Browsing by Subject "syanobakteeri"

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Akineettien erilaistuminen ja siihen liittyvien geenien ekspression seuranta Anabaena 1TU33s10 –kannalla

Jäppinen, Sanni (2013)

Filamentous cyanobacteria taxa Nostocales and Stigonematales cells can differentiate into heterocysts nitrogen fixing cells when nitrogen is limiting the growth and into resting cells akinetes when nutrients decrease or the growth conditions become unfavorable for growth. Akinetes overwinter in the water sediments during the unfavorable growth time. When the growing condition improves akinetes germinate and can start a new cyanobacterial bloom. Akinete differentiation remains unclear. It is known from the literature that only a few akinete specific genes exist. First described akinete specific gene was avak. The morphological changes of akinete differentiation are known but the changes at molecular genetics level in regulation and differentiation remains unclear. The aim of this study was to design a method for akinete differentiation-related genes, avak, hepA and hap, for an Anabaena 1TU33s10 strain and to monitor the gene expression changes in a seven-week growth experiment. Primers for the differentiation related genes were designed based on the known whole genome sequence of the Anabaena 1TU3310 strain. In this study it was managed to design a quantitative reverse transcriptase polymerase chain reaction, qRT-PCR method, based on the genes involved in the akinete differentiation process. It was observed that gene expression changed when akinetes began to differentiate into the filaments. In the growth experiment II avaK-gene expression was increased 2-fold between the 14. and 30. days, and hap-gene showed 1.5 fold growth between 14. and 30. days. The number of akinetes was also increasing at the same time. In the growth experiment I heap-gene showed 1-8 fold growth between the days 21. and 27. –30. days when the number of heterocysts were also increasing. The number of akinetes was relatively low compared to number of vegetative cells which also explains the small expression fold-differences in the cultures during the experiment time when compared to expression fold-differences described in the literature. Designed method can thus also detect minor changes in gene expression. The designed and built qRT-PCR method can be used in the future for monitoring gene expression changes also for new akinete specific genes, and the method can be further optimized for screening natural water samples.
Anabaena 90 –syanobakteerikannan proteomin muutokset ja bioaktiivisten yhdisteiden määrän vaihtelu kasvualustan eri fosforipitoisuuksissa

Teikari, Jonna (2013)

Cyanobacteria are phototrophic organisms. They usually occur in water but many species also live in terrestrial habitats, e.g. in symbiotic relationships with fungus. Inorganic phosphorus (Pi) is usually considered to be a limiting factor for the growth of cyanobacteria living in water, since cyanobacteria can use only Pi as a direct source of phosphorus. It has been shown that cyanobacteria have pho-regulon similar to that of Escherichia coli. Pho-regulon can transport and assimilate inorganic phosphate. Cyanobacteria usually produce a wide range of bioactive compounds, which can e.g. be toxic or prevent growth of other bacteria, fungi or yeast. Many of these compounds are produced by non-ribosomal peptide synthetases (NRPS). The aim of this study was to investigate changes in Anabaena sp. 90 proteome, and differences in amounts of bioactive compounds produced by the strain, while growing it in media with high (5,5 mg/l) or low (0,05 mg/ml) Pi concentration. Before moving the culture into two different Pi media, phosphorus storages of the culture were emptied by growing the strain in the media without Pi. In this study, 2D differential gel electrophoresis (2D-DIGE) coupled with LC/MS was used to study proteomes of the organism. Bioactive compounds were also analyzed by LC/MS. Anabaena sp. 90 was chosen because of its fully sequenced and annotated genome. The strain has been found to produce microcystins, anabaenopeptins and anabaenopeptilides. Eleven protein spots with significantly increased or decreased protein quantities were identified in the low Pi media. Ten of them were identified as proteins, which participate in bacterial stringent response. Stringent response is activated when culture is achieving the stationary phase. These stringent response proteins participate in the amino-acid metabolism and translation. One of the proteins was shown to be a ribosomal protein. In addition, the identified proteins included ribulose-bisphosphate carboxylase oxygenase (RuBisCO), which had a significantly lower concentration in the cells in low-phosphorous media. There were no significant differences in amounts of bioactive compounds when growing the culture in low and high Pi media. More replicates could be used, when the study of bioactive compounds is repeated.
Anabaena 90 -syanobakteerin villityypin ja apd--mutantin proteomien ja anabaenopeptilidigeenien ilmentymisen vertailu

Partanen, Asta (2013)

Cyanobacteria are aquatic and terrestrial organisms that have photosynthetic cababilities. Cyanobacteria are classified to five orders according to their morphology. These are Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales and Stigonematales. Anabaena sp. 90 is a filamentous cyanobacterium in the order of Nostocales. A sample of this cyanobacterium was isolated from the lake Vesijärvi in 1986. Anabaena sp. 90 produces a variety of bioactive peptides such as microcystins, anabaenopeptins and anabaenopeptilides which are the most commonly studied of the peptides produced by the strain. Majority of the bioactive peptides are produced by non-ribosomal peptide synthetases (NRPS). The way these compounds affect cyanobacteria is currently unclear. Anabaenopeptilide deficient mutant (apd-) variety has been developed from the Anabaena sp 90 wild type (WT). The mutant is unable to produce anabaenopeptilides. The aim of my Master´s thesis was to study the changes in proteomes of WT and apd- strains by conducting a six day cultivation experiment. The purpose was to evaluate the possible differences in the amount of proteins that the different cultivation stocks were able to produce. The secondary purpose was to examine the transcription of anabaenopeptilide synthetase genes. Proteomic study was performed using a procedure called two dimensional differential gel electrophoresis (2D DIGE). The differences in the proteomes´ statistics were examined by using the Student´s t test. Transcription of anabaenopeptilide synthetase genes were studied using a reverse transcriptase polymerase chain reaction (RT-PCR). The data collected from these studies concluded that there were differences in the proteomes of WT and apd- strains. In eighteen cases there were significant differences in the protein amounts between the strains. The single protein counts were often higher in the apd- than in the WT strain. Anabaenopeptilide synthetase genes were transcribed in both strains which suggests that the mutation in the apd- strain did not hinder the ability of the strain to produce anabaenopeptilide transcripts. The identification of significant proteins would yield more detailed knowledge of the metabolical functions of WT and apd- strains.
Sticta-suvun jäkälien fotobionttien geneettinen monimuotoisuus Kenian Taitavuorilla

Parviainen, Anna (2020)

Jäkälät ovat sieniosakkaan ja yhteyttävän levän muodostamia symbiooseja, joista suurin osa elää maaekosysteemeissä ympäri maapalloa. Jäkälät toimivat ekosysteemeissä tärkeinä indikaattorilajeina, sillä ne ovat usein hyvin herkkiä elinympäristössä tapahtuville muutoksille, esimerkiksi ilmanlaadun huononemiselle. Sticta-suvun syanojäkäliä on tähän mennessä kuvattu noin 200 lajia erityisesti subtrooppisilta ja trooppisilta alueilta. Todellisuudessa lajimäärän arvioidaan olevan paljon suurempi. Afrikan jäkälistä tiedetään vielä suhteellisen vähän, ja tietomme Itä-Afrikan Sticta-lajeista pohjautuvat pääasiassa 1980-luvulla tehtyyn tutkimukseen. Tässä tutkimuksessa tavoitteenani oli tuottaa DNA- analyysiin perustuva selvitys Sticta-suvusta Kenian Taitavuorilla. Tutkimuksessa selvitin myös Sticta-lajien symbioottisten syanobakteerigenotyyppien levinneisyyttä Taitavuorten eri metsäsaarekkeissa ja eri Sticta-lajeissa. Tutkimukseni aineistona oli 176 jäkälänäytettä, jotka ovat kerätty Taitavuorten alueelta sekä Elgonvuoren rinteeltä Länsi-Keniasta Jouko Rikkisen ja Ulla Kaasalaisen toimesta vuosina 2009- 2017. Alustava lajinmääritys morfologian perusteella tapahtui mikroskopoimalla. Neljänkymmenenkahdeksan näytteen tarkempi lajinmääritys tehtiin DNA-menetelmin, selvittämällä jokaisesta näytteestä sienen sekä syanobakteerin genotyyppi. DNA-eristyksen, PCR:n sekä elektroforeesin tein Helsingin luonnontieteellisen museon laboratoriossa kesällä 2018. DNA- sekvensointi tehtiin Sanger-menetelmällä Saksassa GATC Biotech-yrityksen toimesta. Laboratoriotöiden tuloksena sain sieniosakkaan DNA-sekvenssin kaikkiaan 48 jäkälänäytteistä. Sekvenssien perustella 46 näytettä voitiin määrittää ennestään tunnetuiksi Sticta-lajeiksi, ja kaksi näytettä osoittautui Sticta pseudosylvatica -lajille sukua olevaksi, toistaiseksi kuvaamattomaksi lajiksi. Näytteistä löytyi kaikkiaan 21 erilaista syanobakteerigenotyyppiä. Eri metsäsaarekkeista kerättyjen näytteiden syanobakteerikoostumus ei näyttänyt poikkeavan toisistaan, mutta Sticta- lajien syanobakteerikannat poikkesivat toisistaan.
Sticta-suvun jäkälien fotobionttien geneettinen monimuotoisuus Kenian Taitavuorilla

Parviainen, Anna (2020)

Jäkälät ovat sieniosakkaan ja yhteyttävän levän muodostamia symbiooseja, joista suurin osa elää maaekosysteemeissä ympäri maapalloa. Jäkälät toimivat ekosysteemeissä tärkeinä indikaattorilajeina, sillä ne ovat usein hyvin herkkiä elinympäristössä tapahtuville muutoksille, esimerkiksi ilmanlaadun huononemiselle. Sticta-suvun syanojäkäliä on tähän mennessä kuvattu noin 200 lajia erityisesti subtrooppisilta ja trooppisilta alueilta. Todellisuudessa lajimäärän arvioidaan olevan paljon suurempi. Afrikan jäkälistä tiedetään vielä suhteellisen vähän, ja tietomme Itä-Afrikan Sticta-lajeista pohjautuvat pääasiassa 1980-luvulla tehtyyn tutkimukseen. Tässä tutkimuksessa tavoitteenani oli tuottaa DNA- analyysiin perustuva selvitys Sticta-suvusta Kenian Taitavuorilla. Tutkimuksessa selvitin myös Sticta-lajien symbioottisten syanobakteerigenotyyppien levinneisyyttä Taitavuorten eri metsäsaarekkeissa ja eri Sticta-lajeissa. Tutkimukseni aineistona oli 176 jäkälänäytettä, jotka ovat kerätty Taitavuorten alueelta sekä Elgonvuoren rinteeltä Länsi-Keniasta Jouko Rikkisen ja Ulla Kaasalaisen toimesta vuosina 2009- 2017. Alustava lajinmääritys morfologian perusteella tapahtui mikroskopoimalla. Neljänkymmenenkahdeksan näytteen tarkempi lajinmääritys tehtiin DNA-menetelmin, selvittämällä jokaisesta näytteestä sienen sekä syanobakteerin genotyyppi. DNA-eristyksen, PCR:n sekä elektroforeesin tein Helsingin luonnontieteellisen museon laboratoriossa kesällä 2018. DNA- sekvensointi tehtiin Sanger-menetelmällä Saksassa GATC Biotech-yrityksen toimesta. Laboratoriotöiden tuloksena sain sieniosakkaan DNA-sekvenssin kaikkiaan 48 jäkälänäytteistä. Sekvenssien perustella 46 näytettä voitiin määrittää ennestään tunnetuiksi Sticta-lajeiksi, ja kaksi näytettä osoittautui Sticta pseudosylvatica -lajille sukua olevaksi, toistaiseksi kuvaamattomaksi lajiksi. Näytteistä löytyi kaikkiaan 21 erilaista syanobakteerigenotyyppiä. Eri metsäsaarekkeista kerättyjen näytteiden syanobakteerikoostumus ei näyttänyt poikkeavan toisistaan, mutta Sticta- lajien syanobakteerikannat poikkesivat toisistaan.
Syanobakteerien antileukeemisten yhdisteiden tunnistus ja bioaktiivisuuksien seulonta

Renner, Niklas (2017)

Cyanobacteria produce several secondary metabolites which can be assorted into larger groups like peptides, polyketides and alkaloids based on their structure and synthesis pathways. Though the importance of these compounds to the cyanobacteria is under discussion, some compounds are detected to express bioactivity towards the cells of other organisms. The most known of these compounds are probably toxins like microcystins and saxitoxins which pose a health risk to humans and animals. However, some compounds, when modified, might have a positive affect towards human health. These compounds could for example inhibit the growth of pathogens in human body or harm cancer cells. Therefore, cyanobacteria have risen among the most promising organisms when it comes to finding new drug leads. Unfortunately only a few cyanobacteria strains seem to produce compounds with high medical potential and these compounds of interest are among the hundreds of others metabolites produced by the strain. Therefore, efficient screening and purification methods are needed. This study aimed to isolate antileukemic fractions from cyanobacteria strains, which had expressed cytotoxic activity in previous studies. The compounds of interest would be identified and purified out of these fractions. Fractions were made using solid phase extraction and high performance liquid chromatography. The antileukemic activity of the fractions were tested using human leukemia patient-derived cell line. Using described methods we found apparently cytotoxic compounds resembling carotenoids from one of the strains. We also performed screening based on plate diffusion. The tests were done in order to determine whether the compounds of cyanobacteria strains would be able to cause hemolysis or prevent growth of different microbes. One of the strains tested seemed to express α-hemolytic activity.
Syanobakteerien antileukeemisten yhdisteiden tunnistus ja bioaktiivisuuksien seulonta

Renner, Niklas (2017)

Cyanobacteria produce several secondary metabolites which can be assorted into larger groups like peptides, polyketides and alkaloids based on their structure and synthesis pathways. Though the importance of these compounds to the cyanobacteria is under discussion, some compounds are detected to express bioactivity towards the cells of other organisms. The most known of these compounds are probably toxins like microcystins and saxitoxins which pose a health risk to humans and animals. However, some compounds, when modified, might have a positive affect towards human health. These compounds could for example inhibit the growth of pathogens in human body or harm cancer cells. Therefore, cyanobacteria have risen among the most promising organisms when it comes to finding new drug leads. Unfortunately only a few cyanobacteria strains seem to produce compounds with high medical potential and these compounds of interest are among the hundreds of others metabolites produced by the strain. Therefore, efficient screening and purification methods are needed. This study aimed to isolate antileukemic fractions from cyanobacteria strains, which had expressed cytotoxic activity in previous studies. The compounds of interest would be identified and purified out of these fractions. Fractions were made using solid phase extraction and high performance liquid chromatography. The antileukemic activity of the fractions were tested using human leukemia patient-derived cell line. Using described methods we found apparently cytotoxic compounds resembling carotenoids from one of the strains. We also performed screening based on plate diffusion. The tests were done in order to determine whether the compounds of cyanobacteria strains would be able to cause hemolysis or prevent growth of different microbes. One of the strains tested seemed to express α-hemolytic activity.

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