica????es n??o cient??ficas sobre a realidade, tornando, de maneira acess??vel, objetos familiares e comuns a todo um grupo atrav??s da percep????o hist??rica repleta de vieses cognitivos e distor????es interpretativas de indiv??duos sobre objetos. A literatura analisada indica que os estereotipos criados sobre o profissional cont??bil s??o predominantemente negativos, dificultando a capta????o de bons estudantes. Os ??rg??os de classe profissionais s??o os principais responsaveis pela profiss??o e almejam o status profissional. Diante disso, esse trabalho teve como objetivo identificar e analisar os estere??tipos no discurso do ??rg??o de classe profissional cont??bil brasileiro, no per??odo de 1962 a 2014. Atrav??s de uma pesquisa qualitativa e explorat??ria criou-se uma sele????o pr??via de estere??tipos selecionados atrav??s da literatura analisada que foram pesquisados nos 285 boletins informativos publicados pelo CRC SP. Os dados foram analisados a partir de uma recapitula????o hist??rico-econ??mica e em paralelo com os principais marcos da contabilidade no Brasil. Os resultados obtidos confirmam a afirma????o de Jeacle (2008) de que h?? rela????o entre os estere??tipos negativos e positivos, j?? que em diversas d??cadas ambos tiveram o mesmo comportamento, quando houve confirma????o dos negativos, os positivos tamb??m se confirmaram. N??o foi poss??vel estabelecer a mesma rela????o com o grupo de estere??tipos neutros. Tamb??m foi poss??vel confirmar a ruptura da representa????o ocorrida nas d??cadas de 80/90 mencionadas nos trabalhos de Bougen (1994); Dinmik e Felton (2006); Cotsa, Weffort; Cia (2011). E que, a partir de 2010, houve uma nova ruptura que retoma a imagem do profissional retratada nas primeiras d??cadas desse estudo (60/70). Os resultados tamb??m indicam que os profissionais se adaptam ??s mudan??as externas alterando os signos de representa????es expostos ?? sociedade conforme contexto econ??mico, pol??tico e social.

Thesis (Ph. D.)--West Virginia University, 2002.
Title from document title page. Document formatted into pages; contains xvii, 276 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 199-208).

Abstract Cancer patients do not always respond in the same way to the same drug treatment: the administration of the same dose of an antiblastic drug in a population of patients induces the manifestation of a wide range of toxicity, which sometimes can even be deadly. The intersubject variation, that is observed in efficacy and toxicity of anticancer drugs, can be determined by complex interactions among components of the physiological, environmental and genetic factors. The activities carried out in the framework of the PhD project have focused the attention on the influence of genetic factors on the outcome of drug treatment, identifying possible prognostic and predictive genetic biomarkers. To evaluate the correlation between genotype and phenotype of the patient, we considered a pharmacogenetics study which defined the relationship between a specific polymorphism (UGT1A1 * 28) and the effects of the drug. A phase Ib clinical trial was conducted at the CRO, Aviano (PN). The purpose of this study was to modulate the dosage of CPT-11 in the presence of the inhibitor angiogenetic BV for each individual patient, based on genotype of UGT1A1, in order to obtain an improvement of the therapeutic index . To do this, it was considered necessary to develop a specific method of pharmacokinetic analysis and we proceeded as follows : * Development of a quantitative method for the analysis of the drug of interest * Validation of the method in accordance with FDA guidelines * Measurement of plasma concentrations of the drug * Determination of the pharmacokinetics of the drug * Correlation of pharmacokinetics and pharmacogenetics We enrolled patients with a histological diagnosis of metastatic colorectal adenocarcinoma, naïve or treated with adjuvant chemotherapy (excluding irinotecan), and corresponding to the criteria for eligibility/exclusion described in the protocol. The patients were assigned to their treatment group based on genotype (*1/*1 or *1/*28) until the completion of recruitment for each dose level in each group of patients. Patients with genotype *28/*28 were excluded because of high risk of toxicity. The starting dose of irinotecan administered in patients carrying the *1 (wild-type and heterozygous) was 260mg/m2. The dose of BV was 5mg/kg and it was administered in infusion after two weeks. In the treatment group with lower dose, the irinotecan dose was increased to 310 and 370mg/m2 in case of lack of toxicity. The evaluation of pharmacokinetic and pharmacodynamic interactions between bevacizumab (BV) and irinotecan (CPT-11) was conducted describing the pharmacokinetic profile of the CPT -11 (and its metabolites) in the absence and in the presence of BV in the same patient. The obteined pharmacokinetic parameters exclude an effect of BV on the pharmacokinetics of CPT-11. However, the registered values ??of MTD were lower than those determined by previous works with FOLFIRI alone, and that might suggest that the addition of BV results in changes in the manifestation of toxicity in the high-dose regimens. The drugs that have been investigated in this protocol have been present in the clinical practice since several years, and the high variability of the response and toxicity makes it essential the definition of useful criteria for treatment personalization. Basic criteria for personalization of therapy are clinical parameters, like gender and age of the patients. Many of the elderly subjects, in fact, receive reduced treatments comparing standard clinical protocols, with a considerable reduction of the dosage of the drug or of the number of therapy cycles. Due to their physiological characteristics, the older cancer patients reported greater toxicity effects associated with the treatment. In clinical trials, elderly patients are under-represented because they don’t satisfy all the inclusion criteria, like comorbid conditions and baseline functional status of the patient. Similarly, women are often excluded from some protocols especially for the variability that characterizes the hormonal status. In a second part of the PhD project, we considered a very large series of patients with CRC treated with other medications associated with fluoropyrimidine (FOLFOX or FOLFIRI) at the CRO, Aviano (PN), and other centers participating in the program. We performed a pharmacogenetic study to see if there are genetic biomarkers useful to define a personalization of therapy for subjects considered elderly (age at diagnosis >70 years). In particular, the aim was to identify possible pharmacogenetic (PG) determinants which influence drug effects (toxicity and response) in a different way between young and elderly subjects, and between the two genders (male and female). We analyzed several polymorphisms of genes coding for proteins involved in the mechanisms of activation, metabolism and elimination of drugs mainly used in therapies against CRC and other polymorphisms in genes that regulate the cell cycle and involved in the process of carcinogenesis and tumor progression. Many allelic variations confirm an association with the risk of developing cancer risk, while others with the survival of patients. In particular, the variation in the copy number of GSTM1 and GSTT1 genes, showed a correlation with the age of diagnosis of the patients, as well as with the gender. The increase in copies of GSTM1 or GSTT1 reduction are associated with greater survival in male patients older than 70 years (PGSTM1=0.047, HR=3.937, 95% CI=0.89-0:31; PGSTT1=0.039, HR=4.246). Moreover, we further found a specific association of genotype GSTnull with an increased risk of tumor in young subjects, but not in the elderly. It can therefore be assumed that there is a deficiency in the enzymatic activity of glutathione S-transferase deficiency resulting in reduced cell detoxification mechanisms that involve an increase in damage to the DNA damnage. Additional data allowed us to conclude that genetic characteristics may be associated with both gender and age of the patients: there are polymorphisms predictive of risk related to one of the two parameters (MTHFR 1298A>C in young women); polymorphisms localized in repair genes are mainly associated with a greater risk and a lower survival in the elderly population. Translational research is a fundamental step for the application of experimental research to clinical practice. One of the most important aspects in this area is the customization of the therapy that should be considerate important to get treatment optimization especially in the field of oncology.
Riassunto Pazienti oncologici, con lo stesso tipo di tumore, non sempre rispondono in modo uguale al medesimo trattamento farmacologico: la somministrazione della stessa dose di un dato farmaco antiblastico in una popolazione di pazienti implica spesso la manifestazione di un vasto range di tossicità, che in alcuni casi può risultare addirittura mortale. La variabilità intersoggettiva che si osserva nell’efficacia e nella tossicità dei farmaci antiblastici impiegati nella chemioterapia può pertanto essere determinata da interazioni complesse tra le componenti fisiologiche, ambientali e fattori genetici individuali. L’attività svolta in ambito del progetto di dottorato ha puntato l’attenzione su come i fattori genetici possano influenzare l’esito di un trattamento farmacologico, individuando dei possibili biomarcatori genetici prognostici e predittivi. Per valutare la correlazione tra genotipo e fenotipo del paziente siamo partiti da uno studio di farmacogenetica che ha definito la relazione esistente tra un determinato polimorfismo (UGT1A1*28) e l’alterazione dell’effetto del farmaco che ne consegue. Successivamente, ci si è indirizzati verso il trasferimento e l’applicazione nella pratica clinica attraverso uno studo clinico di fase Ib (“Studio di fase I guidato dal genotipo dell’irinotecano in combinazione con 5-fluorouracile/leucovorina (FOLFIRI) e bevacizumab in pazienti con carcinoma colonrettale”), condotto presso il Centro di Riferimento Oncologico di Aviano (PN). Lo scopo di tale studio è stato quello di modulare il dosaggio dell’irinotecano (CPT-11) in presenza dell’inibitore angiogenetico bevacizumab (BV), non essendo ancora nota l’interazione tra i due farmaci, in base al genotipo di UGT1A1, al fine di ottenere un miglioramento dell’indice terapeutico per ogni singolo paziente. Per fare ciò, si è ritenuto necessario sviluppare un metodo di analisi di farmacocinetica specifico e si è pensato di procedere nel seguente modo: * Sviluppo di un metodo quantitativo per l’analisi del farmaco d’interesse * Validazione del metodo in accordo con le linee guida dell’FDA * Misurazione delle concentrazioni plasmatiche del farmaco * Determinazione della farmacocinetica del farmaco * Correlazione dei dati di farmacocinetica e farmacogenetica Sono stati arruolati pazienti con diagnosi istologica di adenocarcinoma colorettale (CRC) metastatico, non pretrattati con chemioterapia o trattati con terapia adiuvante (escluso irinotecano), e rispondenti ai criteri di eleggibilità/esclusione previsti dal protocollo. I pazienti sono stati assegnati al loro gruppo di trattamento in base al genotipo (*1/*1 o *1/*28) fino al completamento del reclutamento per ogni livello di dose in ogni gruppo di pazienti. I pazienti con genotipo *28/*28 sono stati esclusi perché ad alto rischio di tossicità. La dose iniziale di irinotecano somministrata nei pazienti portatori dell’allele *1 (wild type ed eterozigoti) è di 260 mg/m2. La dose di BV è di 5 mg/kg, somministrata anch’essa in infusione ogni due settimane. Il dosaggio dell’irinotecano è stato incrementato a 310 e 370 mg/m2 qualora nel gruppo di trattamento con dosaggio più basso non vi sia stata tossicità. La valutazione delle interazioni farmacocinetiche e farmacodinamiche tra bevacizumab (BV) ed irinotecano (CPT-11) è stata condotta descrivendo il profilo farmacocinetico del CPT-11 (e dei suoi metaboliti) in assenza ed in presenza di BV nello stesso paziente. Da una prima analisi dei parametri farmacocinetici sembra che si possa escludere un effetto del BV sulla farmacocinetica dell’irinotecano. La sovrapponibilità del dato farmacocinetico con o senza BV, è valida per entrambi i dosaggi di irinotecano considerati. Tuttavia, sono stati riscontrati dei valori di Dose Massima Tollerata (MTD) inferiori rispetto a quelli determinati da lavori precedenti, e ciò potrebbe suggerire che l’aggiunta del BV comporti una variazione nella manifestazione di tossicità in regimi ad alto dosaggio. Una percentuale non trascurabile di pazienti ha ottenuto una riduzione del numero e delle dimensioni delle lesioni secondarie epatiche tanto da renderle aggredibili chirurgicamente o tramite termoablazione, facendo pertanto concludere che il regime terapeutico si configuri quindi come una conversion therapy. Poiché i farmaci che sono stati studiati in questo protocollo sono presenti nella pratica clinica da diversi anni, la notevole variabilità riscontrata nella risposta e nello sviluppo di tossicità rende indispensabile trovare dei criteri utili alla personalizzazione del trattamento. Criteri fondamentali per la personalizzazione della terapia sono i parametri clinici, principalmente il genere e l’età dei pazienti. Molti dei soggetti anziani, infatti, ricevono dei trattamenti ridotti poiché tollerano meno le terapie dei protocolli clinici standard, con una notevole riduzione del dosaggio di farmaco o del numero di cicli di trattamento. Proprio per le loro caratteristiche fisiologiche, i pazienti oncologici anziani riportano maggiori effetti di tossicità associati al trattamento rispetto ai soggetti definiti giovani. Analogamente, anche le donne sono spesso escluse da alcuni protocolli soprattutto per la variabilità ormonale che caratterizza l’età fertile e il periodo della menopausa. A tale proposito, in una seconda parte del progetto di dottorato, abbiamo considerato una casistica molto ampia di pazienti affetti da CRC trattati con fluoropirimidine associate ad altri farmaci (FOLFIRI o FOLFOX) presso il Centro di Riferimento Oncologico di Aviano (PN) e altri centri aderenti al programma. Lo scopo è stato quello di individuare dei possibili determinanti farmacogenetici (PG) che condizionano gli effetti del farmaco (tossicità e risposta) in maniera differente tra soggetti giovani e anziani, e tra i due generi (maschio e femmina), nonché dei possibili marcatori di rischio tumorale specifici. Sono stati analizzati diversi polimorfismi di geni codificanti per proteine coinvolte nei meccanismi di attivazione, metabolizzazione ed eliminazione dei farmaci principalmente usati nelle terapie contro il CRC e altri polimorfismi di geni che regolano il ciclo cellulare in quanto coinvolti nel processo di carcinogenesi e di progressione tumorale. Molte delle variazioni alleliche analizzate confermano un’associazione con il rischio d’insorgenza del tumore, altre con la sopravvivenza dei pazienti. In particolare, la variazione del copy number dei geni codificanti per l’enzima Glutatione S-trasferasi, GSTT1 e GSTM1, ha messo in evidenza una correlazione con l’età di diagnosi dei pazienti, nonché con il genere. L’aumento delle copie di GSTM1 o la riduzione del GSTT1 sono associati a una maggiore sopravvivenza nei soggetti con età superiore a 70 anni e di sesso maschile (PGSTM1=0.047, HR=3.937, 95% CI=0.31–0.89; PGSTT1=0.039, HR=4.246). Abbiamo ulteriormente riscontrato una specifica associazione del genotipo GSTnull con un aumentato rischio d’insorgenza tumorale nei soggetti giovani, ma non nei soggetti anziani. Si può quindi ipotizzare che vi sia una riduzione dell’attività enzimatica della Glutatione-S Transferasi con conseguente deficit ai meccanismi di detossificazione cellulare che comportano un incremento dei danni a carico del DNA. Gli ulteriori dati ottenuti, ci permettono di concludere che ci possono essere delle differenze associate alle caratteristiche genotipiche relative sia al genere che all’età dei pazienti: ci sono polimorfismi predittivi di rischio prevalentemente associati ad uno dei due generi (MTHFR 1298A>C nelle giovani donne); altri polimorfismi localizzati nei geni del riparo del DNA sono prevalentemente associati ad un rischio maggiore e ad una sopravvivenza minore nella popolazione anziana. È pertanto fondamentale che, grazie alle nuove conoscenze nell’ambito della medicina molecolare e alle nuove tecnologie, si arrivi alla personalizzazione della terapia, al fine di massimizzare la risposta e ridurre gli effetti tossici in ciascun paziente.

The aim of this research is to ask questions as to why child right laws for the protection of child soldiers have failed to protect children in the Darfur region of Sudan despite the
fact that Sudan is a member to many of these children&rsquo
s rights instruments. Can conflict transformation therefore be of any help? This is research seeks to address the question posed above.

Colorectal cancer (CRC) is one of most common cancer diseases worldwide. In Swedenapproximately 5,000 new cases of CRC are generated each year, which makes it the thirdmost common cancer disease among both men and women. The past decades ofimproved treatment strategies have considerably increased the five-year survival for CRCpatients. However more could be achieved in this area, in particular for metastatic CRC,which is the cause of most CRC-related deaths. Therefore it is important to study thebiological response to certain treatments induced in CRC to find valuable predictiveand/or prognostic factors to select patients for better suited treatments. The aim of this thesis was to gain insight into the molecular changes that occurfollowing irradiation or treatment with SN-38 in rectal cancer patients or colon cancercell lines by studying the RNA expression, protein expression, DNA cell cycledistribution and apoptotic response. The expression of phosphatase of regenerating liver(PRL) proteins was investigated in rectal cancers from 125 patients included in arandomized clinical trial of preoperative radiotherapy (RT). Increased expression of PRLswas seen at the invasive margin of primary and metastatic cancers compared with theinner area of the tumors. Moreover, strong PRL staining at the invasive margin correlatedto distant recurrence and worse survival of patients in the RT group but not in non-RTgroup (Paper I). Radiosensitivity was studied by treating KM12C, KM12SM andKM12L4a colon cancer cell lines with radiation. KM12C is of low metastatic naturecompared with the highly metastatic KM12SM and KM12L4a. Upregulation of ΔNp73and PRL-3 might contribute to the radioresistant phenotype in KM12C. In contrast,KM12L4a shows a high frequency of apoptosis and lack of upregulation of ΔNp73, PRL-3 and survivin, which might explain its radiosensitive phenotype (Paper II). KM12C,KM12SM and KM12L4a were treated with SN-38 which inhibits topoisomerase 1 (topo-1). The results show that SN-38 induces G2/S arrest and possess the capacity to triggerapoptosis in the three cell lines (Paper III). To further elucidate SN-38 effect on these celllines, the gene expression profile following SN-38 treatment was studied. Oligonucleotidearrays consisting of ~27,000 spots were hybridized with sample and reference cDNA.Both unsupervised and supervised hierarchical clustering analysis, and functional analysiswere performed. Supervised hierarchical clustering gives a strong signal of 1453discriminated genes, the vast majority being upregulated. Both upregulated anddownregulated genes point toward a favorable impact of SN-38 regarding the apoptoticpathways. For example RhoB and Bax are upregulated together with downregulation ofKras and survivin, which promotes apoptosis (Paper IV). In conclusion, PRLs may be valuable biomarkers for RT resistance, predicting apoor prognosis in rectal cancer patients. Targeting radio-resistance factors, such asΔNp73 and survivin may contribute to an increased sensitivity to RT. SN-38 affects cellproliferation and apoptosis.

The Seamless Stressometer® roll (Seamless STR) is used to measure the flatness of aluminum and steel strip when there is an extreme demand on the surface finish. To protect the roll and strip, the roll is coated with two layers deposited by high velocity oxygen fueled spraying (HVOF), Cr-Ni(Si,B) closest to the roll and WC-Co on top. This solution has several disadvantages; high cost and complicated logistics, corrosion sensitivity and high residual stresses creates the need for two coatings which in turn complicates the process. Cobalt is, in addition, sensitive to low pH coolants and environmentally unfriendly. These problems have given rise to the idea of switching both the method and material of the coating. In the first part of this work, high velocity air fueled spraying (HVAF) was evaluated as an alternative deposition method. Three materials, Cr3C2-NiCr, WC-Co and WC-CrC-Ni were deposited on steel coupons with varying chamber pressure, powder feed rate and distance from the nozzle, in order to evaluate if HVAF can be a valid technique for use in this application and to optimize the spraying recipe. The objectives were to get a sufficiently high thickness per sweep, to be able to make the depositions in a manageable number of sweeps, and to get low porosity, since the coatings need to be dense to be hard and possible to polish smooth. The tests showed that all three materials can be sprayed with the high settings on the parameters to obtain coatings that exceeded the set limits of the objectives. In the second part of this work, the recipe obtained from the first part was used to deposit samples for further analysis. The coatings were compared regarding cost, hardness, friction, wear and pick-up properties to evaluate if a switch in material from WC-Co was possible. The coatings showed both similarities and differences. The friction was very similar for the three materials. Cr3C2-NiCr was substantially cheaper than the other two, had lower hardness and higher porosity, but still probably acceptable values, and was satisfactory regarding wear and pick-up. WC-Co and WC-CrC-Ni were very similar to each other regarding cost, hardness and porosity but WC-Co was the best regarding wear and pick-up, where WC-CrC-Ni was the worst. The only clear advantage of WC-CrC-Ni over WC-Co is the lack of cobalt. Taking everything into consideration, including the fact that the wear and pick-up tests in this work was quite exaggerated, Cr3C2-NiCr is an interesting option for this application due to its low cost and acceptable test results, WC-Co had the best results but is expensive and contains cobalt and WC-CrC-Ni had as good results as WC-Co except for the wear and pick-up tests and does not contain cobalt.

The research accomplished in this dissertation is focused on the design of effective solutions to the problem that error codes occur in the ZigBee-based meeting attendance registration system. In this work, several different check algorithms are compared, and the powerful error-detecting Cyclic Redundancy Check (CRC) algorithm is studied. In view of the features of the meeting attendance registration system, we implement the check module of CRC-8. This work also considers the data reliability. We assume use retransmission mechanism to ensure the validity and completeness of transmission data. Finally, the potential technical improvement and future work are presented.

Purpose: The study that has been conducted is a comparative one, where the group compared different innovation management strategies used by three different globally- known Swedish firms that are in the manufacturing industry. The study is aimed at describing, analyzing and making conclusions of the innovation strategies used during the process of product development in the chosen companies bycomparing their similarities and differences. Method: The Study was carried out using a comparative study drawing on the qualitative data. Conclusion: Volvo CE and ABB CRC have similar strategies in internal idea generation because both firms have formalized systems, by using strong online data bases for idea sharing and evaluation. Volvo CE uses a pronounced forum called “Innovation Jams” for online idea sharing among Volvo Group employees whereas ABB CRC uses a strong data base called “ABB Inside” to evaluate ideas within the group. On the other hand, Scania R&D’s internal idea generation process is more informal as it is based on “person-to-person”. When it comes to external idea generation, Scania R&D has a more established strategy of using suppliers and customers for inspiration of ideas. However, ABB CRC generates inspirations from customers through its business centers, whereas Volvo CE has no customer system in place. But one thing that is common in all the three companies is that they are highly collaborating with universities for idea generation and human resource.

Canada and Sweden receives unaccompanied minors and implements the CRC in a similar and different way for them. This work deals with what rights there are written for unaccompanied minors. How do the countries live up to the CRC for unaccompanied minors? This work gives a little understanding how it can look like in two countries that have different political regimes.

It has been demonstrated that intercellular communication via cell- released vesicles is very important both for normal and tumor cells, and specifically to determine tumor development and progression as well as invasion and angiogenesis. Cell communication could involve the exosomes, small vescicles of endocytic origin, which are released from different kinds of donor cells; they can transfer molecular signals as proteins and RNAs through the extracellular environment to specific recipient cells in a autocrine, paracrine or endocrine way. Exosomes can strongly influence the recipient cells phenotype by transferring oncogenes that could influence the response of cells to drugs or immune reactions. Recently, it has been demonstrated the presence of miRNAs inside the exosomes and their potential involvement in cancer development. Considering the important role of miRNAs in colorectal cancer, one of the most diffused and studied tumor, it was considered interesting to investigate the role of exosomal miRNAs and associated proteins in the response of CRC cells to Cetuximab (an anti-EGFR therapeutic antibody). The EGFR signaling pathway is very importantly in relationship both to CRC and miRNA biogenesis and expression, and recently also to the exosomal communication system. Therefore, one of the major aims of this thesis was to analyze the possible involvement of exosomes in the response to Cetuximab of two CRC cell lines (wild-type KRAS Caco-2 cells and KRAS mutated HCT-116 cells) through the transfer of specific miRNAs and proteins to recipient cells. To carry out this analysis, we performed cellular and exosomal miRNA profiling after Cetuximab treatment, for 745 miRNAs by using Real-Time PCR. The results of the analysis showed that exosomal miRNA profiles globally reflect those of whole cells at steady-state, but there exists an important quantitative asymmetrical distribution. After Cetuximab treatment, Caco-2 sensitive cells showed several exosomal differentially expressed (DE) miRNAs in comparison to HCT-116 cells. Many DE miRNAs are involved in cancer and immunity. These data could be explained by considering that the EGFR pathway can regulate miRNA biogenesis via the MAPK/ERK cascade. Exosomal proteins analysis was performed for 741 cancer-related proteins through a specific antibody microarrays platform. Also the profile of exosomal proteins from Caco-2 cells showed important alterations after Cetuximab treatment. Globally, several DE miRNAs and proteins from Caco-2 exosomes were related to cancer, stimulation of immunity and inflammation. Interestingly, exosomes transfection experiments between Caco-2 and HCT-116 cell lines (performed to investigate their effect on cell viability) showed that the transfection of steady state Caco-2 exosomes in the HCT-116 cell line determined a decrease of cell viability of recipient cells, while Cetuximab-treated Caco-2 cells exosomes, transfected in HCT-116 cells, increased their viability. These data could be explained considering that exosomes from Cetuximab-treated cells are enriched in oncogenic- and immune stimulation-related miRNAs. Finally, DE proteins were searched to find potential RNA-binding proteins. Globally, the results of this thesis could be useful to: (1) verify the existence of horizontal transfer of genetic informations in eukaryotes; (2) search for potential miRNAs and proteins biomarkers of Cetuximab response in CRC in vivo. Eventually, it will be interesting to perform the characterization of exosomal miRNAs and proteins expression profiles of plasma from CRC patients after Cetuximab treatment. Moreover, the characterization of the asymmetrical distribution of miRNAs between cells and exosomes could be important to further investigate the potential and specific mechanism of miRNA sorting within exosomes.

It is well known that aberrant activation of the Notch pathway plays a critical role in the pathogenesis of T cell acute lymphoblastic leukaemia (T-ALL) and of certain solid tumors including lung, breast and ovarian cancer. In particular, increased Notch1 activity has been observed in intestinal adenoma, partially accomplished by β-catenin-mediated up-regulation of the Notch ligand Jagged-1. Whether further mechanisms of Notch activation exist and other Notch receptors might be involved in colorectal cancer (CRC) has not been investigated so far. In this study we investigated the possible involvement of Notch3 signaling in CRC, and the possible therapeutic implications. Intrigued by the observation that Notch3 mRNA and protein are over-expressed in a subset (20%) of human CRC, we sought to investigate how Notch3 modulates oncogenic features of CRC cells. By exploiting xenografts of CRC cells with different tumorigenic properties in mice, we found that the aggressive phenotype was associated with altered expression of components of the Notch pathway, including augmented expression of Delta-like 4 (DLL4) and Jagged-1 ligands and increased levels of the Notch3 transcript and intracellular domain (ICD). Stimulation with immobilized recombinant DLL4 dramatically increased Notch3 expression and Notch signaling. Moreover, forced expression of an active form of Notch3 mirrored effects of DLL4 stimulation and increased tumor formation. Conversely, blocking Notch3 signaling resulted in perturbation of the cell cycle followed by reduction of cell proliferation and inhibition of tumor growth. Moreover, we observed that these CRC cells have also different metastatic potential in terms of number and dimension when injected intravenously in mice. Lung metastases formed by CRC cells expressed Notch3, but the number or size was not significantly reduced by anti-Notch2/3 treatment. Overall, these findings indicate that Notch3 receptor can modulate the tumorigenic properties of CRC cells, and that DLL4 contributes to sustain Notch activity in DLL4-expressing tumors. Further studies are necessary to clarify the role of Notch3 in metastasis and design effective target therapies.
E’ ormai noto da alcuni anni che un’aberrante attivazione della via di segnalazione di Notch gioca un ruolo critico nella patogenesi della leucemia linfoblastica acuta a cellule T (T-ALL) e di alcune neoplasie solide come il cancro del polmone, della mammella e dell’ovaio. Inoltre, una marcata attivazione del recettore Notch1 è stata osservata negli adenomi intestinali ed è correlata all’aumentata espressione del ligando Jagged-1 indotta dall’attivazione del pathway Wnt-APC-β-catenina. E’ stato inoltre dimostrato che questa pathway, insieme a quella di Notch, interviene nella regolazione della proliferazione e del differenziamento delle cellule epiteliali della mucosa intestinale normale. Attualmente, non si sa ancora se altri meccanismi di attivazione della pathway di Notch, oltre a quello ligando-dipendente, siano operativi nel CRC e nemmeno se possano essere coinvolti altri recettori della stessa famiglia. In questo studio, abbiamo cercato di chiarire il possibile coinvolgimento di Notch3 nel CRC. Basandoci sull’osservazione che Notch3 risulta essere frequentemente overespresso sia a livello di mRNA che di proteina nei campioni umani di CRC, abbiamo cercato di chiarire come il recettore Notch3 potesse modulare le proprietà tumorigeniche delle cellule di CRC. A tale scopo, si sono rivelati particolarmente utili alcuni xenotrapianti di cellule umane di CRC che presentano una diversa aggressività in topi NOD/SCID. Infatti, utilizzando i tumori sperimentali, abbiamo potuto dimostrare che l’espressione dei diversi componenti del pathway di Notch risulta essere significativamente elevata nella variante aggressiva rispetto a quella dormiente. In particolare si è osservata un’ aumentata espressione dei ligandi DLL4 e Jagged-1 ed un incremento dei livelli del trascritto di Notch3 e della forma attiva del recettore nei tumori che crescono più rapidamente. Una simile up-regolazione di Notch3 con un’aumentata attivazione della via di segnalazione si è osservata in seguito a stimolazione delle cellule di CRC in vitro con il ligando DLL4 ricombinante. Analogamente, anche l’overespressione di una forma attiva del recettore Notch3 in queste stesse cellule conferisce loro una maggiore capacità proliferativa e favorisce la formazione di tumori in vivo. Al contrario, si è visto che l’inattivazione del pathway mediante silenziamento genico di Notch3 nelle cellule aggressive di CRC, determina significative alterazioni del ciclo cellulare con conseguente riduzione nella proliferazione in vitro e un ritardo nella crescita dei tumori in vivo. Complessivamente, questi risultati dimostrano che il recettore Notch3 può modulare le proprietà tumorigeniche delle cellule di CRC, in particolare contribuendo a mantenere elevata l’attivazione della via di Notch nei tumori esprimenti nel loro microambiente tumorale alti livelli di DLL4. Inoltre, l’inoculo della variante più aggressiva rispetto a quella dormiente per via endovenosa in topi NOD/SCID ha mostrato che le cellule di CRC non hanno solo una diversa capacità tumorigenica, ma anche una differente capacità metastatica a livello polmonare, sia in termine di numero che di dimensioni delle metastasi osservate. Basandoci sui risultati fin qui ottenuti, e su uno studio recentemente pubblicato in cui è stato dimostrato come il pathway di Notch sembra essere coinvolto anche nel processo di metastatizzazione, abbiamo condotto un primo esperimento pilota che prevedeva, in topi portatori di metastasi polmonari, il blocco dei recettori Notch3 e Notch2 mediante l’impiego di un anticorpo neutralizzante. Sfortunatamente i risultati ottenuti non hanno fino ad ora mostrato una riduzione significativa nel numero e nelle dimensioni delle metastasi analizzate: l’analisi dei livelli di alterazione di Notch dopo il trattamento è in corso. A conclusione dello studio le nostre osservazioni rappresentano un punto di partenza per un futuro sviluppo di terapie che abbiano Notch3 come bersaglio per il trattamento di un sottogruppo di casi di CRC.

Colorectal cancer (CRC) is the third most common form of cancer and the second cause of cancer-related death in the Western world. Despite the emergence of new targeted agents and the use of various therapeutic combinations, none of the treatments options available is curative in patients with advanced cancer. In the last decade, several studies have shown that a small population of “stem cell like” cells, with the capacity for self-renewal, multipotency and high tumorigenic potential, could be isolated from tumors with different histological origin, including human CRC. These peculiar stem cells are believed to be responsible for tumor initiation, progression and resistance to therapeutic agents. Therefore, treatments that are design to eradicate tumor masses should be also targeted to CSCs. Recently in our laboratory cancer stem cells (CSCs) lines and their fetal bovine serum (FBS)-cultured non-CSC pair lines were in vitro isolated from surgical specimens of patients with primary CRC cancer. In this study we performed an immunobiological characterization of CRC CSCs and investigated whether these cells can represent suitable targets for immunotherapy protocols. The phenotype and functional characterization of these cell lines was examined by IF and cytofluorimetric analysis. Firstly the expression levels of CRC cancer and/or stem cell-associated molecules such as: Ep-CAM, CEA, HCAM, CD44, Aldefluor, CD133, SOX-2, Nanog and Oct-4 was assessed. Notably, the tumorigenic potency of CSCs was higher than that of FBS cells as shown by the serial dilutions and serial transplantations cell injection in immunodeficient mice. Moreover the xenografts generated by CRC CSC showed large areas of necrosis than tumor tissues generated by FBS cells and better matched the phenotype of the original tumor, as revealed by an IHC analysis of CRC and CSC-associated molecules. In addition, the expression of MHC-I and MHC-II molecules, NKG2D ligands, immune regulatory molecules, antigen-processing machinery (APM) and tumor associated antigens (TAA) was examined. Both CRC CSC and FBS lines were found weakly positive for MHC-I and negative for MHC-II, while NKG2D ligands were commonly detected in both cells types. The APM was mainly defective in CRC CSC versus FBS cell lines. We also investigated whether the expression of immune-regulatory molecules (CTLA-4, PD-1, B7-H3) can affect the immunogenicity of these cells. All CSCs and FBS cell lines were homogeneously positive for CTLA-4, PD-1 and, although at higher levels for PD-L1 and B7-H3. All these molecules acts as negative modulator of T-cells response because of the inhibition of the vital ‘second signal’ required for optimal T cell recognition and activation. Interestingly, IL-4 was expressed by all the cell lines both in the cytoplasm and in the membrane while IL-4R was weakly detectable on the membrane of CSC and was absent in FBS lines counterparts. These data may confirm the possibility of an autocrine production by CRC CSC and also FBS line of IL-4, that may thus protect themselves by apoptosis as recently described by Stassi’s group (Todaro M et al, 2007), this citockine may also induce an immunomodulatory role for T-cells mediated responses against CRC. Unexpectedly FBS tumor lines released in the supernatants high levels of prostaglandin-E2 (PGE2) that, on the contrary was weakly or absent in the supernatant of CSCs. In addition, T lymphocytes isolated from the peripheral blood of CRC patients were stimulated in vitro with autologous CSCs or FBS, in order to assess of anti-CRC reactivity. In patient #1076 we could isolate mostly TH1-mediated responses (detected by INF-γ release), showing weak reactivity against CSCs. On the contrary a more efficiently INF-γ release was detectable when T-lymphocytes were stimulated with FBS tumor cells. In a second patient (#1247), a MHC-independent recognition of CSCs was observed, suggesting an NK-mediated response. This result was also confirmed by the phenotype analysis of these lymphocytes that revealed the presence of 20-30% NK cells (CD3-CD16*CD56+).These findings correlate with the defective expression of molecules involved in antigen presenting and processing machinery observed in CSCs. Taken together our data we can conclude that we have isolate from CRC tissues cells with stemness properties, thus showing a lower immunogenicity compared with the FBS counterpart. T cell responses could be obtained in CRC patients directed against CSC and FBS cells, though with higher reactivity for FBS tumor cells, while in another patient NK-mediated responses could be isolated These findings may be useful for the identification of new agents that can efficiently rescue the immunogenicity of CSCs in order to be targeted by T cell-mediated immune response.

Previous research in our laboratory established that pyrB, pyrC or pyrD knock-out mutants in Pseudomonas aeruginosa required pyrimidines for growth. Each mutant was also discovered to be defective in the production of virulence factors. Moreover, the addition of exogenous uracil did not restore the mutant to wild type virulence levels. In an earlier study using non-pathogenic P. putida, mutants blocked in one of the first three enzymes of the pyrimidine pathway produced no pyoverdine pigment while mutants blocked in the fourth, fifth or sixth steps produced copious quantities of pigment, just like wild type P. putida. The present study explored the correlation between pyrimidine auxotrophy and pigment production in P. aeruginosa. Since the pigment pyoverdine is a siderophore it may also be considered a virulence factor. Other virulence factors tested included casein protease, elastase, hemolysin, swimming, swarming and twitching motilities, and iron binding capacity. In all cases, these virulence factors were significantly decreased in the pyrB, pyrC or pyrD mutants and even in the presence of uracil did not attain wild type levels. In order to complete this comprehensive study, pyrimidine mutants blocked in the fifth (pyrE) and sixth (pyrF) steps of the biosynthetic pathway were examined in P. aeruginosa. A third mutant, crc, was also studied because of its location within 80 base pairs of the pyrE gene on the P. aeruginosa chromosome and because of its importance for carbon source utilization. Production of the virulence factors listed above showed a significant decrease in the three mutant strains used in this study when compared with the wild type. This finding may be exploited for novel chemotherapy strategies for ameliorating P. aeruginosa infections in cystic fibrosis patients.

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence for a model in which Crc and Hfq co-target multiple RNA motifs to bring about a two-tiered regulation to subvert catabolism of phenols in the face of preferred substrates – one at the level of the regulator DmpR and another at the level of translation of the catabolic enzymes.

Background: The prognosis of early colorectal cancer differs according to the stage of disease. Presently, TNM staging is the main tool to predict risk of relapse and potential benefit derived from adjuvant treatment. Adjuvant treatment is indicated according to pathological staging and risk stratification, but a significant proportion of patients who receive chemotherapy derive no benefit from treatment. Therefore, biomarkers to improve risk stratification are needed to reduce the number of patients treated unnecessarily. Additionally, it is proven that radical resection of metastatic lesions may give survival benefit in candidate cases; however, metastatic spread is not always timely detected during post-surgical follow up. Biomarkers of early detection of disease relapse would therefore be extremely clinically relevant. We plan to use, in a prospective study performed at the Hospital of Prato, the metabolomic profile of patients with CRC as a biomarker to identify those who are at highest risk of recurrence, and therefore may be more likely to benefit from adjuvant chemotherapy (METCOLON Study). Methods: A population of 41 patients with histologically proven stage I-III CRC, candidate for radical resection were prospectively enrolled in the study. Blood samples were taken from each patient before surgery (T0) and after surgery (T1) and serum samples were analyzed using 1H-NMR spectroscopy to characterize the metabolomic profile. To investigate the differences of metabolomic profiles before and after surgery, OPLS-DA, mPLS) were applied. Results: Using the mPLS, serum samples were correctly classified between pre-operative (T0) and post operative (T1), and showed significant differential clustering, with good separation of the two groups with the all 3 spectra CPMG, NOESY and DIFFUSION Three metabolites (3-Hydroxybutyric_acid, pyruvic acid, acetone) were found to differ significantly (p < 0.05) between the pre-operative and post-operative metabolomic profiles. In particularly pyruvic acid was found high in the post operative samples and the acetone and 3-Hydroxybutyric_acid in the pre-operative samples. Four patients experienced disease relapse in our cohort. For the first ten patients interestingly the pre-operative spectra of two of these relapsed clustered separately from those of disease-free patients suggesting it may be possible to distinguish the metabolomic profile of patients who then presented disease recurrence . Conclusions: Metabolomic analysis seems to correctly classify pre- and post- operative serum samples of patients with CRC. It also seems that metabolomic analysis may be able to correctly identify patients with recurrence from those who have not presented it, even if the events are still numerically few. Although interesting this data are based on a small number of patients with a relatively few relapse events and a short follow up and require further validation.

DNA methylation is an epigenetic mechanism that regulates the gene transcription. Folate is used in cellular synthesis of methyl groups, nucleic acids and amino acids. In complex diseases like cancer and neural tube defects (NTD), various genetic and epigenetic alterations can be found that disrupt the normal cell function. The main goals of this thesis were to analyze whether the genes responsible for the folate transport (FOLR1, PCFT, and RFC1) could be regulated by DNA methylation in placenta, blood leukocytes and colorectal cancer. We also addressed the genome-wide DNA methylation changes in colorectal cancer andcervical cancer.We found that changes in the methylated fraction of the RFC1 gene were dependent on the RFC1 80G>A polymorphism in placental specimens with NTDs and blood leukocytes from subjects with high homocysteine (Paper I). In colorectal cancer, the greatest difference in DNA methylation was observed in the RFC1 gene and was related to a lower protein expression (Paper II).In Paper III and IV we studied the DNA methylated fraction using a high-density array. Paper III was focused on genes in the DNA repair pathway and frequently mutated in colorectal cancer. We found that aberrant methylation in the DNA mismatch repair genes was not a frequent event in colorectal cancer and we identified five candidate biomarker genes in colorectal cancer, among them the GPC6 and DCLRE1C genes. In Paper IV, we found hypomethylation of genes involved in the immune system in cervical cancer specimens compared to healthy cervical scrapes and we identified twenty four candidate genes for further evaluation ofclinical value.In conclusion, the work of this thesis filled a relevant knowledge gap regarding the role of differential methylation of the folate transport genes in NTD and colorectal cancer. This thesis work also provided insights into the functional role of DNA methylation in cancer specific pathways and identified potential novel biomarker genes.

This essay is about children's rights in one African country namely Kenya. It includesinternational documents like the United Nations Convention of the Rights of the Child (CRC) andthe regional African Charter on the Rights and Welfare of the Child (ACRWC). The thesis alsoanalyzes the differences between these documents and also the Sustainable development goals arediscussed and how they directly touch children's rights.This thesis also deals with the topic of how children's rights are protected by the Kenyanconstitution . It will mention the international laws and conventions that Kenya is obligated toimplement and follow in order to guarantee the kenyan children their rights. During the work wewill take a look at legislations and policies that Kenya has established to promote and protect therights of the children in Kenya.I will analyze some of the violations of the rights of children in Kenya and will be mainly focusingon laws and policies that the country established. The thesis also includes an investigation ofwhich children's rights are violated in Kenya. It consists of statistics and what the laws in Kenyasay about these violations.

Colorectal cancer (CRC) is one of the deadliest cancers in the world. The early stage of the disease is usually asymptomatic and therefore screening methods for colorectal cancer need to improve. There is a need for early detection of CRC as treatment is less effective in the advanced stage of the disease.  The current standard screening methods are endoscopy and fecal immunochemical blood tests. Endoscopy is a commonly used method to diagnose the patient, but it is costly, time consuming, and rather unpopular for the patients. An alternative could be to develop targeted molecular imaging probes that specifically deliver agents for example magnetic resonance imaging to colon adenomas and adenocarcinomas. This alternative would be non-invasive and able to detect the disease before morphological changes become evident. Biomarkers are used as an objective indicator of an altered biological process. Here, a literature study was conducted to identify protein biomarkers that are overexpressed in early stages of CRC. This study has focused on biomarkers that could be used to target imaging agents to cancerous lesions. Thus, the biomarkers need to be membrane-bound and expressed on the luminal side of the gastrointestinal tract. This will help future research to develop orally administered targeted imaging probes. Furthermore, a smaller literature search was conducted to identify cell and mouse models representing early stages of CRC. This was done to facilitate translational research going from in vitro to in vivo. Ideally the same protein is available in cell lines, mouse models and humans to enable translational research. This work has resulted in the selection of 7 different proteins that are upregulated during early stages of CRC. These proteins are potentially apically located and therefore possible targets for monoclonal antibodies. These findings might lead to a novel way for preventive patient screening and hopefully reduce the mortality for colorectal cancer.