267 research outputs found
Effects of environmental factors on growth and development of Phaeodactylum tricornutum
No full textDiatoms are eukaryotic photosynthetic organisms found in marine and freshwater environments as primary producers accounting for ~20% of total world photosynthetic output and ~50% of oceanic biomass. Diatoms are of industrial interest as they are lipid accumulating, which could be used in the production of animal feed or biofuels without occupying agricultural space. An important model diatom is P. tricornutum which is a resilient, fast-growing organism. Our knowledge of diatoms is not proportional to their importance and lags behind that of plants and green algae. It is important to better understand them so that we might predict how they will adapt to a changing world climate.
Through this thesis we have looked at how three environmental factors, salinity, pH and presence of herbicides (glyphosate derived from roundup) impact the growth of P. tricornutum. Salinity treated cells were RNA sequenced, underwent lipid extraction with fatty acid analysis. Our findings indicate that high salinity led to a decrease in growth rate, in contrast to low salinity conditions which led to an increase in growth rate. Analysis of fatty acid composition showed that the low salinity treatment had a higher percentage of polyunsaturated fatty acids (PUFAs) with high salinity treatment showing the opposite response. RNA sequencing showed many genes to be differentially expressed, several of which were associated with sodium and chloride. However, a large portion of the differentially expressed genes coded for proteins of unknown function.
The pH treatments showed a decrease in P. tricornutum growth as the pH decreased from normal (pH 8.1), with growth ceasing entirely at pH 5.1. Cell death staining using propidium iodide was performed on the glyphosate treatments. From the glyphosate treatments we saw rapid cell death staining at the highest concentrations (0.8 and 0.4 g/L) while the lower concentration treatments showed a slowly increasing cell death stain percentage, reaching ~20% following 96 hours.
In conclusion, our results show that alterations in environmental conditions can dramatically impact the growth of P. tricornutum depending on the degree of environmental change. Additionally, we saw several opportunities to further develop the knowledge of P. tricornutum and diatoms in general
Effect of environmental factors on growth and development of Phaeodactylum tricornutum
No full textDiatoms are eukaryotic photosynthetic organisms found in marine and freshwater environments as primary producers accounting for ~20% of total world photosynthetic output and ~50% of oceanic biomass. Diatoms are of industrial interest as they are lipid accumulating, which could be used in the production of animal feed or biofuels without occupying agricultural space. An important model diatom is P. tricornutum which is a resilient, fast-growing organism. Our knowledge of diatoms is not proportional to their importance and lags behind that of plants and green algae. It is important to better understand them so that we might predict how they will adapt to a changing world climate.
Through this thesis we have looked at how three environmental factors, salinity, pH and presence of herbicides (glyphosate derived from roundup) impact the growth of P. tricornutum. Salinity treated cells were RNA sequenced, underwent lipid extraction with fatty acid analysis. Our findings indicate that high salinity led to a decrease in growth rate, in contrast to low salinity conditions which led to an increase in growth rate. Analysis of fatty acid composition showed that the low salinity treatment had a higher percentage of polyunsaturated fatty acids (PUFAs) with high salinity treatment showing the opposite response. RNA sequencing showed many genes to be differentially expressed, several of which were associated with sodium and chloride. However, a large portion of the differentially expressed genes coded for proteins of unknown function.
The pH treatments showed a decrease in P. tricornutum growth as the pH decreased from normal (pH 8.1), with growth ceasing entirely at pH 5.1. Cell death staining using propidium iodide was performed on the glyphosate treatments. From the glyphosate treatments we saw rapid cell death staining at the highest concentrations (0.8 and 0.4 g/L) while the lower concentration treatments showed a slowly increasing cell death stain percentage, reaching ~20% following 96 hours.
In conclusion, our results show that alterations in environmental conditions can dramatically impact the growth of P. tricornutum depending on the degree of environmental change. Additionally, we saw several opportunities to further develop the knowledge of P. tricornutum and diatoms in general
Integrative systems approaches to study plant stress responses
No full textThe world’s population is growing at an alarming rate. As of 2010, out of 7 billion people in the world, 925 million are hungry. It represents 13.1 percent of the total world population, or almost 1 in 7 people are hungry (FAO). Climate change is increasingly viewed as a current and future cause of hunger and poverty. In the scenario of global climatic change, different biotic and abiotic stresses are severe threats to the agricultural production worldwide. In nature, plants are continuously stressed by exposure to multiple adverse conditions. The combined effect of multiple biotic and abiotic stresses is a major yield-limiting factor in agriculture. In such a situation, it is of utmost importance to take initiatives for genome scale molecular understanding of stress response mechanisms in plants, so that new stress resistant crop varieties can be developed. Recent developments in omics technologies (metabolomic, proteomic, transcriptomic, phenomics and more) have opened up a new dimension for conducting genome scale molecular studies to understand stress response mechanisms in plants. These studies have led to the revelation of extremely complex and interacting networks of various stress response processes. Statistical, mathematical and informatics driven analysis and integration of the enormous amount of data produced is a challenge. The combination of high throughput profiling techniques, bioinformatics tools and the knowledge of genetics will provide the ways by which to achieve a comprehensive understanding of biological processes related to stress responses in plants. Such knowledge can be translated further to develop better crop varieties.
This thesis presents a few such integrated studies, exploring different aspects of plant stress responses at the molecular and systems levels. I believe that the works presented in this thesis will significantly contribute towards a molecular understanding of plant stress response mechanisms at the systems level. The entire thesis has been divided into seven chapters.
Chapter 1 gives a brief introduction about the adverse effect of global climatic change on plant productivity due to intensified effects of various stress factors and its negative socioeconomic impact on human society. This chapter also briefly summarises the background of seven research papers presented in this thesis along with a review of contemporary works.
Chapter 2 (Paper I) describes why systems biology is useful to study plant stress biology, reviewing various approaches and computational tools available to plant biologists till date.
Chapter 3 (Paper II) explores common and stress specific response signatures by the host plant to two different biotic stresses. It provides a comparative understandings of Arabidopsis – Brevicoryne brassicae (aphid) and Arabidopsis –Pseudomonas syringae(bacteria) interactions at the systems level.
Chapter 4 (Paper III) uncovers the molecular stress response patterns in plants during the co-occurrence of multiple abiotic and biotic stresses. The main outcome is that transcriptome changes in response to combined stresses could not be predicted from the responses to single stress treatments. This chapter also presents a modular network topology based approach to identify functionally related stress responsive gene modules.
Chapter 5 (Paper IV) presents the intraspecific variation in stress response patterns among 10 Arabidopsis ecotypes during cold stress exposure. Using an in silico transcriptional regulatory network model during cellular responses to cold stress in Arabidopsis thaliana, a hypothesis is presented that differentially evolving regulatory networks play a crucial role in climate adaptation of plants.
Chapter 6 (Paper V) presents an in silico transcriptional regulatory network model in responses to 11 stresses (5 single and 6 combined) conditions in Arabidopsis thaliana reconstructed from microarray data using a robust algorithm - Network Component Analysis (NCA).
Chapter 7 presents two application cases as examples of translational research, how knowledge developed in lab can be used in crop plants.
a. (Paper VI) demonstrates how the omics and systems biology approach is useful in improving crop productivity and abiotic stress tolerance in cultivated Fragaria.
b. (Paper VII) presents a case study on developing transgenic Brassica napus MINELESS as a new model system to study plant insect interactions. During this study, activation of plant defense in Brassica napus L. cv. Westar and transgenic MINELESS plants after attack by Mamestra brassicae (cabbage moth) were analysed.PhD i biologiPhD in Biolog
Studying attractiveness and defense responses of Brassica napus transgenic MINELESS plants to insects for pest management
No full textGlucosinolate myrosinase system is well-established defense mechanism against insect herbivores feed on plants from Brassicaceae family. This defense mechanism is also known as the mustard oil bomb which bring myrosinase enzyme and glucosinolate together upon insect attack and tissue disruption and cause glucosinolate hydrolysis by myrosinase enzyme activity. Glucosinolate and its hydrolysis products have a deterring effect on herbivores, and protect plant parts from insect damage. The modified plants have been named MINELESS due to lack of myrosin cells.
The aim of this study was to get an insight into defense responses of MINELESS and wild- type Brassica napus plants after being challenged by insects Delia radicum, Delia floralis, Mamestra brassicae and Meligethes aeneus. The hypothesis behind this work were due to lack of myrosin cells and the enzyme myrosinase, the glucosinolate-myrosinase system is modulated and MINELESS plants possess more glucosinolates in plant tissues. Glucosinolate have adverse and deterring effects on insect herbivores, and larvae of generalist herbivore, Mamestra brassicae preferred to feed on wild-type cotyledons of Brassica napus plants, which contain lower amounts of glucosinolates than those of MINELESS. Role of glucosinolates on attraction of pollen beetles, Meligethes aeneus, is not exactly known, however, pollen beetles are attracted by glucosinolates hydrolysis products, isothyocyanates. Wild-type plants were preferred by pollen beetles for feeding over MINELESS plants which possess high levels of glucosinolates, but low levels of isothiocyanates due to lack of myrosinase activity.
Four glucosinolates were detected with the highest amount of indol-3-ylmethyl- (I3M) glucosinolate both in control and insect challenged aboveground tissues, with slightly higher amounts in MINELESS plants than wild-type. However total glucosinolate levels did not show exact difference between MINELESS and wild-type plants. I3M glucosinolates levels increased upon herbivore feeding in all treatment days. Increasing days of treatment increased levels of aliphatic glucosinolate, 5-methylsulphinylpentyl- (5MSOP) glucosinolate and decreased levels of indole glucosinolate, I3M glucosinolate.
In both no-choice and free choice experiments, M. brassicae larvae consumed more wild-type cotyledons than those of MINELESS. In no-choice experiments consumed area of cotyledons increased in correlation with increasing number of days of feeding, and wild-type plants were consumed more than MINELESS plants.
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The ablation of myrosin cells and consequent lack of myrosinase activity did not affect feeding by pollen beetles in both feeding experiments. Wild-type buds were damaged more than MINELESS buds, however the difference was not significant. Also in Y-tube olfactometer experiment beetles moved mostly towards odour of oilseed rape flowers rather than fresh air. Due to the small number of individuals tested, we did not consider this result significant
Studying attractiveness and defense responses of Brassica napus transgenic MINELESS plants to insects for pest management
No full textGlucosinolate myrosinase system is well-established defense mechanism against insect herbivores feed on plants from Brassicaceae family. This defense mechanism is also known as the mustard oil bomb which bring myrosinase enzyme and glucosinolate together upon insect attack and tissue disruption and cause glucosinolate hydrolysis by myrosinase enzyme activity. Glucosinolate and its hydrolysis products have a deterring effect on herbivores, and protect plant parts from insect damage. The modified plants have been named MINELESS due to lack of myrosin cells.
The aim of this study was to get an insight into defense responses of MINELESS and wild- type Brassica napus plants after being challenged by insects Delia radicum, Delia floralis, Mamestra brassicae and Meligethes aeneus. The hypothesis behind this work were due to lack of myrosin cells and the enzyme myrosinase, the glucosinolate-myrosinase system is modulated and MINELESS plants possess more glucosinolates in plant tissues. Glucosinolate have adverse and deterring effects on insect herbivores, and larvae of generalist herbivore, Mamestra brassicae preferred to feed on wild-type cotyledons of Brassica napus plants, which contain lower amounts of glucosinolates than those of MINELESS. Role of glucosinolates on attraction of pollen beetles, Meligethes aeneus, is not exactly known, however, pollen beetles are attracted by glucosinolates hydrolysis products, isothyocyanates. Wild-type plants were preferred by pollen beetles for feeding over MINELESS plants which possess high levels of glucosinolates, but low levels of isothiocyanates due to lack of myrosinase activity.
Four glucosinolates were detected with the highest amount of indol-3-ylmethyl- (I3M) glucosinolate both in control and insect challenged aboveground tissues, with slightly higher amounts in MINELESS plants than wild-type. However total glucosinolate levels did not show exact difference between MINELESS and wild-type plants. I3M glucosinolates levels increased upon herbivore feeding in all treatment days. Increasing days of treatment increased levels of aliphatic glucosinolate, 5-methylsulphinylpentyl- (5MSOP) glucosinolate and decreased levels of indole glucosinolate, I3M glucosinolate.
In both no-choice and free choice experiments, M. brassicae larvae consumed more wild-type cotyledons than those of MINELESS. In no-choice experiments consumed area of cotyledons increased in correlation with increasing number of days of feeding, and wild-type plants were consumed more than MINELESS plants.
iii
The ablation of myrosin cells and consequent lack of myrosinase activity did not affect feeding by pollen beetles in both feeding experiments. Wild-type buds were damaged more than MINELESS buds, however the difference was not significant. Also in Y-tube olfactometer experiment beetles moved mostly towards odour of oilseed rape flowers rather than fresh air. Due to the small number of individuals tested, we did not consider this result significant
Regulation of photosynthesis in heterokont algae
No full textSeveral microalgae belonging to the phylum of Heterokontophyta have recently been studied with increasing interest, as these microalgae are able to produce very high amounts of fatty acids which are both interesting for biodiesel production and in a nutritional perspective. The production of these fatty acids depends on photosynthesis, which fuels the uptake of inorganic carbon. Their photosynthetic processes have, however, not been studied as extensively as their ability to produce lipids.
The work underlying this thesis has been focusing on the regulation of photosynthetic electron transport in heterokont algae, and also on how the different processes involved in photosynthesis affect the algae’s ability to fix inorganic carbon and thus be efficient lipid producers.
The work has focused on the microalgae Nannochloropsis oceanica and Phaeodactylum tricornutum, and it has been determined that photosynthesis is differently regulated in these algae compared to in green algae and higher plants.
First of all, high light, which is known to reduce the photosynthetic electron transport chain in green algae and plants, have been shown to have the opposite effect in N. oceanica and P. tricornutum. And since several spectroscopic techniques aimed at investigating different aspects of photosynthesis are based on the assumption that high light reduces the electron transport chain, this might lead to misinterpretations of data obtained using such techniques.
It has also been determined that heterokont algae, in contrast to what was previously thought, most likely use state transitions as a mode of photoprotection in conditions where their photosystems are in need of protection
Genome scale transcriptional response diversity among ten ecotypes of Arabidopsis thaliana during heat stress
No full textCopyright © 2013 Barah, Jayavelu, Mundy and Bones. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms
Focused analysis of genomic alterations in primary breast carcinomas and corresponding metastases
No full textBreast cancer is one of the leading causes of cancer mortality worldwide. Distant metastases are nearly always the direct cause of death, and understanding its relationship to the primary tumor is of great importance. Today, the choice of treatment is, in most cases, based upon histological and molecular analysis of the primary tumor. However, recent studies suggest that the cancer cells may spread to distant organs earlier in tumor progression than previously believed and that genetic alterations can evolve independently in the primary tumor and metastasis. Some studies report of a close resemblance between primary tumors and their corresponding metastasis, while others report of genetic divergence. The conflicting results reflect the need for a deeper understanding of the molecular mechanisms underlying metastatic disease, to be able to improve treatment strategies and clinical outcome.
The objective of this thesis was to investigate whether the somatic mutations found in a lymph node metastasis differ from those in the corresponding primary breast tumor. Targeted sequencing of 20 primary breast tumors and their matched lymph node metastases was performed by the use of Ion Torrent Personal Genome Machine. The Ion AmpliSeq Cancer Hotspot Panel v2 was utilized, targeting 207 regions in 50 genes found to be frequently mutated in cancer. The genetic variants were compared to public databases such as dbSNP, 1000 genomes and COSMIC. 55% of the tumors were found to harbor at least one somatic mutation (median 0,8, range 0-3). Frequently mutated genes included TP53 (45%) and PIK3CA (25%). The vast majority of the metastases seemed to retain the somatic mutations detected in the primary tumor, but the variant frequencies were slightly different. In three of the patients a TP53 mutation unique to the primary tumor and/or the lymph node metastasis were revealed. The differences indicate that dissemination may occur at different time points during disease progression, and that analysis of metastatic tumors could provide additional insight affecting treatment decisions. However, as only a small piece of each tumor was analyzed and only selected regions of the cancer genomes were sequenced, no conclusion can be drawn about the time of dissemination or the level of heterogeneity of the tumors. Further analysis of the samples, including copy number analysis and whole genome sequencing, should lead to a broader understanding of breast cancer progression from a local to a metastatic disease
Uttrykk og betydning av GRP78 i myelomceller
No full textMyelomatose, også kjent som beinmargskreft, er en hematologisk malign sykdom i antistoffproduserende plasmaceller i beinmargen. Kreftsykdommen er fortsatt uhelbredelig, og resistensutvikling mot medikamenter er en stor utfordring. Myelomceller karakteriseres av en opphopning av monoklonale immunglobuliner og er utsatt for ER-stress. De er derfor avhengige av en streng kontroll av ER-stress for å overleve. GRP78 er et viktig ER-chaperon som hjelper med folding av proteiner. Hos pasienter med visse typer kreft er proteinet GRP78 funnet på celleoverflaten hos kreftceller, men ikke hos friske celler. Den fysiologiske funksjonen og hvordan GRP78 uttrykkes på celleoverflaten er uklar. Nyere studier viser at GRP78 på celleoverflaten kan danne komplekser med spesifikke proteiner som er med på å regulere både proliferasjon og viabilitet. Det er også vist at hemming av GRP78 på celleoverflaten ved hjelp av spesifikke antistoff kan føre til celledød.
I denne oppgaven er ulike humane myelomcellelinjer undersøkt for uttrykk av GRP78 mRNA (HSPA5) og protein, både intracellulært og på celleoverflaten. Myelomcellelinjene som ble testet ut i denne oppgaven viste ulikt, men generelt høyt uttrykk av HSPA5 mRNA og totalt proteinnivå av GRP78, mens overflateuttrykk av GRP78 varierte fra negativt til ulik grad av positivt uttrykk. Det ble ikke oppdaget en sammenheng mellom intracellulært uttrykk og overflateuttrykk av GRP78.
Det ble forsøkt å se om endring av nivået av ER-stress kunne påvirke uttrykket av GRP78. Bruk av proteasomhemmere, som potensielt gir økt ER-stress, ga økt total proteinmengde av GRP78, men det ble ikke observert noen form for endring av overflateuttrykket til GRP78. Det ble også gjort forsøk med siRNA mot HSPA5 for å se om det påvirket overflatenivået av GRP78 og om det påvirket cellenes følsomhet for proteasomhemmere. Bruk av siRNA nedregulerte mRNA-nivået av HSPA5 og den totale proteinmengden av GRP78, mens overflateuttrykket av GRP78 forble uforandret. Nedregulering av HSPA5 ble heller ikke funnet å være av betydning for overlevelse eller respons til proteasomhemming for INA-6 celler. Det ble undersøkt om GRP78 kunne binde heparin, som en mulig forklaring på GRP78 sin evne til å binde til overflaten. Overflatenivået av GRP78 ble redusert med tilsats av heparin, noe som kan tyde på at GRP78 er delvis avhengig av binding til heparansulfatkjeder på celleoverflaten. Hemming av GRP78 på celleoverflaten med antistoffene Mab159- og N-20 ga ingen endring av viabiliteten
Expression profiles and initial RNAi experiments with tyrosine hydroxylase and tryptophan 2,3-dioxygenase encoding genes in the salmon louse (Lepeophtheirus salmonis)
No full textLakselus er en ektoparasittisk copepode på laksefisk. Den fører til betydelige økonomiske tap i oppdrettsindustrien, samtidig som den representerer en velferdstrussel for oppdrettslaksefisker og en mulig trussel mot ville anadrome laksefiskpopulasjoner. Eksperimentell posttranskripsjonell nedregulering av spesifikke gener ved RNA interferens (RNAi) er en metode for å karakterisere funksjonen til gener i mange organismer, inkludert lakselus, og eksperimentell RNAi har blitt oppnådd i flere av lakselusas utviklingsstadier. RNAi kan, sammen med andre teknikker, øke kunnskapen om interaksjoner mellom parasitt og vert, samt bidra til å identifisere molekylære mål for kjemisk behandling og mulige vaksineantigen.
I dette prosjektet ble de to genene LsPale og LsTDO, som koder for henholdsvis tyrosin hydroksylase (TH) og tryptofan 2,3-dioksygenase (TDO) hos lakselus, studert. De to enzymene er viktige i metabolismen av flere nevrotransmittere, pigmenter og andre metabolitter av tyrosin og tryptofan. Fylogenetiske analyser viste at begge genene hører til sine artropode genfamilier, og at de er nært beslektet med tilsvarende gener i andre hoppekreps. En nedregulering av de to genene ved RNAi ble også forsøkt i lakselusas nauplii-larver. LsPale ble nedregulert suksessfullt, og nedreguleringen var detekterbart i mindre enn syv dager. Ingen morfologiske eller adferdsmessige endringer ble observer tre og syv dager etter behandling, men larvene var inaktive rett etter at dobbeltrådet RNA (dsRNA) ble introdusert. Genutrykksprofiler av de to genene viste at LsPale er sterkt oppregulert før og gjennom den metamorfologiske forvandlingen til copepoditt og at LsTDO er utrykt i både i lakselusas planktoniske og parasittiske utviklingsstadier, med en topp i preadult og adult.
Siden resultatene indikerer at genene er involvert i flere av lakselusas utviklingsstadier, burde det gjøres mer arbeid for å karakterisere de to genene. Videre medvirker sannsynligvis LsTDO i øyepigmentering, og kan potensielt fungere som en seleksjonsmarkør om CRISPR/Cas9-basert genredigering skal utvikles for lakselus. Dette prosjektet illustrerer også at RNAi i lakselus er utfordrende, til tross for flere publikasjoner der lignende RNAi-basert metodikk er benyttet.The salmon louse is an ectoparasite copepod on salmonids. It causes substantial economic loss in the salmon industry and represents a welfare threat to farmed salmonids and a potential threat to the sustainability of several wild salmonid populations. Experimental post-transcriptional knockdown of specific genes by RNA interference (RNAi) is a method used to characterize the function of genes in many organisms, including the salmon louse, and experimental RNAi have been achieved in several of the salmon louse’s developmental stages. RNAi, in combination with other techniques and methods, might expand the understanding of parasite-host interactions, as well as help identify molecular targets for novel chemical treatments and vaccine antigens.
In this project, the two genes LsPale and LsTDO in the salmon louse, encoding tyrosine hydroxylase (TH) and tryptophan 2,3-dioxyganease (TDO), respectively, were studied. The two enzymes represent key steps in the metabolism of several neurotransmitters, pigments, and other metabolites of tyrosine and tryptophan. Phylogenetic analysis revealed that both genes belong to their arthropod gene families and are closely related to corresponding copepode genes. Further, the two genes were knocked down by RNAi in the salmon louse nauplii stages. A successful knockdown was only obtained for LsPale, and the knockdown was detectable for less than seven days. No morphological or behavioral changes were observed at three or seven days after treatment, but larvae were inactive immediately after double-stranded RNA (dsRNA) delivery. Expression profiling of the two genes revealed that the expression of LsPale is strongly increased before and through the metamorphotic molt to copepodite and that LsTDO is expressed in both the planktonic and the parasitic stages of the salmon louse, with a peak in preadult and adults.
As the results presented here indicate that the genes are involved in important developmental phases in the life cycle of the salmon louse, more work should be conducted to characterize the function of the two genes. It is also suggested that LsTDO might play a role in eye pigmentation, and therefore could serve as a suitable selection marker when developing CRISPR/Cas9 based genome editing method for salmon louse. Further, the project illustrates that, despite several publications where similar RNAi-based methods are used in salmon louse, RNAi studies in salmon louse are challenging
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