Abstract Biomass conversion methods represent bioeconomic solutions for the sustainable production of value added commodities (chemicals and materials) as well as for energy purposes, either in solid (pellets), liquid (transport fuels) or gaseous (combustion gases e.g. biomethane) form. Lignocellulosic biomass as a renewable source available in immense quantity, is considered to be one of the most promising natural sources, with high potential in the replacement of conventional transportation fuels and reduction of greenhouse gas emissions. This thesis provides new insights into mechanocatalysis, which as yet is a novel technique in catalytic biomass conversion. The mechanocatalytic approach combines chemical catalysis and mechanical assisted processing driven by ball milling. Lignocellulosic barley straw was impregnated or merely mixed with the catalyst (formic acid, acetic acid, sulfuric acid, oxalic acid dihydrate and potassium pyrosulfate) and ball milled under various conditions yielding the selective depolymerization of lignocellulose into water-soluble xylo-oligosaccharides. Subsequent hydrolysis at moderate temperatures resulted in the formation of valuable reducing sugars, mainly xylose, galactose, arabinose and glucose, which constitute the basic materials for transportation fuel and chemical production. Reducing sugar release of 53.4 wt% with low by-product formation was observed within short milling durations using sulfuric acid as a catalyst in mechanocatalysis. Likewise, oxalic acid dihydrate and potassium pyrosulfate as a novel catalyst, successfully converted barley straw to reducing sugars (42.4 wt% and 39.7 wt%, respectively), however longer milling durations were required. In comparison, lower saccharification (<10 wt%) was obtained by employing formic acid and acetic acid in mechanocatalysis. Harsh milling conditions initiated a temperature increase within the reaction vessel resulting in enhanced sugar release. Likewise, greater sugar release was observed with increased catalyst amount and acidity. The results revealed that the balance of these factors is crucial for efficient catalytic conversion of barley straw. / Tiivistelmä Biomassan konvertointimenetelmät mahdollistavat biotalouden hengen mukaisesti uusia ratkaisuja kemikaalien ja materiaalien kestävään tuotantoon sekä biomassan energiakäyttöön eri muodoissa (kuten pelletit, biopolttoaineet ja biokaasu). Lignoselluloosapohjaista, uusiutuvaa biomassaa, kuten tässä työssä tutkittua ohran olkea, on runsaasti saatavilla. Lignoselluloosa onkin yksi lupaavimmista raaka-aineista korvaamaan fossiilisia polttoaineita ja vähentämään kasvihuonekaasupäästöjä. Väitöskirjatutkimus antaa uutta tietoa ohran oljen mekaanis–katalyyttisestä käsittelystä, mikä on suhteellisen uusi menetelmä biomassan katalyyttisessä muokkauksessa. Menetelmässä yhdistetään kemiallinen katalyysi ja mekaaninen muokkaus (jauhatus) kuulamyllyllä. Lignoselluloosa (ohran olki) impregnoitiin tai sekoitettiin tutkitun katalyytin (muurahaishappo, etikkahappo, rikkihappo, oksaalihappodihydraatti, kaliumpyrosulfaatti) kanssa ja käsiteltiin erilaisissa mekaanis–katalyyttisissä olosuhteissa. Lignoselluloosan selektiivinen depolymerointi muodosti vesiliukoisia oligosakkarideja ja edelleen hydrolyysin kautta pelkistyneitä sokereita (pääasiassa ksyloosia, galaktoosia, arabinoosia ja glukoosia), joita voidaan käyttää biopolttoaineiden ja -kemikaalien valmistuksessa. Tutkimuksen tulosten perusteella rikkihappokatalyytillä saatiin 53,4 massa-% ohran oljen sisältämistä pelkistyneistä sokereista vapautettua lyhyillä käsittelyajoilla. Lisäksi sivutuotteiden muodostuminen oli vähäistä. Vastaavasti oksaalihappodihydraatti (sokerisaanto 42,4 massa-%) ja kaliumpyrosulfaatti (sokerisaanto 39,7 massa-%) toimivat uusina katalyytteinä hyvin, mutta vaativat rikkihappokatalyyttiä pidemmät jauhatusajat. Sen sijaan muurahaishapolla ja etikkahapolla sokerisaanto oli erittäin alhainen (alle 10 massa-%) mekaanis–katalyyttisessä käsittelyssä. Tutkimuksessa todettiin, että voimakas jauhatus vaikutti selkeästi reaktiolämpötilan nousuun käsittelyn aikana, mikä edisti korkeampaa sokerisaantoa. Vastaavasti sokerisaantoa voitiin parantaa katalyyttimäärällä ja happamuudella. Tulokset osoittavat, että näiden muuttujien tasapaino on ratkaisevaa ohran oljen tehokkaan katalyyttisen muuntamisen kannalta.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-12-21T13:19:49Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Pamela Thaisa.pdf: 1195732 bytes, checksum: 0da9cde7a5b7b6d64fcef7d3e8a47ff0 (MD5) / Made available in DSpace on 2018-12-21T13:19:49Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Pamela Thaisa.pdf: 1195732 bytes, checksum: 0da9cde7a5b7b6d64fcef7d3e8a47ff0 (MD5) Previous issue date: 2018-09-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A germinação das sementes de cevada é essencial para a produção do malte e a degradação enzimática do amido em açúcares solúveis é parte do processo de germinação dessas sementes. O objetivo desse trabalho foi avaliar o parâmetro fisiológico de sementes de cevada, através de testes de germinação e de vigor, em função de diferentes momentos de colheita, armazenadas ou não, e a influência da expressão diferencial dos genes, que codificam as enzimas α-amilase e β-amilase. Assim, as sementes das cultivares BRS Cauê e Irina foram produzidas em 2017 e colhidas aos 99 (32% de água), 104 (19,18% de água), 107 (17,92% de água) e 114 (15,89% de água) dias após a semeadura (DAS) para BRS Cauê e 102 (32,76% de água), 107 (20,27% de água), 121 (13,63% de água) e 127 (17,43% de água) DAS para a Irina. A qualidade das sementes foi avaliada logo após a colheita e após o armazenamento por 111 dias (BRS Cauê) e 107 dias (Irina). As sementes mantiveram dormência, principalmente na avaliação inicial; a qualidade das sementes foi superior quando colhidas aos 114 DAS (15,89% de água) para as sementes da cultivar BRS Cauê e aos 107 (20,27% de água) para Irina, sem variação da qualidade das sementes avaliadas após o armazenamento. O nível da expressão diferencial do gene da α-amilase para as sementes da cultivar BRS Cauê colhidas aos 114 DAS foi superior, independentemente da época de avaliação e aos 99 DAS inicialmente. Para Irina os maiores níveis de expressão foram obtidos quando as sementes foram colhidas aos 127 DAS. O nível de expressão do gene da β-amilase foi superior para as sementes colhidas aos 99 DAS para BRS Cauê e aos 102 DAS para Irina avaliadas logo após a colheita, com redução após o armazenamento. A dormência das sementes favoreceu a manutenção da qualidade fisiológica, independentemente do momento em que foram colhidas. Não é possível estabelecer correlação entre os resultados dos testes de germinação e de vigor das sementes de cevada e a expressão gênica para este experimento, evidenciando a complexidade dessa relação e a necessidade de mais estudos que possam melhor elucida-la. / The germination of the barley seeds is essential for the production of malt and enzyme degradation of the starch to soluble sugars, makes part of this seed germination process. The objective of this study was to evaluate the physiological parameters of barley seeds, from germination and vigor tests, depending of the harvests close to physiological maturity, stored or not, and the influence of the differential expression of the genes which codify to α amylase and β-amylase enzymes. The seeds of BRS Cauê and Irina were cultivate in 2017 and harvested at 99 (32% water), 104 (19.18% water), 107 (17.92% water) and 114 (15.89% water) days after sowing (DAS) to BRS Cauê and 102 (32.76% water), 107 (20.27% water), 121 (13.63% water) and 127 (17.43 % water) DAS to Irina. The seed quality was evaluated immediately after harvesting and storage for 111 days (BRS Cauê) and 107 days (Irina). The seeds kept dormancy, mainly in the initial evaluation; (15.89% water) for seeds from BRS Cauê and 107 (20.27% water) for Irina, without variation in the quality of the seeds evaluated after storage. The differential expression level of gene coding to α-amylase, to the seeds of BRS Cauê harvested at 114 DAS, was higher, regardless of the evaluation time and the 99 DAS. For Irina the highest levels of expression were obtained when the seeds were harvested at 127 DAS. The level of expression of the gene coding to β-amylase was higher for seeds harvested at 99 DAS for BRS Cauê and 102 DAS for Irina initially evaluated, with decrease after storage. Seed dormancy favored the maintenance of seed quality, regardless of when they were harvested. It is not possible to establish a correlation between the results of germination and vigor tests of barley seeds and gene expression for this experiment, evidencing the complexity of this relationship and the requirement for research that can better elucidate it.

To prove the post-natal depression model, the antidepressant sertraline, was assessed in rat mothers (n=14) divided into Prenatally Stressed (PS) and Non-Stressed (NS) groups. The data failed to support the hypothesis that ‘the progeny of 10mg of sertraline-treated PS mothers displayed less anxiety than the progeny of vehicle-treated PS mothers’. The forced swim test (FST) was used to examine depressive-like behaviour in mice. Barley successfully increased mobility in mice exposed to the FST. Barley was antidepressant at low doses (0.8g/kg and upwards) if used subchronic; and at high doses(6.4g/kg and 12.8g/kg) if used acutely;(n=113,56acute,57 subchronic- treated). Barley (6.4g/kg) was also able to alleviate the depressive-behaviour in mice induced by the Reserpine Test (n=114, 58 reserpinised, 56 non-reserpinised) and Social ‘Defeat’ Test (n=24, 8 vehicle undefeated, 8 barley defeated, 8 vehicle defeated mice). To confirm that the anti-depressant effects of barley(6.4g/kg) were not simply due to increased locomotor activity in the FST, an Open Field Test(OFT) was undertaken (n=14,7 vehicle, 7 barley). Barley had no effect on locomotor activity and also caused no significant changes in weight (n=16, 8vehicle, 8 barley). In mice,Barley(6.4g/kg) significantly delayed the tremorogenic effects of Physostigmine (n=18, 6 control,6 Physostigmine, 6 Physostigmine with barley); reduced bradykinesia induced by reserpine (n=18,6 control, 6 vehicle, 6 barley treated);and was analgesic in nociception tests (n =20, 5 control, 5 barley, 5 pain, 5 pain with barley). Overall, barley was seen to have many useful properties, though its effect in PND remains to be assessed.

One of the challenges in the 21st Century is to overcome the recalcitrance of lignocellulose for the production of liquid biofuels. Lignin is one of the key factors in this recalcitrance. Grasses such as Miscanthus and switchgrass could become major sources of lignocellulose. Barley has potential as a genetically-tractable research model for such novel bioenergy crops and also as a bioenergy crop itself. This thesis concerns the 4CL and the CCR enzymes on the lignin pathway which were chosen as the targets to manipulate lignin in barley. They were selected because there is evidence that suppression of each of them in dicot species can lead to increased saccharification. The 4CL and CCR genes constitute multigene families where members have different expression patterns. RNAi was used to down-regulate 4CL1 and CCR1 using a constitutive promoter via Agrobacterium-mediated transformation of barley. From an extensive screen of the primary transformants for changes in protein level and lignin content, six CCR and four 4CL lines were taken forward for detailed analysis. Antibodies were also raised against barley 4CL and CCR recombinant proteins and these showed substantial reductions in the respective target protein levels in the RNAi lines. Both 4CL and CCR transgenic lines had significant reductions in lignin content, and CCR lines had changes in lignin structure due to changes in the proportions of acid soluble and acid insoluble lignin. No substantial consistent adverse effects on key agronomic traits were apparent in the 4CL and CCR transgenics. Selected 4CL and CCR transgenics had improved saccharification yield after using three different pretreatment methods, which is a desirable feature for biofuel production.

Os cereais representam importantes fontes de proteína para alimentação humana e animal. Entretanto, são caracterizados pela baixa qualidade nutricional de suas proteínas devido à composição desbalanceada de aminoácidos, causada pelo excesso dos aminoácidos prolina e glutamina e deficiência de lisina, treonina e triptofano. As proteínas de reserva prolaminas constituem 50% do conteúdo total de proteínas no endosperma e são as principais responsáveis por tais características nos cereais. As informações sobre o metabolismo de lisina e o acúmulo de proteínas de reserva no endosperma vêm sendo utilizadas para desenvolver e aplicar estratégias em programas de melhoramento de plantas que visam suprir a deficiência de lisina encontrada nos cereais. Lange e colaboradores (2007) relataram a produção de linhagens transgênicas de cevada com padrão de proteínas de reserva alterado e que apresentaram incremento no teor de lisina e outros aminoácidos essenciais. O presente trabalho teve como objetivo identificar os mecanismos responsáveis pelas alterações observadas. Para tanto, avaliou-se a atividade das enzimas envolvidas na síntese e degradação de lisina, além da caracterização das proteínas de reserva e sua composição de aminoácidos. Observou-se redução na fração protéica das prolaminas (5,91 a 18,34%) e incrementos compensatórios na fração protéica das glutelinas (2,16 a 6,52%). As demais frações apresentaram respostas variáveis dependendo do evento avaliado. Além disso, a composição de aminoácidos foi alterada nas diferentes frações protéicas. As prolaminas exibiram incrementos nos teores de lisina (1,79 a 49,13%), treonina (5,04 a 22,60%) e metionina (13,57 a 45,38%), enquanto que as globulinas aumentaram principalmente o conteúdo de metionina (32,30 a 142,56%). Para os aminoácidos solúveis, foram observados incrementos na ordem de duas a três vezes de histidina, lisina, fenilalanina e metionina. A análise das enzimas envolvidas no metabolismo de lisina revelou que ocorreram alterações nas três principais enzimas da via do ácido aspártico. A enzima aspartato quinase (AK) apresentou aumentos na atividade (4,44 a 47,27%), entretanto, foi mais sensível a inibição causada por lisina. A enzima dihidrodipicolinato sintase (DHDPS) também apresentou incremento na atividade (1,50 a 66,32%), mas diferente da AK, foi menos sensível à inibição causada por lisina. A enzima homoserina desidrogenase (HSDH), a qual compete o substrato ASA com a enzima DHDPS, exibiu redução na atividade (3,36% a 28,80%) (exceto um evento de transformação) e foi menos sensível a inibição causada por treonina. Embora as enzimas envolvidas na degradação de lisina também foram alteradas, os resultados foram variáveis para os diferentes eventos. Para aqueles que foram observados redução na atividade da enzima lisina cetoglutarato redutase (LOR), foi também verificado para enzima sacaropina desidrogenase (SDH), mas na ordem de duas vezes, sendo válido para aqueles que apresentaram incremento. Este trabalho mostrou que a alteração no padrão de proteínas de reserva ocasionou mudanças no metabolismo de aminoácidos, neste caso a lisina, para suprir a demanda necessária para incorporação em proteínas de reserva / Cereals represent an important source of protein to human food and animal feed. However, they are characterized by low nutritional quality of proteins due to the unbalanced composition of amino acids, caused by the excess of the amino acids proline and glutamine and deficiency of lysine, threonine and tryptophan. The prolamin storage proteins constitute 50% of the total protein content in the endosperm and is primarily responsible for these characteristics in cereals. Information on the metabolism of lysine and accumulation of storage proteins in endosperm have been used to develop and implement strategies in plant breeding programs that aim to address the deficiencies found in cereals. Lange and coworkers (2007) reported the production of transgenic lines of barley with a pattern of storage proteins that showed altered and increase in the levels of lysine and other amino acids. This study aimed to identify what were the mechanisms responsible for observed changes. For this, we evaluated the activity of enzymes involved in synthesis and degradation of lysine, besides the characterization of storage proteins and their amino acid composition. There was a reduction in the prolamin protein fraction (5.91 to 18.34%) and compensatory increases in the glutelin fractions (2.16 to 6.52%). The other fractions had variable responses depending on the event evaluated. Moreover, the amino acid composition was changed in the different protein fractions. Prolamins exhibited increases in levels of lysine (1.79 to 49.13%), threonine (5.04 to 22.60%) and methionine (13.57 to 45.38%), whereas increases were mainly globulins content of methionine (32.30 to 142.56%). With respect to soluble amino acids, increases were observed in the order of 2-3 fold of histidine, lysine, phenylalanine and methionine. Analysis of enzymes involved in lysine metabolism showed that changes in three key enzymes of the pathway of aspartic acid. The enzyme aspartate kinase (AK) showed increase in activity (4.44 to 47.27%), however, was more sensitive to inhibition by lysine. The enzyme dihydrodipicolinate synthase (DHDPS) also showed increased activity (from 1.50 to 66.32%), but unlike the AK, was less sensitive to inhibition by lysine. The enzyme homoserine dehydrogenase (HSDH) that competes for the substrate ASA with the DHDPS, exhibited reduced activity (3.36% to 28.80%) (an exception one transgenic line) and was less sensitive to inhibition by threonine. The enzymes involved in degradation of lysine were also changed, though the results varied for different events. Those who observed decreased activity of the enzyme lysine ketoglutarate reductase (LOR) was also found for enzyme saccharopine dehydrogenase (SDH), but the order of twice, which was valid for those who had increased. This study showed that the change in the pattern of storage proteins produced changes in amino acid metabolism, in this case lysine, to supply the demand needed for incorporation into storage proteins.

Scald disease (Rhynchosporium secalis) is one of the major plant diseases causing considerable yield loss in barley (Hordeum vulgare) plantations in Turkey. To develop, scald resistant barley varieties, C.R.I.F.C. of Turkey has a large accumulated collection of barley seed sources in hand, but these samples are difficult to be followed and used in the breeding programs due to lack of genetic studies on them. Thus, the objective of this study was to characterize and fingerprint of eighty barley seed sources, and assess the magnitude and pattern of genetic diversity that could be used to have more efficient scald disease resistant breeding programs in the future. Forty scald disease resistant and 40 scald sensitive Turkish barley seed sources were screened using 6 simple sequence repeats (SSR) primers. Each of barley seed source were represented with four seeds, assuming they are genetically uniform since barley is a self-pollinated crop. Estimated genetic parameters indicated that scald disease resistant and sensitive barley seed sources still maintain large amount of genetic diversity. For example, expected heterozygosity was 0.62&plusmn / 0.01 and 0.64&plusmn / 0.01 for resistant and sensitive Turkish barley seed sources, respectively. Thirty-nine percent of total genetic variation was between populations for resistant and 46% for sensitive group, while 61% of total variation was within populations for resistant group and 54% for sensitive group. When overall Turkish barley seed sources were considered, genetic distances between scald sensitive seed source S18 and resistant R1 as well as between sensitive S28 and resistant R1 were large. Scald resistant and sensitive barley seed sources were generally located in different clusters in dendrogram. The presence of R25, R39 and S16 barley seed sources with high genetic diversity parameters among studied seed sources, suggests that this diversity could be important drive in future barley breeding program in Turkey. However, further study is needed to illustrate genetic divergence of Turkish barley seed sources with use of more molecular markers.

In plants, P450 enzymes encoded by P450 genes play a central role in numerous biosynthetic pathways, such as the production of secondary metabolites, stress responses and disease resistance. This thesis reports upon the utilization of molecular biology techniques to study P450 gene(s) in barley (Hordeum vulgare L.).Using several combinations of degenerate primers, a large number of barley P450 gene fragments were cloned and sequenced from two commercial varieties, Chebec and Harrington. Among 247 isolated sequences, twenty six percent were homologous to genes of known function. The abundance of these sequences differed between the two cultivars. Variations in the motif sequences of the cloned genes were also found between these two cultivars. In addition to the cloned fragments, twenty-two putative barley P450 encoding genes were identified from 24,000 cereal sequences in the International Triticeae EST Cooperative (ITEC) database by homology search. Among these Expressed Sequence Tag (EST) sequences, a full-length P450 sequence was selected for further investigation in this study.This novel P450 gene, CYP72A39, was expressed at a very early vegetative stage, but no expression was detected at the reproductive stage. Comparison of expression profiles of this gene and “digital expression” databases confirmed that this gene was homologous to several cereal EST clones with tissue-specific transcripts responding to various environmental stimuli, such as stresses and disease. Among these, many transcripts in barley were obtained from stressed tissues at the vegetative stage, and two transcripts in wheat (Triticum aestivum L.) were expressed after being challenged by barley powdery mildew pathogen (Blumeria graminis f. sp. hordei L.). This suggested that CYP72A39 may play a defence role in the barley seedling.The secondary structure of CYP72A39 was investigated in a comparative analysis using a computational approach. However, attempts to express CYP72A39 in a heterologous system and functional assays of the native protein in barley did not give decisive results, due to the disordered structure of the native protein and limitations of the current method. Screening the 3’ UnTranslated Region (3’UTR) of this gene in 158 genotypes of domesticated, landrace and wild barley revealed two haplotypes, which differed by a 12 base indel positioned between two transversions. The presence of both haplotypes in wild and cultivated barley suggests this polymorphism predates the domestication of barley. This indel was mapped to the long arm of chromosome 6H, less than 10 centi-Morgans (cM) from the gene encoding resistance to powdery mildew in barley (B. graminis). A comparison between haplotype diversity and powdery mildew resistance data for over 102 genotypes showed a weak link between the ‘long’ haplotype and resistance, while the ‘short’ haplotype was associated with susceptibility. There was no evidence for a strong correlation between haplotype and quality type (malt or feed); however, more malting varieties had the long haplotype, suggesting a possible association with some attributes in modern malting barley. There was no evidence for association with other characteristics such as geographic origin, growth habit or row number. Phylogenetic relationship of the CYP72A39 to other CYP72 members was also investigated.

In plants, P450 enzymes encoded by P450 genes play a central role in numerous biosynthetic pathways, such as the production of secondary metabolites, stress responses and disease resistance. This thesis reports upon the utilization of molecular biology techniques to study P450 gene(s) in barley (Hordeum vulgare L.).Using several combinations of degenerate primers, a large number of barley P450 gene fragments were cloned and sequenced from two commercial varieties, Chebec and Harrington. Among 247 isolated sequences, twenty six percent were homologous to genes of known function. The abundance of these sequences differed between the two cultivars. Variations in the motif sequences of the cloned genes were also found between these two cultivars. In addition to the cloned fragments, twenty-two putative barley P450 encoding genes were identified from 24,000 cereal sequences in the International Triticeae EST Cooperative (ITEC) database by homology search. Among these Expressed Sequence Tag (EST) sequences, a full-length P450 sequence was selected for further investigation in this study.This novel P450 gene, CYP72A39, was expressed at a very early vegetative stage, but no expression was detected at the reproductive stage. Comparison of expression profiles of this gene and “digital expression” databases confirmed that this gene was homologous to several cereal EST clones with tissue-specific transcripts responding to various environmental stimuli, such as stresses and disease. Among these, many transcripts in barley were obtained from stressed tissues at the vegetative stage, and two transcripts in wheat (Triticum aestivum L.) were expressed after being challenged by barley powdery mildew pathogen (Blumeria graminis f. sp. hordei L.). This suggested that CYP72A39 may play a defence role in the barley seedling.The secondary structure of CYP72A39 was investigated in a comparative analysis using a computational approach. However, attempts to express CYP72A39 in a heterologous system and functional assays of the native protein in barley did not give decisive results, due to the disordered structure of the native protein and limitations of the current method. Screening the 3’ UnTranslated Region (3’UTR) of this gene in 158 genotypes of domesticated, landrace and wild barley revealed two haplotypes, which differed by a 12 base indel positioned between two transversions. The presence of both haplotypes in wild and cultivated barley suggests this polymorphism predates the domestication of barley. This indel was mapped to the long arm of chromosome 6H, less than 10 centi-Morgans (cM) from the gene encoding resistance to powdery mildew in barley (B. graminis). A comparison between haplotype diversity and powdery mildew resistance data for over 102 genotypes showed a weak link between the ‘long’ haplotype and resistance, while the ‘short’ haplotype was associated with susceptibility. There was no evidence for a strong correlation between haplotype and quality type (malt or feed); however, more malting varieties had the long haplotype, suggesting a possible association with some attributes in modern malting barley. There was no evidence for association with other characteristics such as geographic origin, growth habit or row number. Phylogenetic relationship of the CYP72A39 to other CYP72 members was also investigated.

Diss. (sammanfattning). Alnarp : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 5 uppsatser.