Abstract The plant can be considered a superorganism that consists of the plant per se and numerous populations of pro- and eukaryotic microorganisms. The interactions between the plant and endophytic microorganisms colonizing plant internal tissues are typically commensalistic or mutualistic. However, information on the role of endophytes in plant defense is limited because pathways are only partly known and systemic responses are typically not seen. The aim of this thesis was to study the priming capacities of endophytic Methylobacterium sp. IMBG290 on potato (Solanum tuberosum L.). Priming of plants by non-pathogenic bacteria allows the host to save energy and to reduce time needed for development of defense reaction during a pathogen attack. Priming phenomenon was demonstrated for Methylobacterium sp. IMBG290 as an activation of salicylic acid and jasmonate/ethylene-dependent defense pathways after challenge inoculation with the pathogen. Moderate activation of plant antioxidant system may also contribute to resistance induction by the strain. The viable but nonculturable state is presumably a survival strategy observed for the majority of bacterial endophytes. Pathogen attack or environmental changes can activate these quiescent forms. Thus Methylobacterium+ sp. IMBG290 became cultivable upon plant inoculation by nonpathogenic bacteria. I observed that the composition of the endophyte community changed in response to Methylobacterium sp. IMBG290 inoculation in shoot tissues and correlated with potato disease resistance and growth promotion. Therefore, the activation of endophytic bacterial populations as a putative mechanism of plant disease resistance was proposed. Endophytes have a high agricultural potential. Growth- and resistance-promoting capacities of Methylobacterium sp. IMBG290 on potato were highly variable depending on the cultivar, pathogen, inoculum density, and environmental conditions. Context-dependent efficacy requires more attention when designing complex microbial inoculants capable influencing positively plant growth, resistance, and nutritional properties. / Tiivistelmä Kasvia voidaan pitää superorganismina, joka koostuu kasvista itsestään ja lukuisista pro-ja eukaryottisista mikrobipopulaatioista. Kasvin ja sen sisäosia asuttavien endofyyttisten mikro-organismien väliset vuorovaikutukset ovat yleensä kommensalistisia tai mutualistisia. Endofyyttien rooli kasvin puolustuksessa on kuitenkin huonosti tunnettu, koska reitit tiedetään vain osittain eikä järjestelmällisiä vasteita usein havaita. Tämän väitöskirjatyön tavoitteena oli tutkia endofyyttisen Methylobacterium sp. IMBG290-kannan kykyä vahvistaa perunan (Solanum tuberosum L.) puolustusta. Tautia aiheuttamattomat bakteerit kykenevät vahvistamaan kasvien puolustusta, mikä auttaa isäntäkasvia säästämään energiaa ja nopeuttamaan puolustusreaktiota patogeenihyökkäyksen aikana. Methylobacterium sp. IMBG290-kannan vahvistuskyvyn osoitettiin perustuvan salisyylihappo- ja jasmonaatti/etyleeni-riippuvaisten puolustusreittien aktivoimiseen patogeeni-istutuksen jälkeen. Antioksidanttijärjestelmän lievä aktivoituminen voi myös vaikuttaa kannan aiheuttamaan vastustuskyvyn lisääntymiseen. Suurimmalle osalle bakteeriendofyyteistä ‘elävä mutta viljelemätön’-olotila on luultavasti selviytymisstrategia. Patogeenihyökkäys tai muutokset ympäristössä voivat aktivoida tällaiset hiljaiset olomuodot. Methylobacterium sp. IMBG290-kanta muuttui viljeltävissä olevaan muotoon kun kasviin istutettiin tautia aiheuttamaton bakteeri. Selvitin, että endofyytti-yhteisön koostumus muuttuu vasteena Methylobacterium sp. IMBG290-kannan istuttamiseen kasvin verson solukoissa, korreloiden lisääntyneen perunan vastustuskyvyn ja kasvun kanssa. Siksi endofyyttisten bakteeripopulaatioiden aktivoitumista esitettiin uutena kasvin puolustusmekanismina. Endofyyteillä on suuret mahdollisuudet maataloudessa. Methylobacterium sp. IMBG290-kannan kasvua ja vastustuskykyä lisäävät ominaisuudet perunalla vaihtelivat lajikkeen, patogeenin, lisätyn bakteeriympin ja ympäristöolosuhteiden mukaan. Suunniteltaessa monimutkaisia bakteeriymppejä kasvien kasvin, vastustuskyvyn ja ravintosisällön lisäämiseksi, täytyy tällainen tilanteesta riippuva tehokkuus ottaa enemmän huomioon.

Abstract Seed potato is the starting point in the potato (Solanum tuberosum L.) production chain. In order to secure potato production in a variety of production conditions, plant diseases must be controlled and the yield characteristics of the used cultivars ensured. In addition, production must be cost-effective. Characteristics particular to northern production conditions include long periods of daylight and a short growing season as well as a several months long seed potato storage period. The focus of the present study is on northern production conditions and methods, including haulm killing and sprout control, which are presumed to affect seed potato quality, as well as the initial stages of the seed potato production chain, i.e. micro- and minituber production, which could influence cost-effectiveness and propagation. Haulm killing is one of the methods used in seed potato production to regulate tuber size. It is often carried out on unsenesced plants. The present results, however, indicate that cultivar properties have a greater effect on the sprouting and crop yield of seed potatoes than production-phase haulm killing or temperature sum accumulation. Nevertheless, haulm killing carried out three weeks after flowering (75 DAP) accelerated emergence. When the effect of haulm killing methods on seed potatoes was compared with natural haulm senescence, haulm killing was shown to increase disease pressure. Black scurf (Rhizoctonia Solani) was present in seed tubers whose haulm had been destroyed by mechanical or mechanical-chemical haulm killing. Naturally senesced haulm had less black scurf, and crop quantity and starch content developed to a level typical of the cultivar. Storage periods lasting several months make controlling seed potato sprouting more challenging. Therefore, use of the plant hormone gibberellic acid (GA) in sprout control was investigated. GA treatments at lower concentration (100 mM) increased the number of tubers in the cultivar Fambo. Thus, the timing of haulm killing and in the case of Fambo, GA treatment influenced the characteristics of seed potatoes. Conventionally, the first tuber generation is produced using microplants to produce minitubers in greenhouses. This production method is, however, labour-intensive, and energy and investment costs are high. With the aim of increasing production efficiency in northern production conditions, the production of minitubers in the laboratory using a novel bioreactor technology and in growth rooms using the hydroponic technique was investigated. The Liquid LabTM Rocker bioreactor system was used in vitro, all the cultivars examined (Asterix, Timo, Van Gogh and Velox) produced microtubers. The quantity of tubers produced per dish varied between 30 (for the cultivar Asterix in eight weeks) and 75 (for the cultivar Velox in 11 weeks). The results showed hydroponic production of minitubers to be successful in indoor conditions: the cultivars Desiree and Van Gogh developed their first tuber three weeks faster than Asterix, and the minituber yield was 4.5 per plant for Desiree, 7.5 for Van Gogh and 4.0 for Asterix. When the results indicate that both the Liquid LabTM Rocker production method and the hydroponic production method are suitable for mass production of seed potatoes. / Tiivistelmä Siemenperuna on lähtökohta perunan (Solanum tuberosum L.) tuotantoketjussa. Jotta perunantuotanto turvataan eri tuotanto-olosuhteissa, on hallittava siemenperunan kasvitaudit, taattava sadontuotto-ominaisuudet ja tuotannon on oltava lisäksi kustannustehokasta. Pohjoisissa tuotanto-olosuhteissa erityispiirteinä ovat valoisuudeltaan pitkät päivät ja kestoltaan lyhyet kasvukaudet. Lisäksi siemenperunoiden varastointijakso kestää useita kuukausia. Tässä tutkimuksessa selvitettiin vaikuttavatko pohjoiset tuotanto-olosuhteet tai tuotannossa käytetyt varsistonhävitys tai itämisen hallinta siemenperunoiden laatuominaisuuksiin. Lisäksi selvitettiin siemenperunoiden ensimmäisen mukulasukupolven (mikro- ja minimukula) tuotantoa eri tekniikoilla. Siemenperunatuotannossa varsistonhävitystä käytetään mukulakoon säätelykeinona. Varsistonhävitys tehdään usein tuleentumattomaan kasvustoon. Saatujen tulosten perusteella lajikeominaisuudet vaikuttivat itämiseen ja sadontuotto-ominaisuuksiin enemmän kuin varsistonhävitys tai mukuloihin kerääntynyt lämpösumma. Varsistonhävitys kolme viikkoa kukinnasta (75 päivää istutuksen jälkeen) nopeutti kuitenkin siemenperunoiden taimettumista. Vertailtaessa varsistonhävitysmenetelmien vaikutusta siemenperunaan verranteena kasvuston luontainen tuleentuminen, varsistonhävitys lisäsi kasvitautipainetta. Mekaanis-kemiallisesti ja mekaanisesti varsistonhävityissä satomukuloissa tuli esiin seittirupea (Rhizoctonia solani). Luontaisesti tuleentuneen kasvuston sadoissa oli seittirupea vähemmän ja myös sadon määrä ja tärkkelyspitoisuus kehittyivät lajikkeelle luontaiselle tasolle. Useiden kuukausien varastointijakso vaikeuttaa siemenperunoiden itämisen hallintaa. Kun tutkittiin gibberelliinihapon (GA) käyttöä itämisen hallintaan, alhaisemman konsentraation (100mM) GA –käsittely lisäsi Fambo –lajikkeen mukulalukumäärää. Tulosten perusteella varsistonhävityksen ajoittamisella ja GA –käsittelyllä (Fambo –lajike) vaikutettiin siemenperunoiden ominaisuuksiin. Siemenperunan ensimmäisen mukulasukupolven eli minimukuloiden tuottaminen tapahtuu perinteisesti kasvihuonekasvatuksena mikrokasveista. Tuotantotapa on työvoima-, energia- ja invetointikustannuksia vaativaa. Tuotannon tehostamiseksi pohjoisissa tuotanto-olosuhteissa tutkittiin mikro- ja minimukuloiden tuotantoa eri teknologioilla. Mikromukuloita tuotettiin bioreaktorimenetelmällä laboratoriossa ja minimukuloita hydroponisella menetelmällä kasvatushuoneissa. Tulokset osoittavat, että kaikki tutkitut lajikkeet (Asterix, Timo, Van Gogh ja Velox) tuottivat bioreaktorissa mikromukuloita. Mikromukuloiden määrä vaihteli 30:sta (Asterix, 8 viikon kasvatus) 75:een (Velox, 11 viikon kasvatus). Myös minimukuloiden hydroponinen tuotanto sisätiloissa on mahdollista; kaikki lajikkeet muodostivat mukuloita, Desiree ja Van Gogh 3 viikkoa nopeammin kuin Asterix. Desiree tuotti minimukuloita 4.5 kpl/kasvi, Van Gogh 7.5 ja Asterix 4.0. Tulokset osoittavat, että molemmat menetelmät (bioreaktori ja hydroponinen) soveltuvat mikro- ja minimukuloiden massatuotantoon.

High temperature limit successful potato cultivation in the lowlands of tropical regions. One effect of high temperature may be an increase in gibberellin activity that is inhibitory to tuberization. Paclobutrazol blocks gibberellin biosynthesis and reduces its level in the plant. The effect of paclobutrazol on potato was examined under non-inductive conditions in a greenhouse and under field conditions in the hot tropical lowlands of eastern Ethiopia. Paclobutrazol was applied as a foliar spray or soil drench at rates equivalent to 0, 2, 3, and 4 kg a. i. per ha. Paclobutrazol increased chlorophyll a and b content, and photosynthetic efficiency, enhanced early tuber initiation, delayed physiological maturity, and increased tuber fresh mass, dry matter content, specific gravity and crude protein content. It reduced the number of tubers per plant and extended the tuber dormancy period. Paclobutrazol reduced shoot growth, and plant height, and increased the partitioning of assimilates to the tubers while reducing assimilate supply to the leaves, stems, roots and stolons. Stomatal conductance and the rate of transpiration were reduced. In addition, paclobutrazol treatment increased tuber N, Ca and Fe content while reducing P, K and Mg content. Growth analyses indicated that paclobutrazol decreased leaf area index, crop growth rate, and total biomass production. It increased specific leaf weight, tuber growth rate, net assimilation rate, and partitioning coefficient (harvest index). Microscopic observations showed that leaves of treated plants developed thicker epicuticular wax layers. The epidermal, palisade and spongy mesophyll cells were larger. It increased the thickness of the cortex and the size of vascular bundles and pith cells of the stem. It also increased the width of the cortex and favoured the formation of more secondary xylem vessels, resulting in thicker roots. Deposition of starch grains in the stem pith cells, and cortical cells of the stem and root, were stimulated in response to paclobutrazol treatment. In most instances the method of application did not affect the efficiency of paclobutrazol. The effect of cultivar and reproductive growth on growth, photosynthetic efficiency, water relations, dry matter production, tuber yield and quality of potato was also the subject of investigation. Non-flowering, flowering and fruiting plants of cultivars Al-624, Al-436, CIP-388453-3(A) and CIP-388453-3(B) were evaluated under field conditions of a sub-humid tropical highland of eastern Ethiopia. Cultivars exhibited differences with respect to leaf stomatal conductance, rate of transpiration, net photosynthesis, biomass production and allocation, tuber yield, tuber size distribution, specific gravity, dry matter content and nutrient composition. Fruiting plants had higher leaf stomatal conductance, and higher rates of transpiration and photosynthesis rates. The leaf area index, tuber growth rate, and partitioning coefficient (harvest index) of the fruiting plants were reduced, but crop growth rates and net assimilation rates were higher. Without affecting total dry matter production, fruit development reduced the amount partitioned to the leaves, stems, roots, and tubers. Fruit development reduced total and marketable tuber mass and tuber numbers. The effect of MCPA and paclobutrazol were studied under greenhouse and field conditions. Single foliar sprays were applied during the early and full bud development stages at rates of 0, 250, 500, and 750 g a.i. ha-1. Both MCPA and paclobutrazol greatly reduced the number of flowers and completely inhibited berry set. MCPA did not affect the number, yield, dry matter content and specific gravity of tubers. Without affecting the number of tubers, paclobutrazol increased tuber yield, dry matter content and specific gravity. / Thesis (DPhil (Horticultural Science))--University of Pretoria, 2007. / Plant Production and Soil Science / unrestricted

Orientador: Leandro Borges Lemos / Banca: Dirceu Maximino Fernandes / Banca: Domingos Fornasieri Filho / Banca: Paulo Cesar Tavares de Melo / Banca: Newton do Prado Granja / Resumo: A cultura da batata tem grande importância econômica para o Brasil e o Estado de São Paulo, sendo Vargem Grande do Sul e Itapetininga os principais pólos produtores paulistas. Nestes, além da cultura da batata, a do feijoeiro destaca-se por sua importância econômica e social. Geralmente os produtores utilizam o feijoeiro em sucessão à batata e objetivando obter alta produtividade de grãos, realizam a adubação de semeadura, desprezando a possível influência residual da adubação da batata. O trabalho objetivou avaliar a influência da adubação de plantio na cultura da batata, da fertilização residual da batata e de semeadura sobre o feijoeiro em sucessão. Para isso foram avaliados por meio de sua contribuição nas características agronômicas e nutricionais das plantas, das alterações nos atributos químicos do solo, bem como a combinação recomendada da adubação de plantio e de semeadura para a batata e o feijão quanto ao retorno econômico. Foram conduzidos dois experimentos, sendo o primeiro em Itapetininga (SP) num Nitossolo Vermelho Distroférrico típico, textura argilosa e o segundo em São Manuel (SP) num Latossolo Vermelho-Amarelo Distrófico típico, textura média. Em ambos foi utilizado o delineamento experimental de blocos casualizados em esquema de parcelas subdivididas, com quatro repetições. As parcelas foram constituídas das adubações de plantio da batata (0, 1.000, 2.000, 3.000 e 4.000 kg ha-1 de 8-28-16) e as subparcelas pela adubação de semeadura do feijoeiro (0 e 330 kg ha-1 de 8-28-16 em Itapetininga e 0, 150, 300 e 450 kg ha-1 de 8-28-16 em São Manuel). Foram utilizadas as cultivares de batata e de feijão Agata e Pérola, respectivamente. Para o solo de textura argilosa a produtividade total e comercial de tubérculos aumentou até a dose próxima de 3.000 kg ha-1 de 8-28-16. A produtividade...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Potato crop has great economical importance for Brazil and São Paulo State. In São Paulo State, Vargem Grande do Sul and Itapetininga are the main producing areas, where beans crop also plays a major economical and social role. Usually, growers plant beans crop after potato crop, aiming high grain yield. They make sowing fertilization and ignore the possible residual influence of potato fertilization. Therefore, the purpose of this research was to evaluate the planting fertilization influence on the potato crop, the residual potato fertilization influence on beans crop in sequence to potato crop, through its contribution in the plant nutrition and agronomic characteristics, and changes in electrical conductivity and chemical soil attributes. This study also tries to find the best economic combination of sowing and planting fertilization recommended for potato and beans crop. Two experiments were carried out, the first one being in Itapetininga (SP) in a typical "Nitossolo Vermelho Distroférrico", clay texture, and the second one in São Manuel (SP) in a typical "Latossolo Vermelho-Amarelo Distrófico", sandy loam texture. The experimental design was in randomized blocks with split plot analyses, with four replicates. The plot treatments consisted of potato fertilization (0, 1,000, 2,000, 3,000 and 4,000 kg ha-1 of 8-28-16) and the split plot of beans sowing fertilization (0 and 330 kg ha-1 of 8-28-16 in Itapetininga, and 0, 150, 300 and 450 kg ha-1 of 8-28-16 in São Manuel). Agata and Pérola varieties for potato and beans, respectively, were used in the experiments. For the clay texture the total and commercial tuber yields increased until near 3,000 kg ha-1 of 8-28-16. No grain yield response to potato fertilization residual was observed. The beans crop fertilization reduced the grain yield. The use of 8-28-16 fertilization increased the P, K and Ca and CE contents in the soil...(Complete abstract click electronic access below) / Doutor

The subject of my thesis was to genetically modify a potato for increased resistance against its pathogens and pests. In developing a resistant plant, it is quite common to use the same type of molecules that plants use themselves in their defense reactions. In this work I used the gene SPI-2 originating from a honeycomb moth (Galleria mellonella). The protein SPI-2 is a member of serine protease inhibitors. Since the previous attempts of the team to detect the protein in transformed plants haven't been successful, the basic form of the gene was modified by adding a Kozak sequence near the start codon, which should have increased the translation initiation and hence increase the level of the protein. Two constructs were prepared for the transformation: SPI-2-T a SPI-2-Y. They differ by one amino acid, which slightly changes their inhibitory activity. First, the construct SPI-2-T was used for a transient transformation of tobacco Nicotiana benthamiana by agroinfiltration of its leaves. Then both constructs were used for a stable transformation of Solanum tuberosum cv. Desireé. The detection of the protein has not been successful, although the inserted gene was transcribed and his sequence was verified by sequencing. It is therefore most likely that the protein has a low stability in the cytoplasm....

Grundlage der vorgelegten Arbeit sind die bei der Alzheimerschen Erkrankung beobachtbaren pathologischen Merkmale, wie die progressive Akkumulation von β-Amyloid-Plaques, cholinerger Dysfunktion und zerebrovaskuläre Abnormalitäten. Die in englischer Sprache verfasste Dissertation ist eine tierexperimentelle Studie, die versucht, den Zusammenhang von β-Amyloid, cholinerger Neurotransmission und zerebralem Gefäßsystem bei der Alzheimerschen Erkrankung näher zu charakterisieren. An Hirnmaterial aus der transgenen Maus Tg2576, die die schwedische Mutation des humanen Amyloidpräkursorproteins als Transgen trägt und ab dem 10. Lebensmonat durch humane β-Amyloid-Plaqueablagerungen in der Hirnrinde imponiert, wurden im Altersverlauf (4 bis 18 Monate) immunhistochemische Untersuchungen zur morphologischen Integrität der zerebralen Mikrogefäße, der kortikalen cholinergen Nervterminalen und der intrazerebralen cholinergen neurovaskulären Innervation durchgeführt. Am somatosensorischen Kortex werden beispielhaft die Expression des Glukosetransporters 1 oder Solanum tuberosum Lektin als Kapillarmarker und des vesikulären Acetylcholintransporters als Marker für cholinerge Fasern mittels Immunfluoreszenz und Laser-Scanning Mikroskopie erfasst, einer semiquantitativen Computer-gestützten Bildanalytischen Auswertung unterzogen und mit dem Ausmaß der kortikalen Plaquebeladung korreliert. So konnte gezeigt werden, dass die Dichte der Blutgefäße und cholinergen Fasern im somatosensorischen Kortex von transgenen Tieren mit dem Alter im Vergleich zu nichttransgenen Kontrolltieren abnimmt, was mit einer Reduktion der perivaskulären cholinergen Innervation einhergeht. Die erhobenen Befunde stützen die von J.C. de la Torre und T. Mussivand schon im Jahre 1993 formulierte „vaskuläre Hypothese“, wonach bei der sporadischen Form der Alzheimerschen Erkrankung alters- und Lebensstil-bedingte Schädigungen des zerebralen Gefäßsystems eine zentrale Rolle bei der Manifestierung der Erkrankung spielen.

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Grundlage der vorgelegten Arbeit sind die bei der Alzheimerschen Erkrankung beobachtbaren pathologischen Merkmale, wie die progressive Akkumulation von β-Amyloid-Plaques, cholinerger Dysfunktion und zerebrovaskuläre Abnormalitäten. Die in englischer Sprache verfasste Dissertation ist eine tierexperimentelle Studie, die versucht, den Zusammenhang von β-Amyloid, cholinerger Neurotransmission und zerebralem Gefäßsystem bei der Alzheimerschen Erkrankung näher zu charakterisieren. An Hirnmaterial aus der transgenen Maus Tg2576, die die schwedische Mutation des humanen Amyloidpräkursorproteins als Transgen trägt und ab dem 10. Lebensmonat durch humane β-Amyloid-Plaqueablagerungen in der Hirnrinde imponiert, wurden im Altersverlauf (4 bis 18 Monate) immunhistochemische Untersuchungen zur morphologischen Integrität der zerebralen Mikrogefäße, der kortikalen cholinergen Nervterminalen und der intrazerebralen cholinergen neurovaskulären Innervation durchgeführt. Am somatosensorischen Kortex werden beispielhaft die Expression des Glukosetransporters 1 oder Solanum tuberosum Lektin als Kapillarmarker und des vesikulären Acetylcholintransporters als Marker für cholinerge Fasern mittels Immunfluoreszenz und Laser-Scanning Mikroskopie erfasst, einer semiquantitativen Computer-gestützten Bildanalytischen Auswertung unterzogen und mit dem Ausmaß der kortikalen Plaquebeladung korreliert. So konnte gezeigt werden, dass die Dichte der Blutgefäße und cholinergen Fasern im somatosensorischen Kortex von transgenen Tieren mit dem Alter im Vergleich zu nichttransgenen Kontrolltieren abnimmt, was mit einer Reduktion der perivaskulären cholinergen Innervation einhergeht. Die erhobenen Befunde stützen die von J.C. de la Torre und T. Mussivand schon im Jahre 1993 formulierte „vaskuläre Hypothese“, wonach bei der sporadischen Form der Alzheimerschen Erkrankung alters- und Lebensstil-bedingte Schädigungen des zerebralen Gefäßsystems eine zentrale Rolle bei der Manifestierung der Erkrankung spielen.:CHAPTER 1: INTRODUCTION 1.1 Alzheimer’s disease 1 1.2 APP processing and β-amyloid production 2 1.3 Cholinergic dysfunction in Alzheimer’s disease 5 1.4 Cerebrovascular abnormalities in Alzheimer’s disease 8 1.5 Cholinergic innervation of intracortical cerebral microvessels 9 1.6 Transgenic Tg2576 mouse model of Alzheimer’s disease 11 1.7 Aim of study 14 CHAPTER 2: MATERIALS AND METHODS 2.1 Materials 15 2.1.1 Chemical reagents used 15 2.1.2 Biological reagents used 15 2.1.3 Preparation of solutions and buffers 15 2.1.4 Antibodies and reagents used for immunohistochemistry 17 2.1.5 Transgenic animals 19 2.2 Methods 20 2.2.1 Tissue preparation and sampling of sections 20 2.2.2 Immunohistochemistry 20 2.2.2.1 Protocol of immunofluorescent labeling 20 2.2.2.2 Protocol of immunoperoxidase labeling (ABC technique) 21 2.2.2.3 Combination of primary and secondary antibodies 22 2.2.2.4 Protocol of β–amyloid immunolabeling (Formic acid epitope retrieval method) 23 2.2.3 Histochemistry 23 2.2.3.1 Thioflavin S staining 23 2.2.3.2 Nissl staining 23 2.2.3.3 Solanum Tuberosum Lectin (STL) staining 24 2.2.4 Double and triple-coloured immuno-/ histochemical staining of brain sections 24 2.2.5 Microscopy and digital image processing 25 2.2.6 Morphological and morphometric analyses 25 2.2.6.1 Cortical microvessels 25 2.2.6.2 Cortical cholinergic innervation 27 2.2.6.2.1 Total density of VAChT-immunoreactivity 27 2.2.6.2.2 Estimation of the density of varicosities on cholinergic fibres 29 2.2.6.3 Estimation of cholinergic perivascular innervation of cortical microvessels 29 2.2.6.4 Three-dimensional-imaging of vessels innervation 30 2.2.7 Statistical analysis 30 CHAPTER 3: RESULTS 3.1 Developmental and amyloid plaque-related changes in cerebral cortical capillaries in transgenic Tg2576 Alzheimer mice 31 3.1.1 Morphological distribution of brain vessels in the cerebral cortex of wild type mice 31 3.1.2 Microvessel density under plaque burden 33 3.2 Developmental and amyloid plaque-related changes in cholinergic neurotransmission in cholinoceptive target regions of transgenic Tg2576 mice 39 3.2.1 Visualisation of cholinergic nerve terminals in mouse brain 39 3.2.2 VAChT-Expression in wild type and transgenic Tg2576 mice 40 3.3 Role of cholinergic system in β-amyloid-related changes in the cerebrovascular system of transgenic Tg2576 mice 46 3.3.1 Solanum tuberosum lectin (STL) histochemistry in visualisation of brain vessels, β-amyloid, and microglia 46 3.3.1.1 Solanum tuberosum lectin and brain vessels 46 3.3.1.2 Solanum tuberosum lectin and β-amyloid plaques 47 3.3.1.3 Solanum tuberosum lectin staining to visualize glial cells 48 3.3.2 Cholinergic perivascular innervation of cerebral cortical microvessels in transgenic Tg2576 and wild type mice 50 CHAPTER 4: DISCUSSION 4.1 β-Amyloid and brain vascular system: the vascular hypothesis of Alzheimer’s disease 55 4.1.1 Evidences of a role of vascular mechanisms in Alzheimer’s disease 55 4.1.2 Effect of β-amyloid on brain vascular system 57 4.1.3 Effect of ischemia and hypoperfusion on APP processing 59 4.1.4 Effect of β-amyloid on cholinergic function in brain vascular system 59 4.2 Aim of study and main results obtained 61 4.3 Age-related changes in cerebral cortical microvessels in the presence and absence of β-amyloid plaque load 62 4.4 Age-related changes of cholinergic terminals in cholinoceptive target regions in the presence and absence of β-amyloid plaque load 64 4.4.1 VAChT – a reliable marker for detection of cholinergic terminals in cerebral cortex 64 4.4.2 The barrel field of the somatosensory cortex 1 (S1BF) as a model region to reveal age-related changes in cholinergic innervation 65 4.4.3 VAChT expression: morphological and morphometric studies 66 4.5 Age-related changes in cholinergic innervation of cerebral cortical microvessels in the presence and absence of β-amyloid plaque load 69 4.5.1 STL – a mono-marker for detection of cortical vessels, senile amyloid plaques and activated microglia in cerebral cortex 69 4.5.2 Cholinergic perivascular innervation of cerebral cortical microvessels in transgenic Tg2576 mice 70 4.5.3 Quantitation of cholinergic input on cerebral microvessels of mouse brain 71 4.6 Summary and conclusions 75 REFERENCES 77

Le fer est un micronutriment important pour la croissance et le développement des plantes. Il agit comme cofacteur pour plusieurs enzymes et il est important pour des processus tels que la photosynthèse et la respiration. Souvent, le Fe dans le sol n’est pas bio-disponible pour la plante. Les plantes ont développé des stratégies pour solubiliser le Fe du sol pour le rendre disponible et assimilable pour elles. Il y a deux stratégies, la première est caractéristique des dicotylédones et la seconde est caractéristique des monocotylédones. Le modèle utilisé dans cette étude est une culture cellulaire de Solanum tuberosum. Une partie de la recherche effectuée a permis la mesure d’activité et d’expression relative de certaines enzymes impliquées dans le métabolisme énergétique et la fourniture de précurseurs pour la synthèse d’ADN : la Nucléoside diphosphate kinase, la Ribonucléotide reductase, la Glucose 6-phosphate déshydrogénase et la 6-Phosphogluconate déshydrogénase dans les cellules en présence ou en absence de Fe. Chez certains organismes, la déficience en Fe est associée à une perte de croissance qui est souvent liée à une diminution de la synthèse d’ADN. Chez les cultures de cellules de S. tuberosum, les résultats indiquent que la différence de biomasse observée entre les traitements n’est pas due à une variation de l’activité ou l’expression relative d’une de ces enzymes. En effet, aucune variation significative n’a été détectée entre les traitements (+/- Fe) pour l’activité ni l’expression relative de ces enzymes. Une autre partie de la recherche a permis d’évaluer l’activité des voies métaboliques impliquées dans la stratégie 1 utilisée par S. tuberosum. Cette stratégie consomme des métabolites énergétiques: de l’ATP pour solubiliser le Fe et du pouvoir réducteur (NAD(P)H), pour réduire le Fe3+ en Fe2+. Des études de flux métaboliques ont été faites afin d’étudier les remaniements du métabolisme carboné en déficience en Fe chez S. tuberosum. Ces études ont démontré une baisse du régime dans les différentes voies du métabolisme énergétique dans les cellules déficientes en Fe, notamment dans le flux glycolytique et le flux de C à travers la phosphoenolpyruvate carboxylase. En déficience de Fe il y aurait donc une dépression du métabolisme chez S. tuberosum qui permettrait à la cellule de ralentir son métabolisme pour maintenir sa vitalité. En plus des flux, les niveaux de pyridines nucléotides ont été mesurés puisque ceux-ci servent à réduire le Fe dans la stratégie 1. Les résultats démontrent des niveaux élevés des formes réduites de ces métabolites en déficience de Fe. L’ensemble des résultats obtenus indiquent qu’en déficience de Fe, il y a une baisse du métabolisme permettant à la cellule de s’adapter et survivre au stress. / Iron is an important micronutrient for plant growth and development. It participates as a cofactor for several enzymes and is important for processes such as photosynthesis and respiration. Often soil Fe is not bioavailable to the plant. Plants have developed strategies to solubilize the Fe in the soil to make it available and easy to assimilate. There are two strategies, the first is characteristic of dicotyledones and the second is characteristic of monocotyledones. The model used in these studies is a cell culture of Solanum tubersoum. A first part of the research involved the study of expression and activity of enzymes required in energy metabolism and the provision of precursors for DNA synthesis: Nucleoside dehydrogenase, Ribonucleotide reductase, Glucose 6-phohate dehydrogenase and 6-Phosphogluconate dehydrogenase. In several organisms, Fe deficiency induces a loss of biomass which is often associated with a decrease in DNA synthesis. In S. tuberosum cell cultures, the results indicate that the loss of biomass observed in Fe deficiency is not linked to a change in the activity or relative expression of these enzymes. Indeed, no significant changes were detected between treatments (+/- Fe) for activity or relative expression. In another part of the research, we evaluated the activity of the metabolism pathways involved in strategy 1, which is used by S. tuberosum. This strategy consumes energetic metabolites: ATP to solubilize Fe and reducing power (NAD(P)H) to reduce the Fe3+ to Fe2+. Metabolic flux studies were done to investigate the alterations of carbon metabolism during Fe deficiency in S. tuberosum. These studies demonstrated that in Fe deficient cells, there is a decrease in the fluxes of some pathways of energy metabolism. Particularly, in the glycolytic flux and the anaplerotic flux of PEPC. Under Fe deficiency there would be a depression of metabolism in S. tuberosum which would allow the cell to slow its metabolism to maintain its vitality. In addition to the fluxes, the levels of pyridine nucleotides were measured since they serve to reduce Fe in the strategy 1. The results show an increase in the reduced forms of these metabolites during Fe deficiency. All results together point out that during Fe deficiency the metabolism decreases, allowing the cell to survive and adapt to the stress.