The Innate immune system is the first line of defense against invading microbial pathogens. It is a fast-acting and non-antigen-specific defense system, which employs germline encoded surveillance systems capable of responding to a broad-spectrum of pathogens. The innate immune system involves a variety of immune cells, which express different profiles of surveillance or detection receptors. Upon sensing pathogens, these receptors trigger cell signalling to turn on transcription of inflammatory cytokines, chemokines, anti-microbial peptides and type I Interferons. These effectors have direct effects on the control of pathogen load and also activate the adaptive immune system, which is ultimately required to clear infections. The type I interferons (IFNs) are the principal cytokines strongly induced during infection with viruses and are required for direct control of viral replication and modulation of cells of the adaptive immune response. The signalling pathways induced in order to activate type I IFNs are dependent on the interferon regulatory factors (IRFs). Striving for survival, microbes have evolved various strategies to subvert/impair these critical defense molecules. In this thesis work, I have used Human Metapneumoviruses (HMPVs), a relatively newly described family of paramyxoviruses as model viruses to explore the role of pattern recognition receptors (PRRs) and the IRF family of transcription factors in the innate immune response. These studies revealed that the recognition of HMPV viral pathogen-associated molecular patterns (PAMPs) by immune cells is different in different cell types. Retinoic acid-inducible gene-I (RIG-I), a cytosolic RNA helicases senses HMPV-A1 virus for triggering type I IFN activation by detecting its 5’- triphosphate viral RNA in most human cells, including cell lines and primary monocytes. An exception to these findings was plasmacytoid dendritic cells (PDCs), where Toll-like receptor (TLR)-7 is the primary sensor involved in detecting HMPV viruses. By comparing the innate immune response to two HMPV strains, we found that these two closely related strains had very different immune stimulatory capabilities. HMPV-1A strain triggered type I IFNs in monocytes, PDCs and cells of epithelial origin. In contrast, a related strain, HMPV-B1 failed to trigger IFN responses in most cell types. Our studies suggested that the phosphoprotein (P) of HMPV-B1 could prevent the viral RNA from being detected by RIG-I, thus inhibiting the induction of type I IFN production in most cell type examined. This finding adds to our understanding of the mechanisms by which viruses are sensed by surveillance receptors and also unveils new means of viral evasion of host immune responses. Although IRFs are extensively studied for their role in regulating type I IFN activation, especially in TLR and RIG-I like receptor (RLR) signalling pathways upon viral infection, a clear understanding of how this family of transcription factors contributes to anti-viral immunity was lacking. Studies conducted as part of this thesis revealed that in addition to IRF3 and IRF7, which play a central role in anti-viral immunity downstream of most PRRs (e.g. TLRs, RLRs, DNA sensors), the related factor IRF5 was also an important component of innate anti-viral defenses. Using IRF5-deficient mice we studied in detail the role of IRF5 in coordinating antiviral defenses by examining its involvement in signalling downstream of TLRs. These studies led us to examine the role of IRF5 in the regulation of type I IFNs as well as inflammatory cytokines in different cell types. While most TLRs that induced IFNβ showed normal responses in IRF5-deficient mice, CpG-B-induced IFNβ production in CD11c+CDCs isolated from mouse spleen but not those generated in vitro from bone marrow required IRF5. This was in contrast to responses with lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid (polyIC), ligands for TLR4 and 3, respectively. Moreover, we found that in contrast to IRF3 and/or IRF7, IRF5 was important in coordinating the expression of inflammatory cytokines such as TNFα downstream of some TLRs. In addition to our studies to examine the requirement for IRF5 in TLR signaling, we also showed that muramyl peptide (MDP) from Mycobacterium tuberculosis (Mtb) could activate type I IFNs via IRF5. This was the first evidence linking IRF5 to a non-TLR-driven pathway. IRF5 activation in this case was downstream of a novel nucleotide-binding oligomerization domain containing (NOD)-2/receptor-interacting serine-threonine kinase (RIP)-2 signaling pathway. Collectively, the studies outlined in this thesis have assisted in providing a framework to understand the role of TLRs, RLRs and IRFs in the immune response to paramyxoviruses and have unveiled new mechanisms of activation of the IRFs as well as new mechanisms by which pathogens subvert or evade these important innate defense mechanisms.

Tese(doutorado)-Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2011. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-06-23T16:27:42Z No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-07-27T20:22:46Z (GMT) No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) / Made available in DSpace on 2012-07-27T20:22:46Z (GMT). No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) Previous issue date: 2011 / A aquicultura tem crescido significativamente nas últimas décadas devido ao aumento da demanda de pescado no mundo e à estagnação do setor pesqueiro. Porém, este crescimento depende do desenvolvimento de novos pacotes tecnológicos que visem o aumento da produtividade. Uma alternativa é a manipulação genética (transgenia), sendo que o hormônio do crescimento (GH) tem sido o principal alvo das pesquisas com peixes transgênicos. Entretanto, está comprovado que o excesso de GH acarreta uma série de efeitos adversos devido a sua ação pleiotrópica. A ativação do eixo somatotrófico de forma tecido-específica e independente do excesso de hormônio circulante pode contornar estes problemas. Neste contexto, o objetivo desta tese foi superexpressar o gene do receptor do GH (GHR) no tecido muscular esquelético do zebrafish (Danio rerio) e estudar os efeitos desta manipulação sobre os mecanismos envolvidos na regulação gênica do crescimento muscular. A linhagem transgênica estável obtida expressa o GHR especificamente no tecido muscular 100 vezes mais do que os não transgênicos. Estes transgênicos não apresentaram aumento significativo no crescimento, provavelmente devido à queda na expressão do fator de crescimento tipo insulina I (IGF-I). Esta queda foi, provavelmente, relacionada à ação dos principais moduladores da sinalização do GH (SOCS1 e 3), os quais apresentaram-se aumentados nos transgênicos. Ainda, foi observada uma queda na expressão das principais proteínas musculares estruturais (Acta1, myhc4 e mylz2), o que explica a ausência de hipertrofia nos transgênicos. Por outro lado, o aumento na expressão dos principais fatores reguladores miogênicos (myod, myf5 e myog) explica a hiperplasia observada nas análises histológicas. Para verificar como a superexpressão do GHR ativou a transcrição dos fatores reguladores miogênicos (MRFs) e, por consequência a hiperplasia, foram estudados os possíveis mecanismos envolvidos neste processo. Dentre estes, tanto a via proliferativa (MEK/ERK) quanto à via relacionada com a síntese protéica (PI3K/Akt), não tiveram alteração na expressão de seus genes. Entretanto, foi observado aumento na expressão das proteínas de transporte para o núcleo (importinas 1, 3 e 1), podendo-se concluir que a ativação dos MRFs está relacionada ao transporte do GHR para o núcleo das células musculares. Desta forma, pode se concluir que hiperplasia e hipertrofia seguem duas vias de sinalização intracelular distintas, ambas desencadeadas pelo GH, mas reguladas por mecanismos diferentes. / Aquiculture practice has been significantly increasing during the last decades due to the fish rising demand and to fishery activity stagnation. However, such increase depends on new technological packages development aiming to productivity rises. Genetic manipulation is an alternative, once growth hormone (GH) has been the main target on transgenic fish researches. Nevertheless, it has been proved that GH excess leads to many adverse effects due to its pleiotropic action. The somatotropic axis activation in a tissue-specific manner and independent on the circulating hormone excess may bypass these problems. Following these ideas, the present thesis objective was overexpressing GH receptor’s gene (GHR), in zebrafish (Danio rerio) skeletal muscular tissue, and studying such manipulation effects over the mechanisms involved in muscular growth gene regulation. The stable transgenic lineage obtained expresses GHR, specifically in muscular tissue, 100 times more than non-transgenic. These transgenic did not present significant growth, possibly due to a gene expression fall in insulin-like growth factor I (IGF-I). The mentioned fall is, probably, related to GH main signaling modulators (SOCS1 and 3) action, which were increased in transgenic. Also, a gene expression fall from the main structural muscle proteins (Acta1, myhc4 and mylz2) was observed, explaining the transgenic hypertrophy absence. However, the main myogenic regulatory factors (myod, myf5 and myog) expression rising explains the observed hyperplasia in histological analysis. Intending to verify how GHR overexpression has activated the myogenic regulatory factors (MRFs) transcription and, consequently, the hyperplasia, the mechanisms possibly involved in this process were studied. Among these, even the proliferative pathway (MEK/ERK) or the pathway related to protein synthesis (PI3K/Akt), did not presented gene expression altering. However, a gene expression rise in transporting proteins into nucleus (importins 1, 3 and 1) was observed, which may be understood as a correlation between MRFs activation and GHR transport into muscular cell’s nucleus. Therefore, it may be understood that hyperplasia and hypertrophy follow two distinct intracellular signaling pathways, both triggered by GH, but regulated by different mechanisms. These data may be important for aquiculture new transgenic lineages development.

Orientador: Maeli Dal Pai Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:12:18Z (GMT). No. of bitstreams: 1 Souza_RodrigoWagnerAlves_M.pdf: 2186081 bytes, checksum: 2a1578159471f249151f3b90ca0b76fb (MD5) Previous issue date: 2010 / Resumo: O treinamento físico pode promover adaptações benéficas ao músculo esquelético. Entretanto, o treinamento de alta intensidade associado com um tempo insuficiente de recuperação, similar às condições de sobretreinamento, pode ocasionar efeitos prejudiciais. Os fatores reguladores miogênicos (MRFs) e o fator de crescimento IGF-1 são importantes reguladores da massa muscular no treinamento físico. Neste contexto, testamos a hipótese que o treinamento de alta intensidade com curto tempo de recuperação, poderia influenciar a morfologia, as isoformas da cadeia pesada de miosina (MHC), e a expressão dos MRFs MyoD e Miogenina e do IGF-1, no músculo esquelético de ratos. Ratos Wistar machos (200-250g) foram divididos em 4 grupos: treinado 8 semanas (T8), controle 8 semanas (C8), treinado 12 semanas (T12) e controle 12 semanas (C12). Os grupos T8 e T12 realizaram um programa de treinamento resistido de alta intensidade (5 dias/semana), envolvendo sessões de saltos em uma cuba contendo água. Ao término de cada período, os animais foram sacrificados e o músculo plantar retirado e submetido às análises morfológica e histoquímica, análises das MHCs e expressão gênica da MyoD, Miogenina e IGF-1. Do início ao final do experimento todos os grupos aumentaram o peso corporal, no entanto, o grupo T12 foi estatisticamente menor em relação ao C12. Com relação à área de secção transversal, observou-se uma redução das fibras IIC e IIAD no grupo T8 e IIA e IID no grupo T12 em relação aos seus controles. O grupo treinado por 12 semanas apresentou um aumento da frequência das fibras IIBD e redução nas frequências das fibras I, IIA e IID, em relação ao grupo controle; esses dados ainda foram corroborados pela redução das isoformas MHCI e MHCIIa e aumento da MHCIIb. A MHCIId não mostrou diferença significativa. A expressão gênica do grupo T12 apontou uma diminuição da MyoD e um aumento do IGF-1 comparado com o grupo C12; já, a expressão da Miogenina foi semelhante entre os grupos. Estes dados mostram que o modelo utilizado, semelhante às condições do sobretreinamento, promoveu a atrofia muscular e a transição das fibras musculares para uma atividade contrátil mais rápida. Estes fatos podem estar associados a uma menor atividade das células satélites. Em adição, o aumento da expressão do IGF-1, decorrente do treinamento, pode ter ocorrido na tentativa de prevenir o processo atrófico. / Abstract: Physical training can promote beneficial changes in skeletal muscle. However, the high-intensity resistance training associated with insufficient recovery time may cause harmful effects. Myogenic regulatory factors (MRFs) and the growth factor IGF-1 are important mediators of muscle mass during physical training. In this context, we tested the hypothesis that high-intensity resistance training with short recovery time, similar to overtraining conditions, could influence the morphology, the myosin heavy chain (MHC) isoforms and the expression of MRFs MyoD and myogenin, and IGF-1 in skeletal muscle of rats. Male Wistar rats (200-250 g) were divided into 4 groups: trained 8 weeks (T8), control 8 weeks (C8), trained 12 weeks (T12) and control 12 weeks (C12). T8 and T12 groups were subjected to a high-intesnsity resistance training program (5 days/week), involving jumps sessions into water, carrying progressive overload equivalent to percentage of body weight. At the end of each period the animals were sacrificed and the plantaris muscles were removed and submited to morphological and histochemical analysis, myosin heavy chain (MHC) analysis and the gene expression of MyoD, Myogenin and IGF-1. From beginning to end of the experiment all groups increased body weight, however, in T12 body weight was lower compared to the C12. Regarding the cross-sectional area, there was a significant reduction of the IIC fibers and IIAD in T8 group and IIA and IID in T12 compared to their controls. The group trained by 12 weeks showed an increase in the IIBD, accompanied by a reduction in the I, IIA and IID muscle fibers frequency, compared to control group; these data have been corroborated by the reduction of MHCI and MHCIIa isoforms and increased of MHCIIb isoform. The MHCIId showed no significant differences. The gene expression of the T12 group showed a decrease in MyoD and an increase in IGF-1 compared with the C12 group; already, the expression of Myogenin was similar between groups. These data show that the model used, similar to the conditions of overtraining, promoted muscular atrophy and muscle fiber transition to a faster contractile activity. These facts may be associated with a lower activity of satellite cells. In addition, increased expression of IGF-1, due to training, may have occurred in an attempt to prevent the atrophic process. / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Chez les scolytes (Curculionidae : Scolytinae) réalisant des pullulations éruptives, les processus conditionnant l’intensité et la durée des pullulations sont généralement mal connus. Nous avons étudié trois facteurs susceptibles de moduler les pullulations d’Ips sexdentatus (Börner) : la compétition intraspécifique, les ennemis naturels et la température. Les densités de colonisation et le seuil critique de densité d’attaques sur arbres ont été estimés lors d’une pullulation, et les effets de ces densités sur la productivité et la fitness des insectes ont été mesurés en laboratoire. L’entomofaune associée au scolyte a été étudiée dans des peuplements de pins présentant différents niveaux de dégâts de l’insecte. Les effets thermiques ont été estimés via des élevages à différentes températures. Les densités de colonisation sur arbres, ou celle correspondant à la valeur du seuil critique, ont fortement affecté la productivité et la fitness d’I. sexdentatus. L’entomofaune associée était peu sensible aux niveaux de population du scolyte, dont la productivité a cependant été réduite par la durée d’exposition aux ennemis naturels. L’estimation des besoins thermiques de l’espèce a permis de calculer qu’un réchauffement de 1°C augmenterait les effectifs et permettrait l’apparition d’une troisième génération annuelle, tout en réduisant la fitness des insectes. La compétition intraspécifique serait un facteur critique de régulation des pullulations d’I. sexdentatus alors que les ennemis naturels joueraient un rôle plus secondaire. Une élévation de température pourrait aggraver les pullulations mais aussi réduire la fitness des insectes et augmenter la compétition intraspécifique. / For eruptive bark beetles (Curculionidae: Scolytinae), the processes affecting the intensity and duration of outbreaks are generally poorly known. We have investigated three factors that may affect the population dynamics of Ips sexdentatus (Börner), namely the intraspecific competition, the natural enemies and the temperature. Colonization densities and the critical threshold of attack densities on trees have been estimated during an outbreak. How such densities affected the productivity and fitness of the beetles has been evaluated under laboratory conditions. The insect community associated with the bark beetle has been characterized in pine stands exhibiting different damage levels. To assess the thermal effects, the insects have been reared at different temperatures. Colonization densities on trees, or equivalent to the critical threshold of attacks, dramatically affected both the productivity and fitness of I. sexdentatus. The associated fauna was loosely related to the population levels of the bark beetle, although the duration of the exposure to the natural enemies affected its productivity. The assessment of thermal requirements allowed calculating that an average warming of 1°C during the activity period would increase the population levels and number of generations per year, but also decrease the beetles’ fitness. Intraspecific competition is probably a critical regulating factor for I. sexdentatus, while natural enemies would rather play a secondary role. A temperature increase could aggravate the outbreaks, but this could be counterbalanced by a fitness reduction and an increase of intraspecific competition.

Changes in cis- and trans-regulatory elements are among the prime sources of genetic and phenotypical variation at species level. The introduction of cis- and trans- regulatory variation has played important roles in driving diversity, phenotypical differentiation, and evolution of humans. Therefore, variation that occurs on cis- and trans- regulatory elements becomes imperative to better understanding of human genetic diversity and its evolution. In this research, around 3360 gene regulatory factors (GRF) from the human genome were catalogued. This catalog includes genes that code for proteins that perform gene regulatory activities such DNA-depending transcription, RNA polymerase II transcription cofactor and co-repressor activity, chromatin binding and remodeling, among other 218 regulatory functions. This GRF catalog allowed us to initially explore how some GRF genes have evolved in humans, archaic humans (Neandertal and Denisovan) and non-human primate species. We discussed the likely phenotypical and medical effects that evolutionary changes in GRF genes may have introduced into the human genome; for instance, traits associated to speech and language capabilities, genomic recombination hotspots, diseases, among others. By using genome-wide datasets, we additionally looked for GRFs likely to be candidates for positive selection in three human populations: Utah Residents with Northern and Western Ancestry (CEU), Han Chinese in Beijing (CHB), and Yoruba in Ibadan (YRI). As result, we produced a set of candidates that gathers genes that may have contributed in shaping the phenotypical diversity currently observed in these populations; for instance, by introducing regulatory diversity at population-specific level. We additionally identified six GRF classes enriched for genes located in regions that are likely candidates for positive selection at population specific level. We found that out of the 41 DNA-binding GRF classes classified so far, six groups exhibited enrichment for genes located on regions that may have been under positive selection: C2H2 zinc finger, KRAB-ZNF zinc finger, Homeo domain, Tryptophan cluster, Fork head/winged helix and, and High-mobility HMG domain. We additionally identified three KRAB-ZNF gene clusters, in the chromosomes one, three, and 16, for the Asian population that exhibit regions with extended haplotype homozygosity EHH (larger than 100 kb). This EHH suggests that these regions have undergone positive selection in CHB population. Finally, considering that a representative fraction of the phenotypic diversity observed between humans and its closely related species are likely explained by changes in cis-regulatory elements (CREs), we investigated putative binding sites for the transcription factor GABPa. Using ChIP-Seq data generated from a human cell line (HEK293T), 11,619 putative GABPa CREs were found, Out of which 224 are putative human-specific. To experimentally validate the transcriptional activity of these human-specific CREs, reporter gene essays and knock-down experiments were performed. Our results supported the functionality of these human-specific GABPa CREs and suggest that at least 1,215 genes are primary targets of GABPa. Finally, further analyses depict scenarios that put together transcriptional regulation by GABPa and the evolution of particular human traits; for instance, cognitive abilities, breast morphology, lipids and glucose metabolic pathways, among others.

Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders. Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease. Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.

CD8+ T cell differentiation is a complex process that requires integration of signals from the TCR, co-stimulatory molecules and cytokines. Ligation of the peptide-MHC complex with the cognate TCR initiates a downstream signaling cascade of which the IL-2 inducible T-cell kinase (ITK) is a key component. Loss of ITK results in a measured reduction in T cell activation. Consequently, Itk deficient mice have defects in thymic selection, CD8+ T cell expansion and differentiation in response to virus infections, and generate a unique population of innate-like CD8+ T cells. The mechanisms that translate TCR and ITK-derived signals into distinct gene transcription programs that regulate CD8+ T cell differentiation are not defined. Our microarray screen identified IRF4 as a potential transcription factor mediating the differentiation of innate-like T cells, and antiviral CD8+ T cell in response to acute and chronic LCMV infections. Innate-like CD8+ T cells are characterized by their high expression of CD44, CD122, CXCR3, and the transcription factor Eomesodermin (Eomes). One component of this altered development is a non-CD8+ T cell-intrinsic role for IL-4. We show that IRF4 expression is induced upon TCR signaling and is dependent on ITK activity. In contrast to WT cells, activation of IRF4-deficient CD8+ T cells leads to rapid and robust expression of Eomes, which is further enhanced by IL-4 stimulation. These data indicate that ITK signaling promotes IRF4 up-regulation following CD8+ T cell activation and that this signaling xii pathway normally suppresses Eomes expression, thereby regulating the differentiation pathway of CD8+ T cells. ITK deficient mice also have reduced expansion of CD8+ T cells in response to acute LCMV infections. We show that IRF4 is transiently upregulated to differing levels in murine CD8+ T cells, based on the strength of TCR signaling. In turn, IRF4 controls the magnitude of the CD8+ T cell response to acute virus infection in a dose-dependent manner. Furthermore, the expression of key transcription factors such as T cell factor 1 and Eomesodermin are highly sensitive to graded levels of IRF4. In contrast, T-bet expression is less dependent on IRF4 levels and is influenced by the nature of the infection. These data indicate that IRF4 is a key component that translates the strength of TCR signaling into a graded response of virus-specific CD8+ T cells. The data from these studies indicated a pivotal role of IRF4 in regulating the expression of T-bet and Eomes. During persistent LCMV infections, CD8+ T cells differentiate into T-bethi and Eomeshi subsets, both of which are required for efficient viral control. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8+ T cells by modulating levels of IRF4. Reduced IRF4 expression results in skewing of this ratio in favor of Eomes, leading to lower proportions and numbers of T-bet+ Eomes- precursors and poor control of LCMV Clone 13 infection. Altering this ratio in favor of T-bet xiii restores the differentiation of T-bet+ Eomes- precursors and the protective balance of T-bet to Eomes required for efficient viral control. These data highlight a critical role for IRF4 in regulating protective anti-viral CD8+ T cell responses by ensuring a balanced ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection.

No description available.

No description available.

Diss. (sammanfattning) Uppsala : Univ., 2009. / Härtill 4 uppsatser.