Methods for construction and analysis of computational models in systems biology : applications to the modelling of the heat shock response and the self-assembly of intermediate filaments

Systems biology is a new, emerging and rapidly developing, multidisciplinary research field that aims to study biochemical and biological systems from a holistic perspective, with the goal of providing a comprehensive, system- level understanding of cellular behaviour. In this way, it addresses one of the greatest challenges faced by contemporary biology, which is to compre- hend the function of complex biological systems. Systems biology combines various methods that originate from scientific disciplines such as molecu- lar biology, chemistry, engineering sciences, mathematics, computer science and systems theory. Systems biology, unlike “traditional” biology, focuses on high-level concepts such as: network, component, robustness, efficiency, control, regulation, hierarchical design, synchronization, concurrency, and many others. The very terminology of systems biology is “foreign” to “tra- ditional” biology, marks its drastic shift in the research paradigm and it indicates close linkage of systems biology to computer science. One of the basic tools utilized in systems biology is the mathematical modelling of life processes tightly linked to experimental practice. The stud- ies contained in this thesis revolve around a number of challenges commonly encountered in the computational modelling in systems biology. The re- search comprises of the development and application of a broad range of methods originating in the fields of computer science and mathematics for construction and analysis of computational models in systems biology. In particular, the performed research is setup in the context of two biolog- ical phenomena chosen as modelling case studies: 1) the eukaryotic heat shock response and 2) the in vitro self-assembly of intermediate filaments, one of the main constituents of the cytoskeleton. The range of presented approaches spans from heuristic, through numerical and statistical to ana- lytical methods applied in the effort to formally describe and analyse the two biological processes. We notice however, that although applied to cer- tain case studies, the presented methods are not limited to them and can be utilized in the analysis of other biological mechanisms as well as com- plex systems in general. The full range of developed and applied modelling techniques as well as model analysis methodologies constitutes a rich mod- elling framework. Moreover, the presentation of the developed methods, their application to the two case studies and the discussions concerning their potentials and limitations point to the difficulties and challenges one encounters in computational modelling of biological systems. The problems of model identifiability, model comparison, model refinement, model inte- gration and extension, choice of the proper modelling framework and level of abstraction, or the choice of the proper scope of the model run through this thesis.

Health Related Quality of Life after Childhood Cancer - A Finnish Nationwide survey

The purpose of this Finnish epidemiological nationwide cross-sectional study was to evaluate the Health Related Quality of Life (HRQL) of young people that have survived childhood cancer at least four years after cancer diagnosis. The study aims were (1) to increase knowledge and understanding about the relationship between childhood cancer and its treatment and HRQL of childhood cancer survivors and (2) to identify survivors who need and could benefit from ongoing long-term follow-up, as well as (3) to identify what kind of aftercare the childhood cancer survivors will possibly need. HRQL and fatigue of currently still young survivors of extracranial childhood malignancies were evaluated with self-reports and parent proxy reports. HRQL was measured with age-appropriate generic instruments: PedsQL™, SF-36, 15D, 16D and 17D. Fatigue for children and adolescents aged below 18 years was measured with the PedsQL™ Multidimensional Fatigue Scale Finnish version. PedsQL™ parent-proxy and the PedsQL™ Multidimensional Fatigue Scale Parentproxy instruments were used to assess the perception of the parents on HRQL and fatigue of their children and adolescents. Postal-survey questionnaires were mailed to 852 childhood cancer survivors aged 11-27 years and their randomly selected gender-, age and living-place matched controls, as well as under 18-year-old children´s parents. A total of 474 survivors, 595 controls, 209 survivor’s parent and 253 control’s parent replied. The mean age of survivors at the time of the study was 18.4 years. The mean length of survival was 12.3 years, and the mean age at diagnosis 5.5 years. The most of the Finnish childhood cancer survivors evaluated that their HRQL as good. Survivors rated their HRQL equal or higher than their controls. The only dimension where the survivors scored poorer than the controls was the 15D mobility dimension. Survivors of childhood cancer did not suffer from significant fatigue. There were subgroups of childhood cancer survivors who had poorer level of HRQL, and suffered from fatigue more than the reference group. The demographic factors that associated with poorer HRQL were female gender, greater weight, living alone, need of remedial education, an additional non-cancer diagnosis, survivors with siblings, and self-reported unhappiness. Disease-related factors that associated with poorer HRQL were higher age at the time of diagnosis, the diagnosis of Wilms tumor, neuroblastoma, or osteosarcoma, and treatment with stem cell transplantation. The factors associated with more fatigue in survivors were male gender, older age at evaluation, the need of remedial education at school, lower overall average grade in the latest school marks report, length of survival more than 10 years, lower HRQL-scores, and a sarcoma diagnosis. However, all the used demographic and disease related factors explained only about one third of the variation in the HRQL scores. In open questions, the survivors were most worried about their physical health, but were also worried about their mental health, cancer inheritance, late-effects, and fertility and relapse issues. It seems that there are subgroups of survivors who need and could benefit from ongoing long-term follow-up. In the future, the survivors of childhood cancer need more information about their physical and mental health, as well as on their cancer inheritance, possible late-effects including fertility issues, and on the risk of relapse.

Nova técnica de alimentação enteral em fístulas esôfago-jejunais

BACKGROUND: A new procedure for the treatment of esophageal fistula, mainly associated to the ebb esophagojejunal in patients submitted to the total gastrectomy and reconstruction with loop jejunal Rouxen-Y anastomosis is present. METHODS: The method is based in the use of "probe standard enteral prolongated with drain to laminate adapted in extremity", which results in advanced positioning inside the jejunum, making the administration of enteral nutrition possible and impeding ebb esophagojejunal. RESULTS: The authors discuss the theoretical advantages of the procedure and they suggest that the treatment of esofagic fistula with probe prolonged enteral would be suitable in the treatment of the fistula esophagojejunal by preventing the ebb esophagojejunal, which would result in smaller period of duration of the fistula esophagojejunal and it would prevent the high mortality rate. CONCLUSIONS: Preliminary studies demonstrated that this is a technically easy, low cost procedure through the endoscopic use. A prospective evaluation for morbility and mortality related to the method is needed.

Sodanuhkaa ja rauhantakeita

11 x 17 cm

Antimicrobial resistance and R-plasmid in Salmonella spp from swine and abattoir environments

Salmonella serovars isolated from swine are of particular interest not only because of the pathogenic potential for this animal species, but also due to its relevance with regard to public health. On basis of the profile of resistance to antimicrobials, 13 Salmonella strains were selected which belonged to the serovars Muenster (7), Derby (4), Typhimurium (1), and Braenderup (1). They were isolated from healthy swine as well as from the abattoir environment in the state of Rio de Janeiro. All strains of Salmonella were subjected to bacterial conjugation, and the E. coli K12 Nal r Lac+ F standard strain was used as receptor, with the purpose to verify the ability to transfer the resistance marks. Gene transfer phenomenon was detected in seven strains, and except SalmonellaTyphimurium which transconjugated to Sm, Tc and Su, the remaining ones were characterized by transferring mark Su only. By plasmidial analysis of strains used and their respective transconjugants, 63 Kb plasmid was found, which was probably related to S. Typhimurium resistance.

Gross anatomical features of the oropharyngeal cavity of the ostrich (Struthio camelus)

Most descriptions of the ostrich oropharynx and oesophagus are superficial and supply little meaningful morphological data. The aim of this investigation is describe the ostrich oropharingeal cavity, in order to supply the deficiency of macroscopic data about this important animal. Five heads of 12 to 14-month-old ostriches of either sex were anatomically dissected to expose the oropharynx. The ostrich oropharynx was "bell-shaped" composed by the maxillary and mandibular ramphoteca. The roof and floor presented two distinct regions different in colour of the mucosa. The rostral region was pale pink contrasting to creamy-pink coloured caudal region. The median longitudinal ridge extended rostrally from the apex of the choana to the tip of the beak in the roof and it is clearly more prominent and rigid than the homolog in the floor that appeared thin and stretched rostrally, continuing caudally surrounding the tongue and the laryngeal mound eventually merging with the oesophageal mucosa. The floor was formed by the interramal region, tongue and laryngeal mound containing shield-shaped glottis. It can be concluded that the present study, in addition to confirming the basic features of the oropharynx previously described for the ostrich, clarified the contradictory information presented in the literature and also provided new, unreported morphological data, some of which may be important when studying nutrition and health in these birds.

Avian pox virus infection in a common barn owl (Tyto alba) in southern Brazil

A young common barn owl (Tyto alba) was referred to the Núcleo de Reabilitação da Fauna Silvestre (Nurfs), Federal University of Pelotas (UFPel), after been found in a barn of a brick factory in the urban area of Pelotas, Rio Grande do Sul, Brazil. The bird was apathic, weak and with crusty lesions in the featherless areas (eyes, beak, legs), and died soon after arrival at Nurfs. Necropsy and histopathological examination of the lesions were carried out. The hyperplasia and hypertrophy of the cutaneous lesions, several eosinophilic intracyto-plasmic inclusion bodies in epithelial cells (Bollinger bodies), as well as particles characteristic of poxvirus, observed by electronic microscopy, confirmed the infection by avian poxvirus, what highlights the importance of Tyto alba as carrier of the virus in the wild.

Detection and characterization of fibropapilloma associated herpesvirus of marine turtles in Rio Grande do Sul, Brazil

Fibropapillomatosis (FP) is a benign tumoral disease that affects sea turtles, hampering movement, sight and feeding, ultimately leading to death. In Brazil, the disease was described for the first time in 1986. Research suggests the involvement of a herpesvirus in association with environmental and genetic factors as causal agents of FP. The objective of the present study was to detect and characterize this herpesvirus in sea turtles living in the coast of state Rio Grande do Sul (RS), Brazil. From October 2008 to July 2010, 14 turtles were observed between the beaches of Torres and Tavares, of which 11 were green turtles (Chelonia mydas) and 3 were loggerhead turtles (Caretta caretta). All turtles were young and mean curved carapace length was 37.71±7.82cm, and varied from 31 to 55cm. Only one green turtle presented a 1cm, papillary, pigmented fibropapilloma. Skin and fibropapilloma samples were analyzed by conventional and real time PCR assays to detect and quantify herpesvirus. All skin samples were negative, though the fibropapilloma specimen was positive in both tests. Viral load was 9,917.04 copies of viral genome per milligram of tissue. The DNA fragment amplified from the fibropapilloma sample was sequenced and allocated in the Atlantic phylogeographic group. This study reports the first molecular characterization of herpesvirus associated with fibropapilloma in turtles from the coast of RS.

Gross morphology of rhea oropharyngeal cavity

The rhea (Rhea americana americana) is an american bird belonging to Ratite's family. Studies related to its morphology are still scarce. This study aims to describe the macroscopic structures of the oropharyngeal cavity. Five heads (2 to 6 months old) formalin preserved were anatomically dissected to expose the oropharynx. The oropharynx of the rhea was "bell-shaped" composed by the maxillary and mandibular rhamphotheca. The roof and floor presented two distinct regions different in colour of the mucosa. The rostral region was pale pink contrasting to grey coloured caudal region. The median longitudinal ridge extended rostrally from the apex of the choana to the tip of the beak in the roof and it is clearly more prominent and rigid than the homolog in the floor that appeared thin and stretched merely along the rostral portion of the regio interramalis. The floor was formed by the interramal region, (regio interramalis) tongue and laryngeal mound containing glove-shaped glottis. This study confirmed the basic morphology of the oropharinx of the rhea. However, important morphological information not previously described is highlighted and contradictory information present in the literature is clarified.

Anti-predator adaptations in aquatic environments

Predation is an important selective force that has led to the evolution of a variety of fascinating anti-predator adaptations, such as many types of protective coloration and prey behaviours. Because the evolution of life has begun in the aquatic environment and many anti-predator adaptations are found already in relative primitive taxa, it is likely that many of these adaptations evolved initially in the aquatic environment. Yet, there has been surprisingly little research on the mechanisms and function of antipredator adaptations in aquatic systems. To understand the function of anti-predator adaptations and natural selection imposed on prey appearance and behaviour, I have investigated how protective coloration can be used, either as such or together with behavioural adaptations, to manipulate predator behaviour and decrease predation risk. To this end I conducted a series of behaviour ecological laboratory experiments in which I manipulated the visual appearance of artificial backgrounds and prey items. In paper I of this thesis, I investigated background choice as an anti-predator strategy, by observing the habitat choice of the least killifish (Heterandria formosa) between pairs of artificial backgrounds, both in the presence and absence of predation threat. It has been suggested that prey could decrease their risk of being detected by predators either by preferring backgrounds into which they blend or by preferring visually complex backgrounds. The least killifish preferred a background that matched their patterning to a background that mismatched it, showing that they are able to respond to cues of visual similarity between their colour pattern and the surrounding environment. Interestingly however, in female least killifish visual complexity of the background was a more important cue for habitat safety and may override or act together with background matching when searching for a safe habitat. It is possible that in females, preference for visually complex backgrounds is associated with lower opportunity costs than preference for matching backgrounds would be. Generally, the least killifish showed stronger preference while under predation threat, indicating that their background choice behaviour is an antipredator adaptation. Many aquatic prey species have eyespots, which are colour patterns that consist of roughly concentric rings and have received their name because they for humans often resemble the vertebrate eye. I investigated the anti-predator function of eyespots against predation by fish in papers II, III and IV. Some eyespots have been suggested to benefit prey by diverting the strikes of predators away from vital parts of the prey body or towards a direction that facilitates prey escape. Although proposed over a century ago, the divertive effect of eyespots has proven to be difficult to show experimentally. In papers II and III, I tested for divertive effect of eyespots towards attacking fish by presenting artificial prey with eyespots to laboratory reared three-spined sticklebacks (Gasterosteus aculeatus). I found that eyespots strongly influenced the behaviour of attacking sticklebacks and effectively drew their strikes towards the eyespots. To further investigate this divertive effect and whether the specific shape of eyespots is important for it, I tested in paper III the response of fish also to other markings than eyespots. I found that eyespots were generally more effective in diverting the first strikes of attacking fish compared to other prey markings. My findings suggest that the common occurrence of eyespots in aquatic prey species can at least partly be explained by the divertive effect of the eyespot shape, possibly together with the relative simple developmental mechanisms underlying circular colour patterns. An eyebar is a stripe that runs through the eye, and this pattern has been suggested to obscure the real eyes of the prey by visually blending parts of the eyes and head of the prey and by creating false edges. In paper III, I show that an eyebar effectively disrupts an eyelike shape. This suggests that eyebars provide an effective way to conceal the eyes and consequently obstruct detection and recognition of prey. This experiment also demonstrates that through concealment of the eyes, eyebars could be used to enhance the divertive effect of eyespots, which can explain the common occurrence of eyebars in many species of fish that have eyespots. Larger eyespots have been shown to intimidate some terrestrial predators, such as passerine birds, either because they resemble the eyes of the predator’s own enemy or because highly salient features may have an intimidating effect. In papers II and IV, I investigated whether the occurrence of eyespots in some aquatic prey could be explained by their intimidating effect predatory fish. In paper IV, I also investigated the reason for the intimidating effect of eyelike prey marks. In paper II, I found no clear intimidating effect of eyespots, whereas in paper IV, using a different approach, I found that sticklebacks hesitated to attack towards eyelike but not towards non-eyelike marks. Importantly, paper IV therefore presents the first rigorous evidence for the idea that eye mimicry, and not merely conspicuousness, underlies the intimidating effect. It also showed that the hesitation shown by fish towards eyelike marks is partly an innate response that is reinforced by encounters with predators. Collectively, this thesis shows that prey colour pattern and the visual appearance of the habitat influence the behaviour of fish. The results demonstrate that protective coloration provides numerous distinctive ways for aquatic prey to escape predation. Thus, visual perception and behaviour of fish are important factors shaping the appearance and behaviours of aquatic prey.

Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases

Induction of apoptosis by tumor necrosis factor (TNF) is modulated by changes in the expression and activity of several cell cycle regulatory proteins. We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 µM), a specific inhibitor of cyclin-dependent kinases (CDK) with high selectivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin condensation and ladder pattern of DNA fragmentation in these cell lines. The TNF-induced apoptosis events were increased in the presence of butyrolactone. In vitro phosphorylation assays for exogenous histone H1 and endogenous retinoblastoma protein (pRb) in the total cell lysates showed that treatment with both TNF and butyrolactone inhibited the histone H1 kinase (WEHI, L929 and HeLa) and pRb kinase (WEHI) activities of CDKs, as compared with the controls. The role of proteases in the TNF and butyrolactone-induced apoptosis was evaluated by comparing the number and expression of polypeptides in the cell lysates by gel electrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular protein bands in the region between 40-200 kDa, and the 110- 90- and 50-kDa proteins were identified as the major substrates, whose degradation was remarkably increased by the treatments. Interestingly, the loss of several cellular protein bands was associated with the marked accumulation of two proteins apparently of 60 and 70 kDa, which may be cleavage products of one or more proteins. These findings link the decrease of cyclin-dependent kinase activities to the increase of protease activities within the growth arrest and apoptosis pathways induced by TNF.

Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

Observatiunculae circa artem antiquarum gentium celebriorum numismata adornandi, in specie numi Sabinae expositio

Variantti B.

Trilling race : the political economy of racialised visual-aural encounters

This thesis investigates the matter of race in the context of Finnish language acquisition among adult migrants in Finland. Here matter denotes both the materiality of race and how race comes to matter. Drawing primarily on an auto/ethno/graphic account of learning the Finnish language as a participant in the Finnish for foreigners classes, this thesis problematises the ontology and epistemology of race, i.e., what race is, how it is known, and what an engagement with race entails. Taking cues from the bodily practices of learning the Finnish trill or the rolling r, this study proposes a notion of “trilling race” and argues for an onto-epistemological dis/continuity that marks race’s arrival. The notion of dis/continuity reworks the distinction between continuity and discontinuity, and asks about the how of the arrival of any identity, the where, and the when. In so doing, an analysis of “trilling race” engages with one of the major problematics that has exercised much critical attention, namely: how to read race differently. That is, to rethink the conundrum of the need to counter “representational weight” (Puar 2007, 191) of race on the one hand, and to account for the racialised lived realities on the other. The link between a study of the phenomenon of host country language acquisition and an examination of the question of race is not as obvious as it might seem. For example, what does the argument that the process of language learning is racialised actually imply? Does it mean that race, as a process of racialisation or an ongoing configuration of sets of power relations, exerts force from an outside on the otherwise neutral process of learning the host country language? Or does it mean that race, as an identity category, presents as among the analytical perspectives, along with gender and class for instance, of the phenomenon of host country language acquisition? With these questions in mind, and to foreground the examination of the question of race in the context of Finnish language acquisition among adult migrants, this thesis opens with a discussion of the art installation Finnexia by Lisa Erdman. Finnexia is a fictitious drug said to facilitate Finnish language learning through accelerating the cognitive learning process and reducing the anxiety of speaking the Finnish language. Not only does the Finnexia installation make visible the ways in which the lack of skill in Finnish is fgured as the threshold – a border that separates the inside from the outside – to integration, but also, and importantly, it raises questions about the nature of difference, and the process of differentiation that separates the individual from the social, fact from fiction, nature from culture. These puzzles animate much of the analysis in this dissertation. These concerns continue to be addressed in the rest of part one. Whereas chapter two offers a reconsideration of the ambiguities of ethnisme/ethnicity and race, chapter three dilates on the methodological implications of a conception of the dis/continuity of race. Part two focuses on the matter of race and examines the political economy of visual-aural encounters, whereas part three shifts the focus and rethinks the possibilities and limitations of transforming racialised and normative constraints. Taking up these particular problematics, this thesis as a whole argues that race trills itself: its identity/difference is simultaneously made possible and impossible.

Síndrome de quebra-nozes (nutcracker) em uma mulher jovem em investigação por quadro de hematúria recorrente: um relato de caso

INTRODUÇÃO: A síndrome de quebra-nozes (SQN) é uma condição na qual há compressão da veia renal esquerda pela aorta e a artéria mesentérica superior, associada a uma sintomatologia característica, porém pouco específica. RELATO DO CASO: Trata-se de uma paciente de 37 anos, apresentando hematúria macroscópica intermitente sem etiologia estabelecida até então. Durante a investigação, uma angiotomografia evidenciou compressão da veia renal esquerda no seu trajeto entre a artéria mesentérica superior e aorta abdominal, associada a importante dilatação de veia ovariana esquerda. DISCUSSÃO: A síndrome desencadeada pela compressão da veia renal esquerda pela aorta e artéria mesentérica superior apresenta prevalência provavelmente aumentada no sexo feminino e postula-se que seja associada a situações particulares, como a nefroptose e a escassez de gordura retroperitoneal. Hematúria, sinal típico, teria como causa a ruptura do fino septo que separa as veias do sistema coletor urinário. Vários exames de imagem podem auxiliar no diagnóstico. O tipo de tratamento que deve ser adotado ainda é controverso, existindo diversas opções cirúrgicas, como o uso de stent intravascular e a conduta conservadora. De diagnóstico costumeiramente tardio, essa síndrome deve ser incluída na investigação de pacientes com quadro de hematúria a esclarecer.