Orientador: Carmen Silvia Passos Lima, Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T11:53:50Z (GMT). No. of bitstreams: 1 Nascimento_Helvia_D.pdf: 1845231 bytes, checksum: 16f227072d706aacb01f06165bd8a553 (MD5) Previous issue date: 2007 / Resumo: Os mecanismos moleculares envolvidos na origem do adenocarcinoma de cólon esporádico (ACE) ainda não estão completamente elucidados. Recentemente, o método da análise seriada da expressão gênica (SAGE) foi descrito como eficaz para identificar a expressão total de genes de tipos celulares diversos, mas esta análise não foi realizada em células epiteliais purificadas do ACE moderadamente diferenciado. Nós caracterizamos pelo método SAGE a expressão gênica total de células epiteliais neoplásicas do cólon de um paciente com ACE moderadamente diferenciado (SAGE CC) e de células epiteliais normais do cólon de um paciente com megacólon chagásico (SAGE CN). Foram geradas, após o seqüenciamento automático, 44.004 e 43.570 tags totais das bibliotecas SAGE CC e SAGE CN, representando 16.484 e 13.479 tags únicas, respectivamente. Na comparação entre as bibliotecas, 171 transcritos diferencialmente expressos foram identificados (P< 0,001; expressão diferencial = 5), incluindo 10% de transcritos que podem representar genes não descritos. As expressões de 10 genes diferencialmente expressos foram quantificadas pela reação em cadeia da polimerase em tempo real (qPCR) na amostra de células epiteliais neoplásicas (SAGE CC), com o intuito de validar os resultados obtidos pelo SAGE, e, posteriormente, em amostras de células epiteliais de outros cinco pacientes com o mesmo tipo de doença. As expressões foram concordantes em 80% dos genes (CEACAM6, KLK6, LYZ, PFN1, S100A8, S100A9, VIL2 e ZFHX1B) e discordantes nos demais 20% (PLA1A e ZNF277). As expressões dos genes de interesse, quantificadas pelos dois métodos, foram similares na amostra SAGE CC e nas amostras dos demais pacientes com a doença. Foram observadas expressões anormais de genes envolvidos com a proliferação e diferenciação celular e com a resposta ao stress em células epiteliais neoplásicas. Foram também visualizadas expressões anormais de genes não relacionados com a doença e de genes ainda não identificados. Em conjunto, os nossos resultados podem contribuir para a identificação de genes relacionados com a origem ou a progressão do ACE moderadamente diferenciado e, ainda, para a descoberta de agentes terapêuticos específicos que controlem a proliferação anormal das células neoplásicas. / Abstract: The molecular mechanisms involved in sporadic colon adenocarcinoma (SCA) are still not completely elucidated. Recently, the serial analysis of gene expression (SAGE) method has allowed the global analysis of genes expressed in diverse cellular types but there are no studies in purified epithelial cells of SCA moderately differenciated. We have characterized through SAGE the global gene expression of neoplastic epithelial cells from a SCA moderately differenciated patient (SAGE CC) and normal epithelial cells from a megacolon patient (SAGE CN). After automatic sequencing, a total of 44.004 tags from SAGE CC and 43.570 tags from SAGE CN profiles were generated, representing 16.484 and 13.479 unique tags, respectively. Comparing both profiles, 171 differentially expressed transcripts were identified (P< 0.001; fold = 5), including 10.0% that may represent novel transcripts. The expression of 10 selected genes was further investigated by realtime polymerase chain reaction (qPCR) in the SCA moderately differenciated epithelial cells sample (SAGE CC), with the purpose of to validate the results obtained by the SAGE method, and also in five epithelial cells samples from the same type of SCA patients. Similar expressions were seen in 80% (CEACAM6, KLK6, LYZ, PFN1, S100A8, S100A9, VIL2 e ZFHX1B) and discordant expressions were seen in 20% (PLA1A e ZNF277) of analysed genes. On SAGE CC sample and samples of the SCA patients, all genes presented similar expressions measured by both methods. We observed abnormal expression of genes involved with cell proliferation and differentiation, and with response to stress in neoplastic epithelial cells. Also, were found abnormal expressions of genes not related with the disease and not identified genes. Together, our results may contribute for the identification of genes involved in the origin or progression of SCA moderately differenciated, as well as for the discovery of new therapeutical agents, with specific action on abnormal proliferation of the neoplastic cells. / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

The usage of women in terrorist bombings have increased in the past years due to security and government's lack of scrutiny of security checks, women have the advantage of slipping through the security nets and perform tasks which have been performed by men. The security put forward by governments and other security organizations have always been focused on the aspect that a man would perform a terrorist attack, the likelihood of a woman performing the task has been very low and now, governments and security details have been caught off guard as there is an increase of women carrying out an attack. There are not that many terrorist profiles out for women for security personnel to follow by, which has made it hard for governments and security organization to know what it is they need to keep an eye on. Motives are an important path to understanding what drives a woman into joining a terrorist organization. This thesis will focus on the motives to see if it is possible to create a profile for women. By going through earlier literature, the motives will be studied, and explain what it is that drives a woman to join. The thesis will also give a brief explanation of what terrorism is for the reader to get more insight knowledge to what terrorism is.

Debugging is an important and critical phase during the software development process. Software debugging is serious and tough practice involved in functional base test driven development. Software vendors encourages their programmers to practice test driven development during the initial development phases to capture the bug traces and the associated code coverage infected from diagnosed bugs. Application’s source code with fewer threats of bug existence or faulty executions is assumed as highly efficient and stable especially when real time software products are in consideration. Due to the fact that process of development of software projects relies on great number of users and testers which required having an effective fault localization technique. This specific fault localization technique can highlight the most critical areas of software system at code as well as modular level so that debugging algorithm can be used to debug the application source code. Nowadays many complex or simple software systems are in corporation with open bug repositories to localize the bugs. Any inconsistency or imperfection in early development phase of software product results in low efficient system and less reliability. Statistical debugging of program source code for visualization of fault is an important and efficient way to select and rank the suspicious lines of code. This research provides guidelines for practicing statistical debugging technique for programs coded in Ruby programming language. This thesis presents statistical debugging techniques available for dynamic programming languages. Firstly, the statistical debugging techniques were thoroughly observed with different predicate base approaches followed in previous work done in the subject area. Secondly, the new process of statistical debugging for programs coded in Ruby programming language is introduced by generating dynamic predicates. Results were analyzed by implementing multiple programs written in Ruby programming language with different complexity level. The analysis of experimentation performed on candidate programs depict that SOBER is more efficient and accurate in bug identification than Cause Isolation Scheme. It is concluded that despite of extensive research in the field of statistical debugging and fault localization it is not possible to identify majority of the bugs. Moreover SOBER and Cause Isolation Scheme algorithms are found to be two most mature and effective statistical debugging algorithms for bug identification with in software source code. / Address: School of Computing Blekinge Institute of Technology SE-371 79 Karlskrona, Sweden Phone: +46-(0)455-385804 Fax: +46-(0)455-385057

Excessive memory use may cause severe performance problems and system crashes. Without appropriate tools, it may be difficult or impossible to determine why a program is using too much memory. This applies even though Python provides automatic memory management --- garbage collection can help avoid many memory allocation bugs, but only to a certain extent due to the lack of information during program execution. There is still a need for tools helping the programmer to understand the memory behaviour of programs, especially in complicated situations. The primary motivation for Heapy is that there has been a lack of such tools for Python. The main questions addressed by Heapy are how much memory is used by objects, what are the objects of most interest for optimization purposes, and why are objects kept in memory. Memory leaks are often of special interest and may be found by comparing snapshots of the heap population taken at different times. Memory profiles, using different kinds of classifiers that may include retainer information, can provide quick overviews revealing optimization possibilities not thought of beforehand. Reference patterns and shortest reference paths provide different perspectives of object access patterns to help explain why objects are kept in memory.

The market needs for embedded or mobile devices were exploding in the last few years. Customers demand for devices that not only have high capacity of managing various complex jobs, but also can do it fast. Manufacturers therefore, are looking for a new field of processors that fits the special needs of embedded market, for example low power consumption, highly integrated with most components, but also provides the ability to handle different use cases. The traditional ASICs satisfied the market with great performance-per-watt but limited scalability. ASIP processors on the other hand, impact the new market with the ability of high-speed optimized general computing while energy efficiency is only slightly lower than ASICs. One essential problem in ASIP design is how to find the algorithms that can be accelerated. Hardware engineers used to optimize the instruction set manually. But with the toolset introduced in this thesis, design automation can be made by program profiling and the development cycle can be trimmed therefore reducing the cost. Profiling is the process of exposing critical parts of a certain program via static code analysis or dynamic performance analysis. This thesis introduced a code profiler that targeted at discovering repetition section of a program through static and dynamic analysis. The profiler also measures the payload of each loop and provides profiling report with a user friendly GUI client.

The aim of this project was to quantitatively measure the resilience of the building water cycle. In order to accomplish this goal, a framework was developed that outlines how building water resilience can be evaluated. The framework presented assumed that resilience describes the fulfillment of system functions; in this case, the system functions considered are those actualized by the building water system. A building water resilience assessment model (WRAM) was developed with the ability to simulate different building water cycles and resilience scenarios. Resilience is dependent on the type and magnitude of a disturbance. Therefore, unique disruption scenarios were developed to test the building water cycle resilience: (1) loss of municipal potable water and (2) loss of both municipal potable water and power. Under each scenario, the building water cycle was tested based on the type of building and the water management strategies utilized by the building. The WRAM requires organization of water demand and source connections, and an explicit prioritization framework was produced based on water source and demand preferences found in literature. The framework gives priority to treated wastewater, stormwater, rainwater, condensate, reclaimed water, and potable water, respectively. The baseline prioritization may be manipulated by restricting demand-source connections, and shifting priorities was shown to affect the potential for potable water offsets as a precursor to resilience. Real building water demand profiles were developed from data collected using smart meters at four building sites (multi-residential neighborhood, commercial building, elementary school, and community center). Water source profiles were developed using hourly climate data for the region. Detailed building water demand and supply profiles were developed for the multi-residential and elementary school building sites for resilience assessment using the WRAM. Each building water profile was adapted into 9 scenarios with each subjected to the two disruption schemes for 5 different disruption durations (1 hour, 6 hours, 24 hours, 72 hours, and 168 hours) at 10 different randomized dates and time throughout the year. The result was 450 model runs for each building subjected to each disruption scheme (potable water loss or potable water and central power loss). The relationship between resilience and sustainability was examined based on sustainable building practices accepted by the U.S. Green Building Council's (USGBC) Leadership in Environmental and Energy Design (LEED) green building rating system. Building WRAM outcomes include unique water demand and supply profiles used to describe resilience in terms of the level of service (LOS) of building water functions. Analysis of water profiles validated redundancy, diversity, capacity, alternative water, passivity, preparation, and adaptation potential indicators as gauges of the resilience of the building water cycle. Results showed that resilience correlates with alternative water building water management strategies, but high resilience values are still attainable using storage of non-renewable, non-sustainable sources. However, building water cycles utilizing alternative water maintained steadier resilience as disruption lengths increase due to the ability of sources to be replenished during disruption events. The strongest correlation with LOS was observed for the diversity, redundancy, alternative water, and capacity indicators when scenarios utilizing only potable water were excluded from analysis. For these scenarios, correlation values were 0.56 for diversity, 0.56 for redundancy, 0.60 for capacity, and 1.00 for alternative water for the multi-residential building subjected to potable water loss; and 0.33 for diversity, 0.24 for redundancy, 0.62 for capacity, and 1.00 for alternative water for the multi-residential building subjected to both potable water and central power disruption. For elementary school scenarios that did not utilize potable water storage, correlation values were 0.67 for diversity, 0.64 for redundancy, 0.06 for capacity, and 0.89 for alternative water when subjected to disruption of potable water; and 0.67 for diversity, 0.64 for redundancy, 0.06 for capacity, and 0.80 for alternative water when subjected to disruption of potable water and central power. Passivity correlation to LOS was between 0.77 and 1.00 for all scenarios, building types, and disruption schemes. Passivity correlation with LOS was lower for potable water disruption scenarios, but higher when building water cycles lost power in addition to potable water. The average of each indicator was also calculated for each scenario for each of the five disruption durations by grouping the individual values from each of the 10 randomized disruption start dates and times. The correlation between the average capacity indicator and LOS greatly increased with this method to a range of 0.41 to 0.78 for all buildings subjected to each disruption scheme. In addition, a positive correlation between the preparation indicator and LOS (and corresponding negative correlation between the adaptation potential indicator and LOS) emerged for scenarios that do not utilize potable water storage. For disruption of potable water, the preparation correlation value was 0.94 for the multi-residential building and 0.78 for the elementary school. For disruption of potable water and central power, the preparation correlation value was 0.32 for the multi-residential building and 0.79 for the elementary school.

Coral reef biodiversity is threatened by rapidly changing anthropogenic activities and natural perturbations, leading to massive ecological and economic consequences ranging from the loss of fisheries to coastal erosion. It is necessary to understand corals responses to environmental changes in order to determine management programs on appropriate spatial and temporal scales to address these issues. Coral larvae are the product of sexual reproduction, have the potential to recruit to new areas, and are fundamental in maintaining genetic diversity. These larvae are subjected to variations in local environmental conditions until they settle, inducing specific larval molecular response patterns. One factor that influences coral health is salinity. Low salinities can alter cell homeostasis creating stress in cells. In the natural environment larvae may be exposed to low salinities due to heavy rainfall or run-off. This study investigated larvae responses to low salinity and characterized gene expression in the reef-building coral Porites astreoides using a coral stress-focused microarray. Nine batches of 250+ larvae from three different colonies were collected and immediately exposed in an acute hyposalinity experiment. Samples from two treatments of 25 and 30 ppt, and a control at 35 ppt were used in this study. After experimental exposure these samples were stored in RNAlater® and molecular analysis was performed. The RNA from the samples was extracted, purified and hybridized to a coral stress-focused microarray. Statistical analysis indicates 72 genes were differentially expressed across treatments (p<0.003, analysis of variance). The hierarchical cluster analysis groups together the larvae exposed to salinities of 30 and 35 ppt indicating both treatments induced similar patterns of gene expression. Larvae responses to 30 ppt are minimal, suggesting larvae can tolerate acute exposures to 30 ppt salinity levels. In contrast, the lower salinity (25 ppt) induced a strong response in both the coral and zooxanthellae. The coral larvae up-regulated stress response genes and down-regulated genes associated with normal cell functioning. Additionally, the zooxanthellae down-regulated genes associated with photosynthesis. These results suggest larvae may be vulnerable to bleaching, which may affect the ability of larvae to successfully undergo metamorphosis and survive at low salinities. However, this has yet to be confirmed with complementary techniques. Long-term studies are recommended to examine the effects of hyposalinity on larvae at different time scales and life history stages.

Background: The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D) J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D) J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D) J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D) J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses. Results: To help address this problem, we propose a standardized file format for representing V(D) J analysis results. The proposed format, VDJML, provides a common standardized format for different V(D) J analysis applications to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format specification is accompanied by a support library, written in C++ and Python, for reading and writing the VDJML file format. Conclusions: The VDJML suite will allow users to streamline their V(D) J analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https:// vdjserver. org/ vdjml/. We welcome participation from the community in developing the file format standard, as well as code contributions.

>Magister Scientiae - MSc / Type 2 diabetes mellitus (T2DM), a metabolic disease characterized by chronic hyperglycemia, is the most prevalent form of diabetes globally, affecting approximately 95 % of the total number of people with diabetes i.e. approximately 366 million. Furthermore, it is also the most prevalent form in South Africa (SA), affecting approximately 3.5 million individuals. This disease and its adverse complications can be delayed or prevented if detected early. Standardized diagnostic tests for T2DM have a few limitations which include the inability to predict the future risk of normal glucose tolerance individuals developing T2DM, they are dependent on blood glucose concentration, its invasiveness, and they cannot specify between T1DM and T2DM. Therefore, there is a need for biomarkers which could be used as a tool for the early and specific detection of T2DM. MicroRNAs are small non-coding RNA molecules which play a key role in controlling gene expression and certain biological processes. Studies show that dysregulation of microRNAs may lead to various diseases including T2DM, and thus, may be useful biomarkers for disease detection. Therefore, identifying biomarkers like microRNAs as a tool for the early and specific detection of T2DM, have great potential for diagnostic purposes. The main focus of this investigation, therefore, is the early detection of T2DM by the identification and validation of novel biomarkers. Furthermore, based on previous studies, the aim of the investigation was to identify differentially expressed miRNAs as well as identify their potential target genes associated with the onset and progression of T2DM. An in silico approach was used to identify miRNAs found to be differentially expressed in the serum/plasma of T2DM individuals. Three publically available target prediction software were used for target gene prediction of the identified miRNA. The target genes were subjected to functional analysis using a web-based software, namely DAVID. Functions which were clustered with an enrichment score > 1.3 were considered significant. The ranked target genes mostly had gene ontologies linked with “transcription regulation”, “neuron signalling, and “metal ion binding”. The ranked target genes were then split into two lists – an up-regulated (ur) miRNA targeted gene list and a down-regulated (dr) miRNA targeted gene list. The in silico method used in this investigation produced a final total of 4 miRNAs: miR-dr-1, miR-ur-1, miR-ur-2, and miR-ur-3. Based on the bioinformatics results, miR-dr-1 and its target genes LDLR, PPARA and CAMTA1, seemed the most promising miRNA for biomarker validation, due to the function of the target genes being associated with T2DM onset and progression. The expression levels of the miRNAs were then profiled in kidney tissue of male Wistar rats that were on a high fat diet (HFD), streptozotocin (STZ)-induced T1DM, and non-diabetic control rats via qRT-PCR analysis. The hypothesis was that similar miRNA expression would be found in the HFD kidney samples compared to serum expression levels of the miRNA obtained from the two databases, since kidneys are involved in cleansing the blood from impurities. This hypothesis proved to be true for all miRNAs except for miR-ur-2. Additionally, miR-ur-1 seemed the most significant miRNA due to it having different expression ratios for T1DM and T2DM (i.e. -7.65 and 4.2 fold, respectively). Future work, therefore, include validation of the predicted target genes to the miRNAs of interest i.e. miR-dr-1: PPARA and LDLR and miR-ur-1: CACNB2, using molecular approaches such as the luciferase assays and western blots.

Le lymphome folliculaire (LF) représente le 2ème lymphome par ordre de fréquence et reste considéré à l’heure actuelle comme incurable. De nombreuses questions sur le processus de lymphomagénèse sont encore non résolues et il n’existe aucun marqueur génomique ou moléculaire unanimement reconnu permettant de prédire l’évolution des patients. Nos travaux de recherche s’inscrivent dans l’objectif de mieux comprendre l’impact des altérations moléculaires identifiées dans ces tumeurs, grâce à une approche intégrative visant à combiner des données génomiques, transcriptomiques et mutationnelles. Ce travail a permis de construire un score, basé sur l’expression d’un panel de gènes, prédictif du risque de progression de la maladie. Ce score a été confirmé sur une seconde cohorte de patients, validant son utilité potentielle en pratique clinique. Par ailleurs, nos résultats suggèrent que les cellules tumorales peuvent acquérir des propriétés évocatrices d’un profil de cellules souches et associées à un pronostic particulièrement défavorable. Une 2ème partie de notre travail a porté sur les altérations touchant le gène EZH2, muté chez 25% des patients. Nous avons démontré qu’un gain génomique au niveau du locus EZH2 pouvait également avoir des conséquences sur le profil transcriptomique et un impact pronostique, soulignant l’importance de prendre en compte l’ensemble des anomalies touchant ce gène. Enfin, nous rapportons qu’un polymorphisme constitutionnel situé dans ce gène est associé au risque de progression des patients traités par un anticorps anti-CD20. L’ensemble de ces résultats apporte un éclairage nouveau sur la biologie du LF et peut contribuer à améliorer la prise en charge des patients / Follicular Lymphoma (FL) is the 2nd most frequent lymphoma subtype and is usually considered incurable with current strategies. Several questions regarding the lymphomagenesis process are still pending, and no molecular or genomic marker has been unanimously recognized yet to predict outcome. We performed an integrative analysis combining genomic, transcriptomic and mutational data in the view to bringing new highlights in the molecular alterations acting in FL. Based on gene-expression profiling data we developed a model able to predict progression-free survival in FL patients. We confirmed its predictive value in another cohort of patients, thereby allowing its potential use in clinical practice. Furthermore, our results highlight that some tumors show a stem-cell-like gene-expression profile that was associated with highly unfavorable outcome. In the second part of our work, we focused on alterations of the gene EZH2. Although mutations have been reported in 25% of FL patients, we questioned whether genomic gains at EZH2 locus could also contribute to lymphomagenesis. We showed that such gain may impact the transcriptional profile and have a prognostic significance, thus highlighting the crucial interest of determining both kinds of alterations. Finally, we report that a germ-line polyporphism in the EZH2 gene was significantly associated with progression-free survival in patients treated by anti-CD20 therapy. Taken together, these results bring new highlights on FL biology and may help to improve the clinical management of FL patients