鱼类疫苗的研究：I: 海水鱼类病原菌乳化口服疫苗的研究；II：迟缓爱德华氏菌和嗜水气单胞菌外膜蛋白疫苗的研究

本论文的目的是研究几种病原菌口服疫苗接种鱼类的免疫效果，并从常见病原菌株中筛选几个具有较好保护效果的蛋白抗原，利用口服免疫的方式，接种养殖动物，并检测免疫效果。 以10号白油为有机溶剂，采用搅拌与均浆方法制备鳗弧菌M3和SMP1的油乳化二价疫苗，用饵料包埋后以口服途径免疫养殖大菱鲆，评价免疫大菱鲆的免疫应答和疫苗的保护效果。结果显示，以油乳化和未油乳化疫苗分别连续口服免疫大菱鲆一周后，在后肠组织，乳化疫苗刺激产生的非特异性活力、特异性抗体水平均高于未乳化疫苗；而在血清，两种疫苗引起的两种酶的活力、SMP1抗体水平没有变化，但在乳化疫苗免疫的大菱鲆检测到明显高于未免疫对照大菱鲆的M3抗体水平。大菱鲆后肠组织原位杂交结果显示，口服免疫的大菱鲆后肠褶皱有IgM抗体的产生和分布。其中，乳化疫苗免疫大菱鲆的IgM抗体的产生和分布水平高于未乳化疫苗免疫的大菱鲆。攻毒实验显示，乳化疫苗免疫的大菱鲆对M3和SMP1的感染分别获得100%和50%的免疫保护率，而未乳化疫苗获得的免疫保护率分别为57.9%和0%，表明乳化疫苗比未乳化疫苗更有效地保护大菱鲆、抵抗病原的感染。在乳化疫苗免疫持续期的研究中，免疫的大菱鲆后肠在免疫后120天仍能检测到抗体效价，在免疫后90天还能观察到一定的免疫保护效果。免疫30天、60天、90天和120天的大菱鲆分别获得100%、66.7%、36.7%和13.3%的免疫保护力。 以鳗弧菌M3和SMP1、链球菌CF、迟缓爱德华菌SMW7作为细菌抗原制备油乳化多价口服疫苗和轮虫携带疫苗，口服途径免疫养殖大菱鲆与大菱鲆初孵仔鱼。结果显示，在免疫大菱鲆后肠可检测到抗M3抗体水平的提高（P<0.05），而在其胆汁、鳃、中肠、体表黏液、前肠与血清中抗体效价变化与对照组没有显示出差异；没有检测到免疫大菱鲆后肠抗SMP1、SMW7、CF抗体效价。M3浸泡攻毒实验显示，口服免疫的大菱鲆获得了100%的免疫保护力；在M3注射攻毒和SMP1、CF、SMW7浸泡攻毒大菱鲆的实验中，在每个攻毒组中，免疫组大菱鲆开始死亡的时间都要比对照组有不同程度的延迟，但攻毒大菱鲆都发生死亡，不能显示出与对照组的差异。轮虫携带免疫的结果显示，免疫的大菱鲆初孵仔鱼并未获得较好的保护效果，与对照大菱鲆没有体现出差异。 从致病性病嗜水气单胞菌（Aeromonas hydrophila）LSA34克隆并表达ahaI基因和gapA基因，从迟缓爱德华菌（Edwardsiella tarda）LSE40克隆并表达eseB，将所得蛋白分别通过腹腔注射途径免疫大菱鲆，检测蛋白的免疫原性和免疫保护。结果在免疫后7天就可以检测到AhaI、GapA蛋白免疫组大菱鲆产生的抗体，至第40天可以检测到明显的保护性抗体，之后抗体效价增加明显，直至第60天时达到最高值。EseB免疫的大菱鲆第一次免疫后15天就有较高的抗体效价产生，明显高于对照组大菱鲆血清抗体效价，到距第一次免疫60天时，抗体效价达到最高值。攻毒实验显示，与对照组相比，AhaI免疫组和GapA免疫组对LSA34感染的免疫保护力分别为80%和100%；AhaI免疫组和GapA免疫组对LSE40感染的免疫保护力分别为30%和10%。，而对照组牙鲆对人工攻毒不具有保护力。以AhaI和GapA作为疫苗免疫大菱鲆，使大菱鲆获得了对嗜水气单胞菌LSA34较高的免疫保护；而对迟缓爱德华氏菌LSE40的交叉保护能力没有明显提高。EseB免疫的大菱鲆在攻毒实验中并没有显示出较好的保护效果，与对照组相比，只是在死亡时间上有所延迟。 以从致病性嗜水气单胞菌中克隆的ahaI和gapA基因表达出的蛋白为蛋白抗原制备油乳化疫苗，用饵料包埋后以口服途径免疫养殖牙鲆，评价免疫牙鲆的免疫应答和疫苗的保护效果。结果显示，以油乳化和未油乳化疫苗分别免疫牙鲆一周后，在后肠组织，AhaI和GapA乳化疫苗免疫组牙鲆检测到抗体，且分别高于AhaI和GapA未乳化疫苗免疫的牙鲆；而在血清，GapA的两种疫苗引起的GapA抗体水平没有变化；但在AhaI乳化疫苗免疫的牙鲆第21天和第35天的血清中检测到高于未免疫对照牙鲆的AhaI抗体水平，AhaI未乳化疫苗免疫牙鲆血清对照组相比没有检测到AhaI抗体水平的变化。

Selective and specific detection of sulfate-reducing bacteria using potentiometric stripping analysis

A fast, sensitive and reliable potentiometric stripping analysis (PSA) is described for the selective detection of the marine pathogenic sulfate-reducing bacterium (SRB). Desulforibrio caledoiensis. The chemical and electrochemical parameters that exert influence on the deposition and stripping of lead ion, such as deposition potential, deposition time and pH value were carefully studied. The concentration of SRB was determined in acetate buffer solution (pH 5.2) under the optimized condition (deposition potential of -1.3 V. deposition time of 250 s, ionic strength of 0.2 mol L-1 and oxidant mercury (II) concentration of 40 mg L-1). A linear relationship between the stripping response and the logarithm of the bacterial concentration was observed in the range of 2.3 x 10 to 2.3 x 10(7) cfu mL(-1). In addition, the potentiometric stripping technique gave a distinct response to the SRB, but had no obvious response to Escherichia coli. The measurement system has a potential for further applications and provides a facile and sample method for detection of pathogenic bacteria. (C) 2010 Elsevier B.V. All rights reserved.

A literary occupation: responses of German writers in service in occupied Europe

This thesis examines the literary output of German servicemen writers writing from the occupied territories of Europe in the period 1940-1944. Whereas literary-biographical studies and appraisals of the more significant individual writers have been written, and also a collective assessment of the Eastern front writers, this thesis addresses in addition the German literary responses in France and Greece, as being then theatres of particular cultural/ideological attention. Original papers of the writer Felix Hartlaub were consulted by the author at the Deutsches Literatur Archiv (DLA) at Marbach. Original imprints of the wartime works of the subject writers are referred to throughout, and citations are from these. As all the published works were written under conditions of wartime censorship and, even where unpublished, for fear of discovery written in oblique terms, the texts were here examined for subliminal authorial intention. The critical focus of the thesis is on literary quality: on aesthetic niveau, on applied literary form, and on integrity of authorial intention. The thesis sought to discover: (1) the extent of the literary output in book-length forms. (2) the auspices and conditions under which this literary output was produced. (3) the publication history and critical reception of the output. The thesis took into account, inter alia: (1) occupation policy as it pertained locally to the writers’ remit; (2) the ethical implications of this for the writers; (3) the writers’ literary stratagems for negotiating the constraints of censorship.

Fas ligand expression and tumour immune evasion; the role of prostaglandin E2 and the EP1 receptor

Background and Aim: During carcinogenesis, tumours develop multiple mechanisms to evade the immune system and suppress the anti-tumour immune response. Upregulation of Fas Ligand (FasL/CD95L) expression may represent one such mechanism. FasL is a member of the tumour necrosis factor superfamily that triggers apoptotic cell death following ligation to its receptor Fas. Numerous studies have demonstrated upregulated FasL expression in tumor cells, with FasL expression associated with numerous pro-tumorigenic effects. However, little is known about the mechanisms that regulate FasL expression in tumours. The cyclooxgenase (COX) signalling pathway may play an important role in colon carcinogenesis, via the production of prostaglandins, in particular PGE2. PGE2 signals through four different receptor subtypes, EP1 – EP4. Thus, the aim of this study was to investigate the effect of targeting the PGE2-FasL signaling pathway. Results: (i) PGE2 induces FasL expression via the EP1 receptor in colon cancer cells. (ii) Suppression of FasL expression in colon tumour cells in vivo significantly delays and reduces tumour growth. (iii) Blocking EP1 receptor signaling, or suppression of the EP1 receptor in colon tumour cells, reduces tumour growth in vivo. Suppression of tumour growth correlates in part with suppression of FasL expression. (iv) The reduction in tumour growth is associated with an improved anti-tumour immune response. Tumour infiltration by Treg cells and macrophages was reduced, and the cytotoxic activity of CTL generated from splenocytes isolated from these mice increased. Conclusion: 1) Targeting FasL expression by blocking PGE2-EP1 receptor signalling reduces tumour development in vivo. 2) The mechanism is indirect but is associated with an increased anti-tumour immune response. Thus, unraveling the mechanisms regulating FasL expression and the pro-tumorigenic effects of the EP1 receptor may aid in the search for new therapeutic targets against colon cancer.

Engineering design of a bioprocess for the production of natural red colourants by submerged fermentation of the thermophilic fungus Penicillium purpurogenum GH2

The development of a new bioprocess requires several steps from initial concept to a practical and feasible application. Industrial applications of fungal pigments will depend on: (i) safety of consumption, (ii) stability of the pigments to the food processing conditions required by the products where they will be incorporated and (iii) high production yields so that production costs are reasonable. Of these requirements the first involves the highest research costs and the practical application of this type of processes may face several hurdles until final regulatory approval as a new food ingredient. Therefore, before going through expensive research to have them accepted as new products, the process potential should be assessed early on, and this brings forward pigment stability studies and process optimisation goals. Only ingredients that are usable in economically feasible conditions should progress to regulatory approval. This thesis covers these two aspects, stability and process optimisation, for a potential new ingredient; natural red colour, produced by microbial fermentation. The main goal was to design, optimise and scale-up the production process of red pigments by Penicillium purpurogenum GH2. The approach followed to reach this objective was first to establish that pigments produced by Penicillium purpurogenum GH2 are sufficiently stable under different processing conditions (thermal and non-thermal) that can be found in food and textile industries. Once defined that pigments were stable enough, the work progressed towards process optimisation, aiming for the highest productivity using submerged fermentation as production culture. Optimum production conditions defined at flask scale were used to scale up the pigment production process to a pilot reactor scale. Finally, the potential applications of the pigments were assessed. Based on this sequence of specific targets, the thesis was structured in six parts, containing a total of nine chapters. Engineering design of a bioprocess for the production of natural red colourants by submerged fermentation of the thermophilic fungus Penicillium purpurogenum GH2.

Membrane binding of plasmid DNA and endocytic pathways are involved in electrotransfection of mammalian cells.

Electric field mediated gene delivery or electrotransfection is a widely used method in various studies ranging from basic cell biology research to clinical gene therapy. Yet, mechanisms of electrotransfection are still controversial. To this end, we investigated the dependence of electrotransfection efficiency (eTE) on binding of plasmid DNA (pDNA) to plasma membrane and how treatment of cells with three endocytic inhibitors (chlorpromazine, genistein, dynasore) or silencing of dynamin expression with specific, small interfering RNA (siRNA) would affect the eTE. Our data demonstrated that the presence of divalent cations (Ca(2+) and Mg(2+)) in electrotransfection buffer enhanced pDNA adsorption to cell membrane and consequently, this enhanced adsorption led to an increase in eTE, up to a certain threshold concentration for each cation. Trypsin treatment of cells at 10 min post electrotransfection stripped off membrane-bound pDNA and resulted in a significant reduction in eTE, indicating that the time period for complete cellular uptake of pDNA (between 10 and 40 min) far exceeded the lifetime of electric field-induced transient pores (∼10 msec) in the cell membrane. Furthermore, treatment of cells with the siRNA and all three pharmacological inhibitors yielded substantial and statistically significant reductions in the eTE. These findings suggest that electrotransfection depends on two mechanisms: (i) binding of pDNA to cell membrane and (ii) endocytosis of membrane-bound pDNA.

Surface area, porosity and water adsorption properties of fine volcanic ash particles

Our understanding on how ash particles in volcanic plumes react with coexisting gases and aerosols is still rudimentary, despite the importance of these reactions in influencing the chemistry and dynamics of a plume. In this study, six samples of fine ash (<100 μm) from different volcanoes were measured for their specific surface area, as, porosity and water adsorption properties with the aim to provide insights into the capacity of silicate ash particles to react with gases, including water vapour. To do so, we performed high-resolution nitrogen and water vapour adsorption/desorption experiments at 77 K and 303 K, respectively. The nitrogen data indicated as values in the range 1.1-2.1 m2/g, except in one case where as of 10 m2/g was measured. This high value is attributed to incorporation of hydrothermal phases, such as clay minerals, in the ash surface composition. The data also revealed that the ash samples are essentially non-porous, or have a porosity dominated by macropores with widths >500 Å All the specimens had similar pore size distributions, with a small peak centered around 50 Å These findings suggest that fine ash particles have relatively undifferentiated surface textures, irrespective of the chemical composition and eruption type. Adsorption isotherms for water vapour revealed that the capacity of the ash samples for water adsorption is systematically larger than predicted from the nitrogen adsorption as values. Enhanced reactivity of the ash surface towards water may result from (i) hydration of bulk ash constituents; (ii) hydration of surface compounds; and/or (iii) hydroxylation of the surface of the ash. The later mechanism may lead to irreversible retention of water. Based on these experiments, we predict that volcanic ash is covered by a complete monolayer of water under ambient atmospheric conditions. In addition, capillary condensation within ash pores should allow for deposition of condensed water on to ash particles before water reaches saturation in the plume. The total mass of water vapour retained by 1 g of fine ash at 0.95 relative water vapour pressure is calculated to be ∼10-2 g. Some volcanic implications of this study are discussed. © Springer-Verlag 2004.

Rotación y fertilización en sistemas de producción de la región semiárida bonaerense II : cambios de algunas propiedades químicas del suelo

p.9-14

Dinámica de la población de semillas de Sorghum halepense (L) Pers. en un suelo arable : II-efecto de las labores y de la manipulación del canopeo

p.11-19

Vibrational spectroscopy and DFT calculations of di-amino acid cyclic peptides. Part I: cyclo(Gly-Gly), cyclo(L-Ala-L-Ala) and cyclo(L-Ala-Gly) in the solid state and in aqueous solution

Investigations of the vibrational spectra of cyclo(Gly-Gly), cyclo(L-Ala-L-Ala) and cyclo(t-Ala-Gly) are reported. Raman scattering and Fourier transform infrared (FTIR) spectra of solid-state and aqueous protonated samples, as well as their corresponding N-deuterated isotopomers, have been examined. In addition, density functional theory (DFT) (B3-LYP/cc-pVDZ) calculations of molecular structures and their associated vibrational modes were carried out. In each case, the calculated structures of lowest energy for the isolated gas-phase molecules have boat conformations. Assignments have been made for the observed Raman and FTIR vibrational bands of the cyclic di-amino acid peptides (CDAPs) examined. Raman polarization studies of aqueous phase samples are consistent with C-2 and C-1 symmetries for the six-membered rings of cyclo(L-Ala-L-Ala) and cydo(L-Ala-Gly), respectively. There is a good correlation between experimental and calculated vibrational bands for the three CDAPs. These data are in keeping with boat conformations for cydo(L-Ala-L-Ala) and cyclo(L-Ala-Gly) molecules, predicted by the ab initio calculations, in both the solid and aqueous solution states. However, Raman spectroscopic results might infer that cyclo(L-AlaGly) deviates only slightly from planarity in the solid state. The potential energy distributions of the amide I and II modes of a cis-peptide linkage are shown to be significantly different from those of the trans-peptides. For example, deuterium shifts have shown that the cis-amide I vibrations found in cyclo(Gly-Gly), cyclo(L-Ala-L-Ala), and cyclo(L-Ala-Gly) have larger N-H contributions compared to their trans-amide counterparts. Compared to trans-amide II vibrations, cis-amide II vibrations show a considerable decrease in N-H character.