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  • Zhao, Xiang (Helsingin yliopisto, 2015)
    Vertebrate brain is one of the most complex and mysterious objects for biological research. Embryonic brain development involves stereotypic brain structure formation, and a vast number of precise intercellular connections are established for the generation of the highly complex circuitry of the brain. This work aims at explaining HMGB1 and AMIGO1 function in modulating vertebrate brain development. Hmgb1 knockdown zebrafish morphants produced by injection of morpholino oligonucleotides display severe defects in the forebrain and gross deteriorated catecholaminergic system. The morphant is also deficient in survival and proliferation of neural progenitors. Similar central nervous system (CNS) developmental defects have been observed in HMGB1 knockout mouse embryo. The HMGB1 null mouse embryonic brain cells showed much lower proliferating and differentiating activities compared to wild type animals. HMGB1 knockdown and knockout model respectively from zebrafish and mouse have confirmed that AMIGO1 expression is directly regulated by HMGB1. AMIGO1 regulates expression of Kv2.1 potassium channel during development, but the colocalization of AMIGO1 and Kv2.1 has only been observed in mouse and zebrafish adult brain. Furthermore, knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. The same defect can be also induced by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. The impaired formation of neural circuitry is reflected in enhanced locomotor activity and attenuated escape responses. Our data demonstrate that HMGB1 is a critical factor for embryonic CNS development involved in many important developmental events. HMGB1 is essential for the neurogenesis and differentiation occurring at the developmental stage when forebrain structures are forming. Amigo1 is required for the development of neural circuits under the regulation of HMGB1. The mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. HMGB1 and AMIGO1 are both crucial for embryonic brain development and neural circuit formation.
  • Susi, Hanna (Helsingin yliopisto, 2014)
    At the very core of the evolution of living organisms lie interactions with other species. Between two coevolving species, a change in one species may generate selection for a change in the other species. In host-pathogen coevolution the central dilemma is to understand how infectivity and virulence evolve. Infectivity is the ability to infect a given host while virulence is the harm the pathogen causes to its host, and therefore they determine the outcome of the interaction between the host and the pathogen. The emergence of new highly virulent pathogen species (e.g. Ash dieback pathogen Hymenoscyphus pseudoalbidus) and single pathogen strains (e.g. Ug99 of wheat stem rust pathogen Puccinia graminis f. sp. tritici) underline the urgent need for a deeper understanding of how virulence evolves. The aim of my thesis is to understand how life-history trade-offs and coinfection where two or more strains of the same pathogen are infecting the same host - are driving host-pathogen coevolution, and how these evolutionary trajectories translate to ecological dynamics in a metapopulation context using the Plantago lanceolata Podosphaera plantaginis interaction as a model system. The study approach ranged from the molecular level to population and metapopulation levels. I studied natural populations of P. lanceolata and P. plantaginis in the Åland islands to measure prevalence of coinfection and its consequences for disease epidemics in the wild. I also investigated variation in resistance in the natural host populations as well as the efficiency and costs of different plant resistance strategies in a common garden setting. Context dependence of evolutionary trade-offs were investigated by accounting for some of the spatial and temporal complexity of the natural pathogen metapopulation. Pathogen life-history trade-offs were studied in the context of local adaptation and costs of resistance in the perennial host were measured across multiple seasons. The pathogen s host exploitation versus transmission strategies were examined on relevant epidemiological time scales to understand factors creating heterogeneity in transmission dynamics. Key findings of the thesis include detection of high, yet variable levels of coinfection across the pathogen metapopulation, with more devastating epidemics measured in populations with higher levels of coinfection. This suggests a major role for coinfection in driving disease dynamics in natural populations. In the dynamic pathogen metapopulation, local adaptation mediates pathogen life history trade-offs and resistance polymorphism can be maintained through costs of resistance and changes in resource allocation under infection. In conclusion, this work contributes to our understanding of the drivers of evolution and maintenance of variation in the host and pathogen populations by linking evolutionary theory with empirical findings.
  • Lappalainen, Tuuli (Helsingin yliopisto, 2009)
    In this thesis, the genetic variation of human populations from the Baltic Sea region was studied in order to elucidate population history as well as evolutionary adaptation in this region. The study provided novel understanding of how the complex population level processes of migration, genetic drift, and natural selection have shaped genetic variation in North European populations. Results from genome-wide, mitochondrial DNA and Y-chromosomal analyses suggested that the genetic background of the populations of the Baltic Sea region lies predominantly in Continental Europe, which is consistent with earlier studies and archaeological evidence. The late settlement of Fennoscandia after the Ice Age and the subsequent small population size have led to pronounced genetic drift, especially in Finland and Karelia but also in Sweden, evident especially in genome-wide and Y-chromosomal analyses. Consequently, these populations show striking genetic differentiation, as opposed to much more homogeneous pattern of variation in Central European populations. Additionally, the eastern side of the Baltic Sea was observed to have experienced eastern influence in the genome-wide data as well as in mitochondrial DNA and Y-chromosomal variation – consistent with linguistic connections. However, Slavic influence in the Baltic Sea populations appears minor on genetic level. While the genetic diversity of the Finnish population overall was low, genome-wide and Y-chromosomal results showed pronounced regional differences. The genetic distance between Western and Eastern Finland was larger than for many geographically distant population pairs, and provinces also showed genetic differences. This is probably mainly due to the late settlement of Eastern Finland and local isolation, although differences in ancestral migration waves may contribute to this, too. In contrast, mitochondrial DNA and Y-chromosomal analyses of the contemporary Swedish population revealed a much less pronounced population structure and a fusion of the traces of ancient admixture, genetic drift, and recent immigration. Genome-wide datasets also provide a resource for studying the adaptive evolution of human populations. This study revealed tens of loci with strong signs of recent positive selection in Northern Europe. These results provide interesting targets for future research on evolutionary adaptation, and may be important for understanding the background of disease-causing variants in human populations.
  • Hokynar, Kati (Helsingin yliopisto, 2007)
    Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.
  • Lappalainen, Ilkka (Helsingin yliopisto, 2004)
  • Shakeel, Shabih (Helsingin yliopisto, 2014)
    Pathogenic human picornaviruses are known to cause a wide variety of diseases ranging from mild colds to severe paralysis. In addition to their importance in causing disease, they also serve as models for understanding the basic mechanisms of host-pathogen interactions, virus entry, viral genome release, viral synthesis and viral assembly. In picornaviruses, the majority of the structural and host-cell interaction studies have been conducted on polioviruses and human rhinoviruses. Picornaviruses like coxsackievirus A 7, coxsackievirus A 9 and human parechovirus 1 have not been so well studied because of difficulties in culturing them. Recently, the number of cases reported for infection by these viruses has increased dramatically due to better detection methods, thus making structural studies of these viruses and their interactions with their host cells important in order to understand their mode of infection so that better therapeutics can be designed against them. I have studied coxsackievirus A 7, coxsackievirus A 9 and human parechovirus 1, which are all pathogenic picornaviruses, in order to understand the mechanism of pathogenesis, tropism, viral entry and assembly for these viruses in particular and for picornaviruses in general. Two studies dealt with determining the structure of coxsackievirus A 7, a Human Enterovirus A species for which there was no structural information available at the time when this study was conducted. The genome-filled and empty structure of coxsackievirus A 7 were determined using cryo electron microscopy to sub-nanometer resolution which helped in building pseudo-atomic models for them using homology modelling and flexible fitting. With the help of these models, the majority of the strain variations in the capsid proteins were identified on the surface of VP1. Such variations are the likely cause of differences in pathogenesis and tropism between strains. Furthermore, superimposition of these models showed that the capsid underwent a conformational change on RNA release. In the process, generalised methods for optimising and comparing results from flexible fitting were developed. The next structural study elucidated the interaction of coxsackievirus A 9, a Human Enterovirus B species, with a cellular receptor. Integrins were found to bind sub-stoichiometrically to the capsid using electron cryo-tomography (cryo-ET). Asymmetric reconstruction indicated that this was probably due to steric hindrance. The affinity of this interaction was calculated to be 1nM using surface plasmon resonance. Additionally, the conformational changes which occur on its RNA release were quantified. The fourth study explained the importance of viral RNA in picornavirus assembly. Pentameric intermediates of human parechovirus 1 were isolated and used to identify packaging signals in the viral RNA required for capsid assembly using aptamer library screening and next generation sequencing analysis. Poly-U was identified as the common motif for these packaging signals present on the stem or the loop of the RNA secondary structure. Overall, this thesis gives an insight into many important aspects of host-virus interactions especially the events occurring on viral RNA exit and during its encapsidation. The work in this thesis could be utilized to identify potential targets for antiviral synthesis and also to define general virus assembly principles.
  • Pellinen, Riikka (Helsingin yliopisto, 2001)
  • Nummela, Pirjo (Helsingin yliopisto, 2011)
    Cancer is becoming the leading cause of deaths in the world. As 90% of all deaths from cancer are caused by metastasis, discovery of the mechanisms behind cancer cell invasion and metastasis is of utmost importance. Only new effective therapies targeting cancer progression can reduce cancer mortality rates. The aim of this study was to identify molecules that are relevant for tumor cell invasion and spreading in fibrosarcomas and melanomas, and to analyze their potential for cancer biomarkers or therapeutic targets. First, the gene expression changes of normal cells and transformed cells showing high invasiveness, S-adenosylmethionine decarboxylase (AdoMetDC)-transfected murine fibroblasts and human melanoma cells, were studied by microarray analyses. The function of the identified candidate molecules were then studied in detail in these cell lines. Finally, the physiological relevance of the identified changes was studied by immunohistochemical analyses of human sarcoma and melanoma specimens or by a mouse xenograft model. In fibrosarcoma cells, the most remarkable change detected was a dramatic up-regulation of the actin-sequestering molecule thymosin beta 4 (TB4), which was shown to be important for the transformed phenotype of the AdoMetDC-transfected cells (Amdc-s and -as). A sponge toxin latrunculin A, inhibiting the binding of TB4 to actin, was found to selectively inhibit the migration and invasion of these cells. Further, Amdc-s-induced mouse tumors and human high-grade sarcomas were found to show intense TB4 immunostaining. In addition to TB4, integrin subunits alfa 6 and beta 7 (ItgA6 and ItgB7) were found to be up-regulated in Amdc-s and -as cells. ItgA6 was shown to dimerize mainly with ItgB1 in Amdc-s. Inhibition of ItgA6 or ItgB1 function with neutralizing antibodies fully blocked the invasiveness of Amdc-s cells, and importantly also human HT-1080 fibrosarcoma cells, in three-dimensional (3D)-Matrigel mimicking tumor extracellular matrix (ECM). By immunohistochemical analyses, strong staining for ITGA6 was detected in human high-grade fibrosarcomas and other sarcomas, especially at the invasion fronts of the tumors. In the studied melanoma cell lines, the expression levels of the adhesion-related ECM proteins tenascin-C (TN-C), fibronectin (FN), and transforming growth factor beta-induced (TGFBI) were found to be highly up-regulated. By immunohistochemistry, intense TN-C and FN staining was detected in invasive and metastatic melanoma tumors, showing co-localization (together with procollagen-I) in tubular meshworks and channels around the invading melanoma cells. In vitro, TN-C and FN were further found to directly stimulate the migration of melanoma cells in 3D-collagen-I matrix. The third candidate protein, TGFBI, was found to be an anti-adhesive molecule for melanoma cells, and knockdown of its expression in metastatic melanoma cells (TGFBI-KD cells) led to dramatically impaired tumor growth in immunocompromized mice. Interestingly, the control tumors showed intense TGFBI immunostaining in the invasion fronts, showing partial co-localization with the fibrillar FN staining, whereas the small TGFBI-KD cell-induced tumors displayed amorphous, non-fibrillar FN staining. These data suggest an important role for TGFBI in FN fibrillogenesis and melanoma progression. In conclusion, we have identified several invasion-related molecules, which show potential for cancer diagnostic or prognostic markers, or therapeutic targets. Based on our previous and present fibrosarcoma studies, we propose the possibility of using ITGA6 antagonists (affecting tumor cell adhesion) in combination with TB4 inhibitors (affecting tumor cell migration) and cathepsin L inhibitors (affecting the degradation of basement membrane and ECM proteins) for the treatment of fibrosarcomas and other tumors overexpressing these molecules. With melanoma cells, in turn, we point to the importance of three secreted ECM proteins, TN-C, FN, and TGFBI, in melanoma progression. Of these, especially the potential of TN-C as a prognostic melanoma biomarker and TGFBI as a promising therapeutic target molecule are clearly worth additional studies.
  • Hilditch, Satu (Helsingin yliopisto, 2010)
    Pectin is a natural polymer consisting mainly of D-galacturonic acid monomers. Microorganisms living on decaying plant material can use D-galacturonic acid for growth. Although bacterial pathways for D-galacturonate catabolism had been described previously, no eukaryotic pathway for D-galacturonate catabolism was known at the beginning of this work. The aim of this work was to identify such a pathway. In this thesis the pathway for D-galacturonate catabolism was identified in the filamentous fungus Trichoderma reesei. The pathway consisted of four enzymes: NADPH-dependent D-galacturonate reductase (GAR1), L-galactonate dehydratase (LGD1), L-threo-3-deoxy-hexulosonate aldolase (LGA1) and NADPH-dependent glyceraldehyde reductase (GLD1). In this pathway D-galacturonate was converted to pyruvate and glycerol via L-galactonate, L-threo-3-deoxy-hexulosonate and L-glyceraldehyde. The enzyme activities of GAR1, LGD1 and LGA1 were present in crude mycelial extract only when T. reesei was grown on D-galacturonate. The activity of GLD1 was equally present on all the tested carbon sources. The corresponding genes were identified either by purifying and sequencing the enzyme or by expressing genes with homology to other similar enzymes in a heterologous host and testing the activities. The new genes that were identified were expressed in Saccharomyces cerevisiae and resulted in active enzymes. The GAR1, LGA1 and GLD1 were also produced in S. cerevisiae as active enzymes with a polyhistidine-tag, and purified and characterised. GAR1 and LGA1 catalysed reversible reactions, whereas only the forward reactions were observed for LGD1 and GLD1. When gar1, lgd1 or lga1 was deleted in T. reesei the deletion strain was unable to grow with D-galacturonate as the only carbon source, demonstrating that all the corresponding enzymes were essential for D-galacturonate catabolism and that no alternative D-galacturonate pathway exists in T. reesei. A challenge for biotechnology is to convert cheap raw materials to useful and more valuable products. Filamentous fungi are especially useful for the conversion of pectin, since they are efficient producers of pectinases. Identification of the fungal D-galacturonate pathway is of fundamental importance for the utilisation of pectin and its conversion to useful products.
  • Tiittanen, Minna (Helsingin yliopisto, 2006)
    Type 1 diabetes (T1D) is considered to be an autoimmune disease. The cause of T1D is the destruction of insulin-producing β-cells in the pancreatic islets. The autoimmune nature of T1D is characterized by the presence of autoreactive T-cells and autoantibodies against β-cell molecules. Insulin is the only β-cell-specific autoantigen associated with T1D but the insulin autoantibodies (IAAs) are difficult to measure with proper sensitivity. T-cell assays for detection of autoreactive T-cells, such as insulin-specific T-cells, have also proven to be difficult to perform. The genetic risk of T1D is associated with the HLA gene region but the environmental factors also play an important role. The most studied environmental risk factors of T1D are enteroviruses and cow's milk which both affect the immune system through the gut. One hypothesis is that the insulin-specific immune response develops against bovine insulin in cow's milk during early infancy and later spreads to include human insulin. The aims of this study were to determine whether the separation of immunoglobulin (Ig)G from plasma would improve the sensitivity of the IAA assay and how insulin treatment affects the cellular immune response to insulin in newly diagnosed patients. Furthermore, the effect of insulin concentration in mother's breast milk on the development of antibodies to dietary insulin in the child was examined. Small intestinal biopsies were also obtained from children with T1D to characterize any immunological changes associated with T1D in the gut. The isolation of the IgG fraction from the plasma of T1D patients negative for plasma IAA led to detectable IAA levels that exceeded those in the control children. Thus the isolation of IgG may improve the sensitivity of the IAA assay. The effect of insulin treatment on insulin-specific T-cells was studied by culturing peripheral blood mononuclear cells with insulin. The insulin stimulation induced increased expression of regulatory T-cell markers, such as Foxp3, in those patients treated with insulin than in patients examined before initiating insulin treatment. This finding suggests that insulin treatment in patients with T1D stimulates regulatory T-cells in vivo and this may partly explain the difficulties in measuring autoantigen-specific T-cell responses in recently diagnosed patients. The stimulation of regulatory T-cells by insulin treatment may also explain the remission period often seen after initiating insulin treatment. In the third study we showed that insulin concentration in mother's breast milk correlates inversely with the levels of bovine insulin-specific antibodies in those infants who were exposed to cow's milk proteins in their diet, suggesting that human insulin in breast milk induces tolerance to dietary bovine insulin. However, in infants who later developed T1D-associated autoantibodies, the insulin concentration in their mother's breast milk was increased. This finding may indicate that in those children prone to β-cell autoimmunity, breast milk insulin does not promote tolerance to insulin. In the small intestinal biopsies the presence of several immunological markers were quantified with the RT-PCR. From these markers the expression of the interleukin (IL)-18 cytokine was significantly increased in the gut in patients with T1D compared with children with celiac disease or control children. The increased IL-18 expression lends further support for the hypothesis that the gut immune system is involved in the pathogenesis of T1D.
  • Piha, Henna (Helsingin yliopisto, 2006)
    Agriculture-mediated habitat loss and degradation together with climate change are among the greatest global threats to species, communities, and ecosystem functioning. During the last century, more than 50% of the world's wetlands have been lost and agricultural activities have subjected wetland species to increased isolation and decreased quality of habitats. Likewise, as a part of agricultural intensification, the use of pesticides has increased notably, and pesticide residues occur frequently in wetlands making the exposure of wetland organisms to pesticides highly probable. In this thesis, a set of ecotoxicological and landscape ecological studies were carried out to investigate pesticide-effects on tadpoles, and species-habitat relationships of amphibians in agricultural landscapes. The results show that the fitness of R. temporaria tadpoles can be negatively affected by sublethal pesticide concentrations, and that pesticides may increase the costs of response to natural environmental stressors. However, tadpoles may also be able to compensate for some of the negative effects of pesticides. The results further demonstrate that both historic and current-day agricultural land use can negatively impact amphibians, but that in some cases the costs of living in agricultural habitats may only become apparent when amphibians face other environmental stressors, such as drought. Habitat heterogeneity may, however, increase the persistence of amphibians in agricultural landscapes. Hence, the results suggest that amphibians are likely to be affected by agricultural processes that operate at several spatial and temporal scales, and that it is probable that various processes related to current-day agriculture will affect both larval and adult amphibians. The results imply that maintaining dense wetland patterns could enhance persistence of amphibian populations in agricultural habitats, and indicate that heterogeneous landscapes may lower the risk of regional amphibian population declines under extreme weather perturbations.
  • Örmälä-Odegrip, Anni-Maria (Helsingin yliopisto, 2015)
    Bacterial viruses (i.e. phages) are ubiquitous intracellular parasites of bacteria, that along with protist grazers account for majority of bacterial mortality in nature. Phages impose strong selection for bacterial phage-resistance, which is often coupled with fitness costs on bacterial traits such as growth ability, virulence or motility. Traditionally phage-host interactions have been studied with two species systems in the laboratory, neglecting the complex web of interactions present in natural communities. The ability of phages to selectively kill bacteria has ignited an interest on phages as alternative antibacterials. However, in order to develop phage therapy, understanding of phage-host interactions in the eco-evolutionary context is essential. In this thesis I studied the implications of lytic phages on opportunistic pathogenic bacteria, as opportunists often have the ability reproduce and reside in outside-host environments, where they are predisposed to a variety of selection pressures. The role of phages in top-down control of bacterial biomass and the evolution of bacterial phage-resistance were studied in the presence of protist predators with differing feeding modes, in low-resource systems mimicking natural pond environment. Hypothesis of coincidental evolution suggests that virulence is a by-product of selection for traits that maximize bacterial fitness in environmental reservoirs. Yet, disease outbreaks by opportunists are relatively rare, suggesting that something constrains the selection for virulence. To assess the role of lytic phages on the evolution of virulence, bacteria were cultured in low-resource environment, accompanied with changes in temperature regime or changes in composition of the community of interacting bacterial enemy species, and the virulence of bacteria was measured in vivo. To study whether the potential phage-resistant bacteria surviving phage therapy would be coupled with lowered virulence, due to costs associated with phage-resistance, a clinical bacterial isolate was exposed to phage cocktails and the virulence of the phage-resistant bacteria was measured in vivo. given the strong selection for phage- resistance, the prospects of phage therapy depend a great deal on whether new phages infecting pathogenic bacteria can be readily isolated from environment. To address this, an attempt was made to isolate phages against clinical bacterial isolates harboring resistance genes to multiple antibiotics. A single lytic phage was shown to be a non- efficient top-down regulator of bacterial biomass. Rapidly emerging phage-resistant bacteria took over the bacterial populations after initial lysis by phages and protist grazers accounted for most of the long-term negative trophic effects on bacterial biomass. The presence of protist predators selected for bacteria that were less susceptible to infection by lytic phages, which suggests an overlap in the bacterial defense against a parasite and predatory protists. In general, the presence of lytic phages selected for lowered virulence in bacteria. High temperature selected for more virulent and more motile bacteria, but this was constrained by the presence of a lytic phage. In the multispecies communities the presence of all bacterial enemies led to decreased virulence in vivo. Altogether, these results contrast the hypothesis of coincidental evolution, and suggest that the presence of phages in natural reservoirs constrains the evolution of virulence, most likely through fitness costs associated with phage-resistance. Exposure to phage cocktails was also shown to be associated with decreased bacterial virulence in the phage-resistant bacteria. However, exposure to some individual phages resulted in more virulent bacteria, suggesting that the outcome of therapy could depend on the identity of the phage cocktail. Finally, a phage cocktail lysing a wide range of clinical strains was isolated from sewage. This, along with geographical patterns of phage infections suggest that new phages are available in environmental reservoirs for therapy, and the emergence of phage-resistance should not hinder the prospects of phage therapy in the global perspective.
  • Gu, Yuexi (Helsingin yliopisto, 2015)
    Women with germline mutations of BRCA1 gene are predisposed to the development of basal-like breast cancer, which is characterized by the absence of the hormonal and growth factor receptors, ER, PR and HER2. Mutation of BRCA1 predicts increased sensitivity to certain DNA-damaging agents, e.g. PARP inhibitors and cisplatin. Yet in clinical trials, some BRCA1-mutant breast tumors show resistance to these drugs. Therefore, it is necessary to understand the molecular mechanisms of the acquired resistance and identify novel therapeutic targets for the treatment of those tumors. In order to better understand the biology of breast cancers caused by BRCA1 mutations, we collected and characterized four BRCA1-mutant breast cancer cell lines as surrogates of BRCA1-mutant tumors. Together with several cell lines expressing wildtype BRCA1, we tested their sensitivities to a panel of DNA-damaging agents. We also carried out a high-throughput chemical compound screen on the BRCA1-mutant and BRCA1-wildtype cell lines, and performed a proteome profiling assay to test the kinase activities of those cell lines. Our results reveal extensive heterogeneity among the BRCA1-mutant breast cancer cell lines, which showed resistance to DNA-damaging agents. Then we created isogenic MDA-MB-231 cells with or without BRCA1 depletion by siRNA transfection, and carried out a high-throughput chemical compound screen on the pair of cells in order to identify potential targets that are synthetically lethal with BRCA1 deficiency. Two proteasome inhibitors, bortezomib and carfilzomib, were found to be able to selectively kill BRCA1-depleted cells. Further studies on mechanisms demonstrate that the proteasome inhibitor bortezomib does not induce DNA damage; rather it inactivates G1 cell cycle checkpoint in BRCA1-deficient cells and leads to the accumulation of these cells at G2/M phase. This is caused by inactivation of the retinoblastoma protein (Rb) through its hyperphosphorylation, which in turn activates its downstream transcription factor E2F1. In addition, bortezomib causes compromised G2/M cell cycle checkpoint in BRCA1-deficient cells, which drives cells to enter mitosis and leads to apoptosis due to uncontrolled cell division. In order to investigate the mechanisms underlying the consistent basal-like phenotype of breast tumors associated with BRCA1 mutations, we established an in vitro assay to study the transition of mouse mammary epithelial cells from luminal to basal lineages. Our results showed that loss of BRCA1 promotes basal-like differentiation by sustaining ΔNp63 activity. In luminal cells, ΔNp63 is not expressed or remains inactive through localization in nucleoli. Depletion of BRCA1 leads to translocation of ΔNp63 into nucleoplasm and promotes transition of luminal cells into a basal state. This study provides a potential link between BRCA1 loss and the basal-like differentiation, which may help to explain why BRCA1-mutant breast cancer tends to bear a basal-like phenotype.
  • Penttilä, Tuula (Helsingin yliopisto, 2004)
  • Heiniö, Raija-Liisa (Helsingin yliopisto, 2003)