Identification of parasitoid species using PCR amplification and restriction enzyme digestion

Spodoptera frugiperda is a pest of great economic importance in the Americas. It is attacked by several species of parasitoids, which act as biological control agents. Parasitoids are morphologically identifiable as adults, but not as larvae. Laboratory rearing conditions are not always optimal to rear out parasitic wasps from S. frugiperda larvae collected from wild populations, and it frequently happens that parasitoids do not complete their life cycle and stop developing at the larval stage. Therefore, we explored ways to identify parasitoid larvae using molecular techniques. Sequencing is one possible technique, yet it is expensive. Here we present an alternate, cheaper way of identifying seven species of parasitoids (Cotesia marginiventris, Campoletis sonorensis, Pristomerus spinator, Chelonus insularis, Chelonus cautus, Eiphosoma vitticolle and Meteorus laphygmae) using PCR amplification of COI gene followed by a digestion with a combination of four restriction endonucleases. Each species was found to exhibit a specific pattern when the amplification product was run on an agarose gel. Identifying larvae revealed that conclusions on species composition of a population of parasitic wasps can be biased if only the emerging adults are taken into account.

Modification of the monomer composition of polyhydroxyalkanoate synthesized in Saccharomyces cerevisiae expressing variants of the beta-oxidation-associated multifunctional enzyme.

Expression by Saccharomyces cerevisiae of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the Brassica napus isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA. The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the B. napus ICL to target foreign proteins to the peroxisome of S. cerevisiae was demonstrated with green fluorescent protein fusions. PHA synthesis was found to be dependent on the presence of both the enzymes generating the beta-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding PEX5 gene, demonstrating the requirement for a functional peroxisome and a beta-oxidation cycle for PHA synthesis. Using a variant of the S. cerevisiae beta-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons.

Angiotensin converting enzyme inhibitor induced angio-oedema: a review of the pathophysiology and risk factors.

Angio-oedema (AE) is a known adverse effect of angiotensin converting enzyme inhibitor (ACE-I) therapy. Over the past several decades, evidence of failure to diagnose this important and potentially fatal reaction is commonly found in the literature. Because this reaction is often seen first in the primary care setting, a review was undertaken to analyse and document the keys to both diagnostic criteria as well as to investigate potential risk factors for ACE-I AE occurrence. A general review of published literature was conducted through Medline, EMBASE, and the Cochrane Database, targeting ACE-I-related AE pathomechanism, diagnosis, epidemiology, risk factors, and clinical decision making and treatment. The incidence and severity of AE appears to be on the rise and there is evidence of considerable delay in diagnosis contributing to significant morbidity and mortality for patients. The mechanism of AE due to ACE-I drugs is not fully understood, but some genomic and metabolomic information has been correlated. Additional epidemiologic data and clinical treatment outcome predictors have been evaluated, creating a basis for future work on the development of clinical prediction tools to aid in risk identification and diagnostic differentiation. Accurate recognition of AE by the primary care provider is essential to limit the rising morbidity associated with ACE-I treatment-related AE. Research findings on the phenotypic indicators relevant to this group of patients as well as basic research into the pathomechanism of AE are available, and should be used in the construction of better risk analysis and clinical diagnostic tools for ACE-I AE.

Embedding, Extraction and Detection of Digital Watermark in Spectral Images

Tässä diplomityössä tutkitaan tekniikoita, joillavesileima lisätään spektrikuvaan, ja menetelmiä, joilla vesileimat tunnistetaanja havaitaan spektrikuvista. PCA (Principal Component Analysis) -algoritmia käyttäen alkuperäisten kuvien spektriulottuvuutta vähennettiin. Vesileiman lisääminen spektrikuvaan suoritettiin muunnosavaruudessa. Ehdotetun mallin mukaisesti muunnosavaruuden komponentti korvattiin vesileiman ja toisen muunnosavaruuden komponentin lineaarikombinaatiolla. Lisäyksessä käytettävää parametrijoukkoa tutkittiin. Vesileimattujen kuvien laatu mitattiin ja analysoitiin. Suositukset vesileiman lisäykseen esitettiin. Useita menetelmiä käytettiin vesileimojen tunnistamiseen ja tunnistamisen tulokset analysoitiin. Vesileimojen kyky sietää erilaisia hyökkäyksiä tarkistettiin. Diplomityössä suoritettiin joukko havaitsemis-kokeita ottamalla huomioon vesileiman lisäyksessä käytetyt parametrit. ICA (Independent Component Analysis) -menetelmää pidetään yhtenä mahdollisena vaihtoehtona vesileiman havaitsemisessa.

Enzyme convertedstarch in pigment coating

Tämän diplomityön tavoitteena oli selvittää entsyymikonvertoinnin mahdollisuudet vaikuttaa sideainetärkkelyksen toiminnallisiin ominaisuuksiin. Tärkein tehtävä oli etsiä vastaukset kysymykseen, kuinka paljon entsyymikonvertointia optimoimalla voidaan maksimoida tärkkelyksen positiivisia vaikutuksia. Tavoitteena oli myös tutkia, voiko lisäaineita käyttämällä ja tärkkelystä plastisoimalla säilyttää tärkkelyksen vaikutus paperin jäykkyyteen ja saada tärkkelysfilmille joustavuutta. Kirjallisuusosassa tarkasteltiin tärkkelyksen entsyymikonvertointiin vaikuttavia tekijöitä, eri tärkkelysraaka-aineiden eroja, sekä konvertoinnissa käytettävien entsyymien ominaisuuksia. Kirjallisuusosassa tarkasteltiin myöstärkkelyksen käyttöä sideaineena pigmenttipäällystyksessä. Kokeellisessa osassakeskityttiin selvittämään entsyymikonvertoinnin olosuhteiden, käytettävän raakatärkkelyksen ja entsyymin vaikutusta konvertoidun tärkkelyksen ominaisuuksiin. Konvertoiduista tärkkelyksistä valmistettiin päällystyspastat, ja tutkittiin niinpastan kuin päällystetyn paperin ominaisuuksia. Myös erilaisten pehmentimien vaikutusta niin päällystyspastaan, kuin paperin pinnalle tutkittiin. Havaittiin, että konvertoimalla tärkkelysketjua entsymaattisesti, voidaan tärkkelysketjun pituutta säädellä. Tarkoituksena oli konvertoida tärkkelystä niin, että tärkkelyksen molekyyliketjujakaumat sisältävät lyhyitä, keskipitkiä sekäpitkiä molekyylejä. Päällystämisen havaittiin olevan vaikeaa Helicoaterilla varsinkin pitkäketjuista tärkkelystä suuren määrän sisältävillä pastoilla. Myös tärkkelys/lateksi-suhde vaihteli eri pastoilla. Päällystyspastojen reologisia ominaisuuksia testattaessa huomattiin, että tärkkelysketjun pituuden kasvaessa pastanviskositeetti lisääntyy ja vesiretentio vähenee. Havaittiin vain muutamia teknisiä paperiominaisuuksia, jotka korreloivat hyvin tärkkelysketjun pituuden kanssa. Näitä olivat kiilto, Gurley-Hill huokoisuus, taivutusvastus, taivutuspituus sekä IGT pintalujuus. Pehmentimien ei havaittu vaikuttavan moneenkaan paperin eri tekniseen ominaisuuteen. Suurimmat erot huomattiin paperin taivutuspituudessa ja taivutusvastuksessa.

Population genetics of Capercaillie (Tetrao urogallus) in the Jura and the Pyrenees: a non-invasive approach to avian conservation genetics

RÉSUMÉ Le Grand tétras est un galliforme de montagne apparenté au faisan et au tétras lyre. Il est distribué de manière continue à travers la toundra et les montagnes de moyenne altitude en Europe de l'ouest. Toutefois, les populations d'Europe de l'ouest ont subi un déclin constant au cours des derniers siècles. Les causes de ce déclin sont probablement liées à l'activité humaine, telle .que l'élevage ou le tourisme, qui ont engendré une modification et une fragmentation de l'habitat de l'espèce. Malheureusement, les populations soumises à de forts déclins démographiques peuvent subir des effets génétiques (augmentation de la consanguinité et perte de diversité génétique) pouvant diminuer leur potentiel de reproduction et conduire irrémédiablement à l'extinction. Cette thèse présente les analyses conduites dans le but d'estimer l'impact du déclin démographique des populations de Grand tétras sur l'étendue et la distribution de leur variabilité génétique dans le Jura et dans les Pyrénées. Du fait de la législation locale protégeant les tétraonidés en général, mais également en raison de la biologie très cryptique du Grand tétras, l'ensemble des analyses de cette étude a été réalisé à partir de matériel génétique extrait des fientes (ou échantillonnage génétique non invasif). Dans la première partie de l'étude, je détaille les protocoles d'extraction. d'ADN et d'amplification par PCR modifiés à partir des protocoles classiques utilisant des échantillons conventionnels, riches en ADN. L'utilisation d'ADN fécal impose des contraintes dues à la mauvaise qualité et à la faible quantité du matériel génétique à disposition dans les fientes. Ces contraintes ont pu être partiellement contournées en réalisant des répétitions multiples du génotypage afin d'obtenir un degré de fiabilité suffisante. J'ai également analysé les causes de la dégradation de l'ADN dans les excréments. Parmi les causes les plus communes, telles que l'activité bactérienne, l'hydrolyse spontanée et la dégradation enzymatique par les DNases libres, c'est ce dernier facteur qui apparaît comme étant la cause majeure et la plus rapide responsable de la dégradation de la qualité des échantillons. La rapidité de l'action enzymatique suggère que les plans d'échantillonnages de excréments sur le terrain pourraient être optimisés en les réalisant dans des conditions climatiques froides et sèches, favorisant ainsi l'inhibition des DNases. La seconde partie de la thèse est une étude par simulation visant à déterminer la capacité du logiciel Structure à identifier les structures génétiques complexes et hiérarchiques fréquemment rencontrées dans les populations naturelles, et ce en utilisant différents types de marqueurs génétiques. Les troisième et quatrième parties de cette thèse décrivent le statut génétique des populations résiduelles du Jura et des Pyrénées à partir de l'analyse de 11 loci microsatellites. Nous n'avons pas pu mettre en évidence dans les deux populations des effets liés à la consanguinité ou à la réduction de la diversité génétique. De plus, la différenciation génétique entre les patches d'habitats favorables reste modérée et corrélée à la distance géographique, ce qui suggère que la dispersion d'individus entre les patches a été importante au moins pendant ces dernières générations. La comparaison des paramètres de la diversité génétique avec ceux d'autres populations de Grand tétras, ou d'autres espèces proches, indique que la population du Jura a retenu une proportion importante de sa diversité originelle. Ces résultats suggèrent que le déclin récent des populations a jusqu'ici eu un impact modéré sur les facteurs génétiques et que ces populations semblent avoir conservé le potentiel génétique nécessaire à leur survie à long terme. Finalement, en cinquième partie, l'analyse de l'apparentement entre les mâles qui participent à la parade sur les places de chant (leks) indique que ces derniers sont distribués en agrégats de manière non aléatoire, préférentiellement entre individus apparentés. De plus, la corrélation entre les distances génétique et géographique entre les leks est en accord avec les motifs d'isolement par la distance mis en évidence à d'autres niveaux hiérarchiques (entre patches d'habitat et populations), ainsi qu'avec les études menées sur d'autres espèces ayant choisi ce même système de reproduction. En conclusion, cette première étude basée uniquement sur de l'ADN nucléaire aviaire extrait à partir de fèces a fourni des informations nouvelles qui n'auraient pas pu être obtenues par une méthode d'observation sur le terrain ou d'échantillonnage génétique classique. Aucun oiseau n'a été dérangé ou capturé, et les résultats sont comparables à d'autres études concernant des espèces proches. Néanmoins, la taille de ces populations approche des niveaux au-dessous desquels la survie à long terme est fortement incertaine. La persistance de la diversité génétique pour les prochaines générations reste en conséquence liée à la survie des adultes et à une reprise du succès de la reproduction. ABSTRACT Capercaillie (Tetrao urogallus) is a large grouse that is continuously distributed across the tundra and the mid-high mountains of Western Europe. However, the populations in Western Europe have been showing a constant decline during the last decades. The causes for this decline are possibly related to human activities, such as cattle breeding and tourism that have both led to habitat modification and fragmentation. Unfortunately, populations that have undergone drastic demographic bottlenecks often go through genetic processes of inbreeding and loss of diversity that decrease their fitness and eventually lead to extinction. This thesis presents the investigations conducted to estimate the impact of the demographic decline of capercaillie populations on the extent and distribution of their genetic variability in the Jura and in the Pyrenees mountains. Because grouse are protected by wildlife legislation, and also because of the cryptic behaviour of capercaillie, all DNA material used in this study was extracted from faeces (non-invasive genetic sampling). In the first part of my thesis, I detail the protocols of DNA extraction and PCR amplification adapted from classical methods using conventional DNA-rich samples. The use of faecal DNA imposes specific constraints due to the low quantity and the highly degraded genetic material available. These constraints are partially overcome by performing multiple genotyping repetitions to obtain sufficient reliability. I also investigate the causes of DNA degradation in faeces. Among the main degraders, namely bacterial activity, spontaneous hydrolysis, and free-¬DNase activities, the latter was pointed out as the most important according to our experiments. These enzymes degrade DNA very rapidly, and, as a consequence, faeces sampling schemes must be planned preferably in cold and dry weather conditions, allowing for enzyme activity inhibition. The second part of the thesis is a simulation study aiming to assess the capacity of the software Structure to detect population structure in hierarchical models relevant to situations encountered in wild populations, using several genetic markers. The methods implemented in Structure appear efficient in detecting the highest hierarchical structure. The third and fourth parts of the thesis describe the population genetics status of the remaining Jura and Pyrenees populations using 11 microsatellite loci. In either of these populations, no inbreeding nor reduced genetic diversity was detected. Furthermore, the genetic differentiation between patches defined by habitat suitability remains moderate and correlated with geographical distance, suggesting that significant dispersion between patches was at work at least until the last generations. The comparison of diversity indicators with other species or other populations of capercaillie indicate that population in the Jura has retained a large part of its original genetic diversity. These results suggest that the recent decline has had so forth a moderate impact on• genetic factors and that these populations might have retained the potential for long term survival, if the decline is stopped. Finally, in the fifth part, the analysis of relatedness between males participating in the reproduction parade, or lek, indicate that capercaillie males, like has been shown for some other grouse species, gather on leks• among individuals that are more related than the average of the population. This pattern appears to be due to both population structure and kin-association. As a conclusion, this first study relying exclusively on nuclear DNA extracted from faeces has provided novel information that was not available through field observation or classical genetic sampling. No bird has been captured or disturbed, and the results are consistent with other studies of closely related species. However, the size of these populations is approaching thresholds below which long-term survival is unlikely. The persistence of genetic diversity for the forthcoming generations remains therefore bond to adult survival and to the increase of reproduction success.

Local and Global Feature Extraction for Invariant Object Recognition

Perceiving the world visually is a basic act for humans, but for computers it is still an unsolved problem. The variability present innatural environments is an obstacle for effective computer vision. The goal of invariant object recognition is to recognise objects in a digital image despite variations in, for example, pose, lighting or occlusion. In this study, invariant object recognition is considered from the viewpoint of feature extraction. Thedifferences between local and global features are studied with emphasis on Hough transform and Gabor filtering based feature extraction. The methods are examined with respect to four capabilities: generality, invariance, stability, and efficiency. Invariant features are presented using both Hough transform and Gabor filtering. A modified Hough transform technique is also presented where the distortion tolerance is increased by incorporating local information. In addition, methods for decreasing the computational costs of the Hough transform employing parallel processing and local information are introduced.

Identifying quantitative operation principles in metabolic pathways: a systematic method for searching feasible enzyme activity patterns leading to cellular adaptive responses

Background: Optimization methods allow designing changes in a system so that specific goals are attained. These techniques are fundamental for metabolic engineering. However, they are not directly applicable for investigating the evolution of metabolic adaptation to environmental changes. Although biological systems have evolved by natural selection and result in well-adapted systems, we can hardly expect that actual metabolic processes are at the theoretical optimum that could result from an optimization analysis. More likely, natural systems are to be found in a feasible region compatible with global physiological requirements. Results: We first present a new method for globally optimizing nonlinear models of metabolic pathways that are based on the Generalized Mass Action (GMA) representation. The optimization task is posed as a nonconvex nonlinear programming (NLP) problem that is solved by an outer- approximation algorithm. This method relies on solving iteratively reduced NLP slave subproblems and mixed-integer linear programming (MILP) master problems that provide valid upper and lower bounds, respectively, on the global solution to the original NLP. The capabilities of this method are illustrated through its application to the anaerobic fermentation pathway in Saccharomyces cerevisiae. We next introduce a method to identify the feasibility parametric regions that allow a system to meet a set of physiological constraints that can be represented in mathematical terms through algebraic equations. This technique is based on applying the outer-approximation based algorithm iteratively over a reduced search space in order to identify regions that contain feasible solutions to the problem and discard others in which no feasible solution exists. As an example, we characterize the feasible enzyme activity changes that are compatible with an appropriate adaptive response of yeast Saccharomyces cerevisiae to heat shock Conclusion: Our results show the utility of the suggested approach for investigating the evolution of adaptive responses to environmental changes. The proposed method can be used in other important applications such as the evaluation of parameter changes that are compatible with health and disease states.

Bioinspired electronic white cane implementation based on a LIDAR, a tri-axial accelerometer and a tactile belt

This work proposes the creation of a bioinspired electronic white cane for blind people using the whiskers principle for short-range navigation and exploration. Whiskers are coarse hairs of an animal's face that tells the animal that it has touched something using the nerves of the skin. In this work the raw data acquired from a low-size terrestrial LIDAR and a tri-axial accelerometer is converted into tactile information using several electromagnetic devices configured as a tactile belt. The LIDAR and the accelerometer are attached to the user’s forearm and connected with a wire to the control unit placed on the belt. Early validation experiments carried out in the laboratory are promising in terms of usability and description of the environment.

Intravoxel incoherent motion diffusion-weighted imaging in the liver: comparison of mono-, bi- and tri-exponential modelling at 3.0-T.

PURPOSE: To determine whether a mono-, bi- or tri-exponential model best fits the intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) signal of normal livers. MATERIALS AND METHODS: The pilot and validation studies were conducted in 38 and 36 patients with normal livers, respectively. The DWI sequence was performed using single-shot echoplanar imaging with 11 (pilot study) and 16 (validation study) b values. In each study, data from all patients were used to model the IVIM signal of normal liver. Diffusion coefficients (Di ± standard deviations) and their fractions (fi ± standard deviations) were determined from each model. The models were compared using the extra sum-of-squares test and information criteria. RESULTS: The tri-exponential model provided a better fit than both the bi- and mono-exponential models. The tri-exponential IVIM model determined three diffusion compartments: a slow (D1 = 1.35 ± 0.03 × 10(-3) mm(2)/s; f1 = 72.7 ± 0.9 %), a fast (D2 = 26.50 ± 2.49 × 10(-3) mm(2)/s; f2 = 13.7 ± 0.6 %) and a very fast (D3 = 404.00 ± 43.7 × 10(-3) mm(2)/s; f3 = 13.5 ± 0.8 %) diffusion compartment [results from the validation study]. The very fast compartment contributed to the IVIM signal only for b values ≤15 s/mm(2) CONCLUSION: The tri-exponential model provided the best fit for IVIM signal decay in the liver over the 0-800 s/mm(2) range. In IVIM analysis of normal liver, a third very fast (pseudo)diffusion component might be relevant. KEY POINTS: ? For normal liver, tri-exponential IVIM model might be superior to bi-exponential ? A very fast compartment (D = 404.00 ± 43.7 × 10 (-3)  mm (2) /s; f = 13.5 ± 0.8 %) is determined from the tri-exponential model ? The compartment contributes to the IVIM signal only for b ≤ 15 s/mm (2.)

Enzymatic technology to improve oil extraction from Caryocar brasiliense camb. (Pequi) Pulp.

The present study aims to compare yield and quality of pequi pulp oil when applying two distinct processes: in the first, pulp drying in a tray dryer at 60ºC was combined with enzymatic treatment and pressing to oil extraction; in the second, a simple process was carried out by combining sun-drying pulp and pressing. In this study, raw pequi fruits were collected in Mato Grosso State, Brazil. The fruits were autoclaved at 121ºC and stored under refrigeration. An enzymatic extract with pectinase and CMCase activities was used for hydrolysis of pequi pulp, prior to oil extraction. The oil extractions were carried out by hydraulic pressing, with or without enzymatic incubation. The oil content in the pequi pulp (45% w/w) and the physicochemical characteristic of the oil was determined according to standard analytical methods. Free fatty acids, peroxide values, iodine and saponification indices were respectively 1.46 mgKOH/g, 2.98 meq/kg, 49.13 and 189.40. The acidity and peroxide values were lower than the obtained values in commercial oil samples, respectively 2.48 mgKOH/g and 5.22 meq/kg. Aqueous extraction has presented lower efficiency and higher oxidation of unsaturated fatty acids. On the other hand, pequi pulp pressing at room temperature has produced better quality oil. However its efficiency is still smaller than the combined enzymatic treatment and pressing process. This combined process promotes cellular wall hydrolysis and pulp viscosity reduction, contributing to at least 20% of oil yield increase by pressing.

Angiotensin converting enzyme inhibitory peptides in Finnish cereals : a database survey

Selostus: Angiotensiini I -muuntavaa entsyymiä estävien peptidien aminohapposekvenssien esiintyminen viljan varastoproteiinien rakenteessa