How to improve the Swedish hardiness system for herbaceous perennials : the development of subcategories

Det svenska A-D härdighetssystemet för perenner har varit i bruk under många år i Sverige men dess användning är inte så utbredd som önskat. Härdighetssystemet togs ursprungligen fram av svenska perennodlare. Därefter vidareutvecklades de från tre till fyra härdighetskategorier. 2012 skrevs ett examensarbete kring perenners härdighet av Fredrika Eklund. I Eklunds arbete gjordes en utredning av det svenska härdighetssystemet för perenner samt inledande förslag på vidareutveckling av systemet med hjälp av underkategorier. Eklunds examensarbete har legat till grund för detta examensarbete som är ett försök att ta vid där hon slutade. Syftet med detta examensarbete är att ta reda på hur det svenska härdighetssystemet för perenner kan förbättras. En förbättring av härdighetssystemet skulle medföra att förutsättningarna förbättras för att både professionella och privatpersoner ska kunna välja rätt perenn till rätt plats. Målet är att med hjälp av underkategorier förtydliga de krav olika perenner har för att övervintra. Detta skulle bidra till en ökad spridning av systemet både hos yrkesverksamma inom trädgårdsbranschen och hos privatpersoner med trädgårdsintresse. Resultatet har tagits fram genom litteraturstudier och semistrukturerade kvalitativa intervjuer. Litteraturstudien visar att de härdighetsfaktorer som påverkar perenner är många och mycket komplexa. De är svåra att separera från varandra samt rangordna sinsemellan. Det har dock framkommit att dränering vintertid är en av de mest avgörande faktorerna för en perenns förmåga att övervintra. Därför ligger perennens behov av dränering till grund för utformandet av underkategorier till det befintliga härdighetssystemet för perenner. De kvalitativa intervjuerna har visat att erfarenhetsbaserad kunskap prioriteras över härdighetsmärkningen en perenn innehar vid val av perenner. Användandet av det befintliga härdighetssystemet är mycket begränsat hos personer med lång erfarenhet inom branschen. Detta kan ställas i kontrast till nybörjare och privata trädgårdsägares brist på sagda erfarenhet för att avgöra perenners härdighet. För dem är ett system som ger riktlinjer en av godo. Intervjuerna visar även att det finns fler härdighetsfaktorer samt sekundära faktorer som påverkar perenners förmåga att utvecklas och som är önskvärda att ta upp i eventuella underkategorier. Trots att dränering vintertid är en viktig faktor finns en önskan att skapa underkategorier som tar hänsyn till övriga faktorer. Exempelvis kan dessa vara mer anpassade till specifika växtmiljöer. Det finns även ett behov av att bearbeta de befintliga A-D kategorierna i härdighetssystemet. Mycket handlar om att förtydliga ett flertal begrepp, t.ex. väldränerat, fuktigt och skyddat men även att avgöra vilka lägen som anses som "hela Sverige", "stora delar av Sverige" samt "landets mest gynnsamma delar". Det är även viktigt att minnas att härdighetssystemet aldrig kommer att skapa den slutgiltiga lösningen på härdighetsproblematiken. Detta pga. att både klimat och soriment kommer att förändras. Därför måste även härdighetssystemet vara föränderligt.Even though the Swedish hardiness system for herbaceous perennials was developed many years ago, the use is not as widespread as one would have hoped. The hardiness system was originally developed by Swedish perennial growers and then enhanced from three to four categories, A-D. In 2012 Fredrika Eklund did her bachelor thesis by doing a review of the Swedish hardiness system for herbaceous perennials and suggestions of modifications to improve it by adding subcategories. My own thesis is an attempt to pick up where she left off. The meaning of this thesis is to find ways to improve the Swedish hardiness system for herbaceous perennials. An improvement would make it easier for both professionals and garden owners to choose the right perennial at the right place. The goal is to clarify what needs perennials have to be able to endure the harsh winter climate by adding subcategories to the hardiness system. This would mean that the hardiness system would more easily be adapted by professionals and others. The results of this thesis has been reached through literature and semi-structured qualitative interviews. The results shows that there are many variables that effects hardiness of herbaceous perennials and that they are hard to separate from each other or rank. But the results do show that winter drainage is one of the most crucial. This is reflected by the proposed subcategories. The semi-structured qualitative interviews shows that experiential knowledge is by far used more often than the hardiness categories when it comes to choosing perennials. The use of the hardiness system is very limited when it comes to people with long experience. But for people that does not yet have that long experience, the hardiness system is a good way to get guidelines when determining perennials hardiness. The interviews also shows that there are more things that affect the hardiness of herbaceous perennials, and their ability to develop as good as possible, that is desirable to include in the subcategories although the winter drainage is very important. One possibility is to developed subcategories by dividing them by habitats. There is also a need to process the existing A-D hardiness categories. Concepts as drainage, moisture and shelter needs to be clarified. The meaning of "the whole country", "big parts of the country" and "the most favorable parts of the country" also needs to be clarified. Maybe the most important thing of all is to remember that the Swedish hardiness system for herbaceous perennials never can be ultimate and everlasting. Due to changes in the climate and the perennial market, the hardiness system must be dynamic and follow the changes

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.</p> <p>Conclusions</p> <p>We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant <it>P. pastoris </it>clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of <it>P. pastoris </it>clones with high expression levels of aquaporins and other classes of membrane proteins.</p

Surface and Inner Deformation during Shape Rolling of High Speed Steels

Shape rolling is a common manufacturing process used to produce long products i.e. bars and wire. One of the problems that might occur during rolling is defect formation leading to rejection of the finished product. This work is a step towards a better understanding of the evolution of some of these defects. The evolution and reduction of cracks during shape rolling is studied in this thesis. To accomplish this, artificial longitudinal cracks are machined along bars of high speed steel. The cracks are positioned at different sites evenly distributed along the periphery in intervals of 45°. Some of the cracks are left open and some are filled with carbon or stainless steel welds. FE simulations are performed using the commercial code MSC.Marc and the results from the simulations are compared with experimental ones. Generally, simulations predict less reduction than observed experimentally. For most positions, the cracks tend to reduce most effectively followed by carbon steel welds and stainless steel welds. To evaluate the inner deformation of a cross section during shape rolling in an oval-round-oval-round series, sample bars of M2 high speed steel are prepared with grids made up by stainless steel wires. After collecting samples after each pass, they are X-rayed to create an image of the grid. The deformation of the wires can favorably be described by FE simulations of a bar originally rotated 10° when entering the first pass. The results suggest that the simulations describe the deformation during shape rolling well.QC 2010111

From Sequence to Structure- Characterizing Human and Plant Aquaporins

Aquaporins constitute a class of membrane proteins that create pores through the lipid bilayer of biological membranes, facilitating the passage of water and other small solutes. Nearly all living organisms possess a more or less intricate set up of aquaporin proteins and the importance of these channels has been implicated in many aspects of health and disease. The aim of the projects summarized in this thesis was to increase the knowledge about the structure and function of aquaporins. A method for obtaining high levels of recombinantly expressed membrane proteins in the yeast Pichia pastoris by screening for clones with multiple recombinant gene inserts was developed. Subsequent to overexpression, a number of different purification schemes for obtaining preparations for molecular characterization were established and three aquaporin isoforms; human HsAQP5 and HsAQP8 and a plant aquaporin, NbXIP1;1, were successfully purified. The two human isoforms were reconstituted into artificial liposomes and by functionality assessments their water transporting capacity was confirmed. The verification of retained functionality after recombinant expression and subsequent purification trials is crucial in order to validate the quality of the protein that goes into crystallization and structure determination trials. By using two different crystallization techniques, diffracting crystals of both HsAQP5 and HsAQP8 were obtained and subsequently, a high resolution structure of HsAQP5 was solved. A different approach was used to identify hypothetical aquaporins in the chloroplast, which is the organelle responsible for the conversion of sunlight into chemical energy in plants. By immunoblotting procedures and mass spectrometry analyses, the presence of several aquaporin isoforms was confirmed, suggesting a possible dependence of the photosynthetic reactions on an assisted trans-membrane water flux

Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes (‘‘peptiplexes’’) enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide- mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the ‘‘chelate effect’’ and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA

Using Ethidium To Probe Nonequilibrium States of DNA Condensed for Gene Delivery

Here we explore the use of ethidium to determine relative affinities of different gene delivery vectors for DNA and describe an improved method for studying the interaction. Specifically, we investigate the binding of poly(amidoamine) dendrimers and show that the DNA-dendrimer-ethidium system is far from thermodynamic equilibrium. Moreover, dendrimer surface modification through PEGylation appears to make the interaction with DNA more reversible, which is favorable from the perspective of vector unpacking. Probing the nonequilibrium state of DNA during condensation processes is thus important for developing novel vectors, and further, it could also be useful in the study of chromatin folding

DNA condensation by PAMAM dendrimers: Self-assembly characteristics and effect on transcription

Electrostatic shielding and steric blocking by histones are two significant factors that participate in the control of the local rates of transcription in chromatin. As a simple model system to determine how the degree of DNA condensation affects enzyme accessibility and gene expression, we have used generation 5 polyamidoamine (G5 PAMAM) cationic dendrimer particles (size 5.4 nm) as a synthetic histone model together with an in vitro transcription assay. The degree of compaction, conformation, and binding availability of the dendrimer-DNA complexes is characterized by linear and circular dichroism, dynamic light scattering, and competitive binding of ethidium. Using ultracentrifugation we are able to show explicitly, for the first time, that dendrimer particles bind to DNA in a highly cooperative manner, and that the dendrimer-induced condensation of the DNA strongly attenuates transcription. Two fractions with different properties can be identified: a low-density fraction which behaves very similar to uncondensed DNA and a high-density fraction which is condensed to a high extent and where binding availability and transcription are strongly reduced. Circular dichroism gives clues to the structure of the condensed DNA indicating long-range order between the helices such as in polymer-salt-induced cholesteric liquid crystalline domains, one possible shape being a toroidal structure. On the basis of the experimental data, we propose a model for the self-assembly of the dendrimer-DNA system

Stimulated endocytosis in penetratin uptake: Effect of arginine and lysine

Cell-penetrating peptides can deliver macromolecular cargo into cells and show promise as vectors for intracellular drug delivery. Internalization occurs predominantly via endocytosis, but the exact uptake mechanisms are not fully understood. We show quantitatively how penetratin, a 16-residue cationic peptide, stimulates fluid-phase endocytosis and triggers its own uptake into Chinese hamster ovarian cells, using a 70 kDa dextran to indicate macropinocytosis. The total cellular endocytotic rate is significantly less affected and we therefore propose up-regulation of macropinocytosis to occur at the expense of other types of endocytosis. By comparing penetratin to its analogs PenArg and PenLys, enriched in arginines and lysines, respectively, we show how these side-chains contribute to uptake efficiency. The degree of peptide and dextran uptake follows similar patterns regarding peptide concentration and arginine/lysine content (PenArg > penetratin > PenLys), indicating that a high content of arginines is beneficial but not necessary for stimulating endocytosis