Browsing by Subject "general microbiology"

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  • Varghese, Finny Symon (Helsingin yliopisto, 2016)
    Chikungunya virus (CHIKV) is an arbovirus spread by the Aedes sp. of mosquitoes. Chikungunya fever results in a sudden onset of a febrile disease with headache, nausea and maculopapular rash. Additionally, a large proportion of the affected individuals experience persistent arthralgia months after all other signs of the disease have vanished. Originally discovered in Tanzania in 1952, it re-emerged with a massive outbreak in several islands of the Indian Ocean in 2004 and spilled over onto the Indian sub-continent and South-east Asia. Later CHIKV invaded Southern Europe and since the last two years has ventured into the western hemisphere, causing more than 1 million suspected infections in the Caribbean islands, Central and Latin America. The explosive nature of these outbreaks has led to a tremendous strain on the public health system of many of the affected countries already burdened with the endemically circulating Dengue virus. So far, no licensed vaccines or antivirals exist to counter this virus. Besides, it is paramount to have an in-depth understanding of the replication mechanisms of this re-emerging pathogen in order to come up with novel and effective therapeutic measures. A previously characterized CHIKV replicon cell line was used to conduct a high-throughput screen of ~3000 bioactive compounds, which are in clinical use or in clinical trials against other diseases. This led to the discovery of abamectin, ivermectin and berberine as novel antivirals effective at low micromolar concentrations and having broad-spectrum anti-alphaviral activity. Deciphering the mode of action of berberine led to the discovery that CHIKV infection robustly activates the three main branches of the mitogen-activated protein kinase (MAPK) signaling extracellular signal regulated kinase (ERK), p38 MAPK and c-Jun NH2-terminal kinase (JNK). Berberine was shown to reduce this virus-induced MAPK activation and also suppressed virus-independent ERK activation. These pathways were shown to be important for CHIKV replication, as specific inhibitors of the ERK and JNK pathways significantly reduced the viral progeny release. Most importantly, berberine reduced CHIKV-induced inflammatory disease in a mouse model and is one of the few compounds reported to show in vivo efficacy. Exploring the antiviral mechanism of obatoclax, an anticancer compound previously reported to be active against different viruses, including influenza A virus and Sindbis virus, revealed the compound to be active against other alphaviruses, including SFV and CHIKV. Further characterization showed that obatoclax inhibits viral fusion by rapidly neutralizing the acidic environment of endolysosomal organelles. Additionally, characterization of escape mutants showed that a single mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. This study has unearthed effective candidate antivirals against alphaviruses, which have served as useful tools to help us gain further insight into alphavirus biology when characterizing their modes of action.
  • Svirskaite, Julija (Helsingin yliopisto, 2016)
    Archaea were discovered only 4-5 decades ago. The majority of archaea and their viruses originate from extreme environments many of which are characterized by thriving in extreme salinities. The number of isolated archaeal viruses is just a small fraction of the known viruses. Such a lack of knowledge warrants further studies on archaeal viruses and their life cycles. The exit of mature progeny viruses from the archaeal cell is the focus of this study. The viruses used in this study represent all known haloarchaeal virus morphotypes: icosahedral-tailed (HHTV-1), icosahedral-tailless (SH1, HCIV-1), spindle-shaped (His1) and pleomorphic (His2). To describe the virion and its life cycle (by using cell culture turbidity and external virus concentration measurements) are established technologies. Factors associated with membrane integrity, the binding of lipophilic anion phenyldicarbaunundecaborane (PCB), oxygen consumption and adenosine triphosphate (ATP) levels were used to extend the traditional methods for the life cycle studies. These technologies were then utilized in the life cycle studies of HCIV-1 a recently isolated haloarchaeal virus with 12 virion structural proteins and an inner membrane. The internal membrane vesicle encloses a linear double-stranded DNA (dsDNA) genome of 31,314 bp. The genome sequence and its organization express a high similarity to the genomes of archaeal viruses in the Sphaerolipoviridae family. A rapid cell culture turbidity drop and increase of virus concentration in the cell culture medium took place when SH1, HHTV-1 and HCIV-1 exited the cell. The data also demonstrated the simultaneously binding of lipophilic PCB anions to cell debris, a lethal decrease in respiration and ATP leakage. All the measured properties support the conclusion that these three viruses have a lytic life cycle. However, His1 and His2 virus release did not affect significantly cell physiology suggesting that these haloarchaeal viruses cross the plasma membrane without depolarizing the cell. These results provide insights into the enigmatic and unique release mechanisms of haloarchaeal viruses and highlights the step forward in our understanding of archaeal viruses and their interactions with their host cell.
  • Mukherjee, Shinjini (Helsingin yliopisto, 2014)
    Ever-increasing urbanization and industrialization have led to contamination of a vast numbers of terrestrial sites with petroleum hydrocarbons. Petroleum hydrocarbon pollution has a deleterious impact on biotic and abiotic properties of ecosystem and can thereby affect some valuable ecosystem services. Microbes have the ability to metabolize various components of these harmful contaminants; this unique ability has been harnessed for decades in form of bioremediation and rhizoremediation, with varying success. Advanced knowledge on ecology of microbes in contaminated ecosystems can pave the way for improved design, optimization and monitoring of suitable bio-/rhizoremediation regimes in the clean-up of polluted sites. Recent developments in the molecular microbiology have enabled us studying microbial community structure, function and dynamics at great resolution and precision. In this thesis, two important dimensions of microbial ecology in polluted ecosystems were explored: temporal and spatial. Succession of microbial communities in contaminated soil and Populus rhizosphere were studied in two different experiments. A short-term greenhouse study was conducted to monitor the immediate response of rhizosphere-associated and soil bacterial communities to oil pollution. We further scaled up our study to monitor the bacterial succession during a 2-year field study which also allowed us to analyse the effect of seasonal variation in boreal climate zone. Finally, a case study on an aged creosote-contaminated site located in South-eastern Finland was carried out in order to investigate the spatial patterns of microbial diversity and activity in relation to the heterogeneity of soil chemical parameters. Dynamics and diversity of microbial communities were accessed by employing T-RFLP fingerprinting and 454 pyrosequencing of structural and functional marker genes. Successional changes in microbial communities could be observed in both our time-series experiments. High resolution sequencing of phylogenetic and catabolic marker genes for microbial community profiling not only enabled us to identify the bacterial groups during different stages of succession but also provided some insights on the structure-function relationship of bacterial communities. A gradual shift from specialist to generalist strategy was observed in the communities of aromatic and aliphatic degraders during the secondary succession in oil pollution. Effect of Populus rhizosphere on the general bacterial community structure was masked by the heavy oil pollution but upon careful examination of catabolic gene communities, rhizosphere-prevalent groups were observed. A significant variation in bacterial community structure was observed during the winter months pointing towards a distinct seasonal effect. Our study on spatial heterogeneity of microbial communities in an aged contaminated site highlighted niche differentiation as the major mechanism regulating bacterial community structure. Geostatistical modelling and spatial prediction brought forward two distinct patterns in geochemical properties - patchy distribution of creosotes and a natural gradient of pH on the polluted site. While most bacterial taxa drastically reduced in abundance in the hotspots of pollution, Proteobacteria clearly dominated these zones. Acidobacteria, on the other hand, responded only to the pH variation irrespective of the differences in pollution levels. Analysing the behaviour of bacterial groups at lower taxonomic levels further clarified the patterns of niche differentiation created by combined effect of pH and contaminants. The spatial profiles of specific microbial taxa could be used as proxies or indicators for monitoring this polluted site. The results obtained in this thesis project are not only scientifically interesting but they also find an application in real-time ecological restoration. Both of our studies on bacterial secondary succession were carried out as a part of a phytoremediation project in collaboration with the Finnish Forest Research Institute. Phytoremediation with hybrid aspen is being implemented at the creosote-contaminated site in Luumäki, Finland (2013- ). Our results on the spatial heterogeneity of microbial diversity and activity played a great role in the pre-evaluation of this site for remediation. This knowledge on the spatial patterns of microbial diversity will be highly useful in the coming years for the monitoring and evaluation of phytoremediation in the creosote-polluted site.