No description available.

No description available.

No description available.

With reduced prevalence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC), direct and field deployable diagnostic tests are needed to implement an effective diagnostic and surveillance algorithm for post-elimination VL control. In this regard, here we investigated the diagnostic efficacies of a loop-mediated isothermal amplification (LAMP) assay (Loopamp™ Leishmania Detection Kit, Eiken Chemical CO., Ltd, Japan), a real-time quantitative PCR assay (qPCR) and the Leishmania antigen ELISA (CLIN-TECH, UK) with different sampling techniques and evaluated their prospect to incorporate into post-elimination VL control strategies. Eighty clinically and rK39 rapid diagnostic test confirmed VL cases and 80 endemic healthy controls were enrolled in the study. Peripheral blood and dried blood spots (DBS) were collected from all the participants at the time of diagnosis. DNA was extracted from whole blood (WB) and DBS via silica columns (QIAGEN) and boil & spin (B&S) methods and tested with qPCR and Loopamp. Urine was collected from all participants at the time of diagnosis and was directly subjected to the Leishmania antigen ELISA. 41 patients were followed up and urine samples were collected at day 30 and day 180 after treatment and ELISA was performed. The sensitivities of the Loopamp-WB(B&S) and Loopamp-WB(QIA) were 96.2% (95% CI 89·43-99·22) and 95% (95% CI 87·69-98·62) respectively. The sensitivity of Loopamp- DBS(QIA) was 85% (95% CI 75·26- 92·00). The sensitivities of the qPCR-WB(QIA) and qPCR-DBS(QIA) were 93.8% (95% CI 86·01-97·94) and 72.5% (95% CI 61·38-81·90) respectively. The specificity of all molecular assays was 100%. The sensitivity and specificity of the Leishmania antigen ELISA were 97.5% (95% CI 91·47-99·70) and 91.95% (95% CI 84·12-96·70) respectively. The Leishmania antigen ELISA depicted clinical cure at day 180 in all the followed-up cases. Efficacy and sustainability identify the Loopamp-WB(B&S) and the Leishmania antigen ELISA as promising and minimally invasive VL diagnostic tools to support VL diagnostic and surveillance activities respectively in the post-elimination era.

Plants maintain organelle genomes that are descended from ancient microbes. Ages ago, these ancient microbes were engulfed by larger cells, beginning a process of co-evolution we now call the endo-symbiotic theory. Over time, DNA from the engulfed microbe was transferred to the genome of the larger engulfing cell, eventually losing the ability to be free-living, and establishing a permanent residency in the larger cell. Similarly, the larger cell came to rely so much on the microbe it had engulfed, that it too lost its ability to survive without it. Thus, mitochondria and plastids were born. Nearly all multicellular eukaryotes possess mitochondria; however, different evolutionary pressures have created drastically different genomes in plants versus animals. For one, animals have very compact, efficient mitochondrial genomes, with about 97% of the DNA coding for genes. These genomes are very consistent in size across different animal species. Plants, on the other hand, have mitochondrial genomes 10 to more than 100 times as large as animal mitochondrial genomes. Plants also use a variety of mechanisms to replicate and maintain their DNA. Central to these mechanisms are nuclear-encoded, organelle targeted replication proteins. To date, there are two DNA polymerases that have been identified in plant mitochondria and chloroplasts, Pol1A and Pol1B. There is also a DNA helicase-primase that localizes to mitochondria and chloroplasts called Twinkle, which has similarities to the gp4 protein from T7 phage. In this dissertation, we discuss the roles of the polymerases and the effects of mutating the Pol1A and Pol1B genes respectively. We show that organelle genome copy number decreases slightly and over time but with little effect on plant development. We also detail the interactions between Twinkle and Pol1A or Pol1B. Plants possess the same organellar proteins found in animal mitochondria, which are homologs to T7 phage DNA replication proteins. We show that similar to animals and some phage, plants utilize the same proteins in similar interactions to form the basis of a DNA replisome. However, we also show that plants mutated for Twinkle protein show no discernable growth defects, suggesting there are alternative replication mechanisms available to plant mitochondria that are not accessible in animals.

No description available.

Marine mammals play an important role in ecosystem stability. However, anthropogenic activity is compounding pressure on many already vulnerable populations. A potential consequence of anthropogenic disturbance is physiological stress, which can impact metabolism, immunity, and reproduction, especially if it occurs repeatedly. Previous studies on marine mammals have focused on acute stress, but the impacts of repeated stress are poorly understood. Due to its accessibility on land during haul-outs, the northern elephant seal (Mirounga angustirostris) is a good system in which to study the effects of stress in marine mammals. Stress stimulates the release of glucocorticoid hormones, primarily cortisol. Elevated cortisol is a good indicator of acute stress, but it is an unreliable proxy for chronic stress, and cortisol measurements alone do not provide information on the downstream physiological consequences of chronic stress. Therefore, additional biomarkers may provide a more comprehensive understanding of the effects of repeated stress on marine mammals. I examined two approaches for assessing stress in response to administration of the hormone that stimulates secretion of cortisol (adrenocorticotropic hormone; ACTH): a non-targeted proteomics approach using blood plasma and a targeted gene expression approach examining blubber expression of glucocorticoid receptor (GR) and two of its regulators, FKBP5 and KLF9. For the first approach, I used the highly sensitive LC-MS/MS technique to detect changes in circulating plasma proteins in juvenile seals in response to repeated ACTH administration. I identified changes in relative abundance of proteins of interest in the plasma proteome that included those with roles in lipid, iron, and redox homeostasis, cortisol and thyroid hormone transport, adipogenesis, oxidative stress, blood pressure regulation, vitamin and mineral transport, and innate immunity. I then measured blubber expression of GR and its regulator genes in blubber of adult female seals undergoing early and late molting to examine changes during different life-history stages in response to acute stress induced by ACTH administration. Using RT-qPCR, I found that GR expression decreased in blubber, while expression of FKBP5 increased, suggesting negative feedback at the tissue level which may reduce sensitivity to cortisol during key life-history stages, such as molting, which require fasting. These data provide insights into the resilience of marine mammals to acute stress and novel biomarkers that may be used to study the effects of prolonged stress in wildlife.

Here, we validate novel protein interactors of TCF7L1 (also known as TCF3), a downstream transcription factor in the Wnt/β-catenin signaling pathway, from an initial protein interaction screen that utilized the BioID system in mouse embryonic stem cells. The BioID-TCF7L1 screen identified multiple proteins including several transcription factors and numerous epigenetic regulators. Notably, SALL4, a key embryonic stem cell factor belonging to the SPALT family of transcription factors was validated to interact with TCF7L1 through Proximity Ligation Assay (PLA), and Co-Immunopreciptation (Co-IP). Analysing mRNA transcriptomic signatures of TCF7L1-null mEScs and SALL4 overexpressing mESCs, we observed similarly increased output of the pluripotency-gene, Tbx3, suggesting a transcriptionally opposing function between TCF7L1 and SALL4. Furthermore, we identified that SALL4 also interacted with TCF7, suggesting that SALL4 may interact with all four members of the TCF/LEF transcription factor family to regulate Wnt targets. This work further validates the utility and effectiveness of screening transcription factor interactors through the BioID system and provides important insights into SALL4 mediated Wnt regulation through the TCF/LEFs. / Thesis / Master of Science (MSc) / The biology of cells is highly complex. The genes within are under tight regulation to promote balance that is critical to the growth and status of the cell. Cells communicate with one another to support this balance through molecule secretion signaling which dictates biology. Understanding the complex biology within cells is critical, and therefore here we study one of many signaling pathways known as the Wnt Signaling Pathway. This work contributes to the knowledge of Wnt signaling by validating the interaction of proteins that dictate the onset or offset of important genes in mouse embryonic stem cells.

[ES] El virus meridional del tomate (Southern tomato virus, STV) es un virus persistente (género Amalgavirus, familia Amalgaviridae) que se ha detectado en diversos países como España e Italia. Inicialmente, fue asociado a síntomas de decoloración y falta de maduración en el fruto de tomate. Sin embargo, la presencia frecuente de virus agudos en las plantas infectadas con el STV y la detección de éste en plantas asintomáticas, ponen en duda su patogenicidad y el impacto que puede tener en el cultivo. En esta tesis doctoral se puso a punto la RT-LAMP y la RT-qPCR para la detección específica y sensible del STV. La RT-LAMP permitió reducir costes y simplificar el procedimiento, siendo útil para la detección en campo. La RT-qPCR nos permitió detectar y cuantificar el STV en distintos tipos de tejidos de tomate, incluyendo semillas individuales. El virus se acumulaba principalmente en las raíces y hojas, y en las semillas se encontraba tanto en cubierta como en embrión, lo que dificulta las tareas de desinfección. La tasa de transmisión por semilla del virus, la incidencia en campo y en viveros era muy elevada, afectando más a variedades comerciales que locales. Los análisis filogenéticos mostraron que el STV tenía una baja diversidad genética con una fuerte presión de selección negativa. Además, no había una correlación entre distancia genética del virus y origen geográfico debido a una rápida dispersión de semillas infectadas y/o una fuerte presión de selección negativa. Se comprobó que el STV en condiciones de infección simple no inducía síntomas, no alteraba la producción, ni afectaba a parámetros fisiológicos como conductividad estomática, fotosíntesis y peso, en condiciones de estrés salino. Tampoco se observaron cambios a nivel tisular ni celular, ni se encontraron partículas virales. Sin embargo, el virus modificaba la expresión de algunos miRNAs con importantes funciones. Se detectaron muy poca cantidad de vsiRNAs derivados del STV, lo cual podría deberse a la supresión del silenciamiento génico de la planta por acción de un supresor codificado por el virus. Los ensayos de expresión transitoria en plantas de N. benthamiana 16C determinaron que la p42 del STV no tenía actividad supresora de silenciamiento génico. Finalmente, estudiamos el efecto del STV en infecciones múltiples con otros virus agudos como el CMV y el PepMV. Se observaron complejas interacciones entre los virus que implicaban variaciones en la severidad de síntomas, en los niveles de acumulación viral y en las poblaciones de siRNAs. El STV y el CMV establecían una interacción sinérgica que producía el adelanto y aumento de la severidad de los síntomas, y de la acumulación del CMV en las primeras fases de la infección. La presencia del STV en plantas infectadas con el PepMV también producía un adelanto de los síntomas sin cambios en la acumulación viral del PepMV. En las plantas coinfectadas con el CMV y PepMV se observó un efecto antagónico que disminuía la concentración del CMV y alteraba los síntomas. El STV era capaz de romper este efecto antagónico restableciendo la concentración del CMV y modificando los síntomas. Los análisis de siRNAs permitieron identificar un total de 78 miRNAs, 47 noveles, que se expresaban diferencialmente en los grupos de plantas infectadas con los diferentes virus respecto a las plantas sin infectar. Estos miRNAs estaban implicados en la regulación de importantes funciones y tanto su número como su nivel de expresión variaba dependiendo de la combinación viral. También se identificaron vsiRNAs de origen viral y se vio que su proporción variaba dependiendo de la combinación viral. La cantidad de vsiRNAs del STV se incrementaba notablemente con la presencia de otros virus. La frecuencia de acumulación de vsiRNAs en los genomas virales no era uniforme y no se veía influenciada por las combinaciones de virus. / [CA] El virus meridional de la tomaca (Southern tomato virus, STV) és un virus persistent (gènere Amalgavirus, família Amalgaviridae) que s'ha detectat en diversos països com Espanya i Itàlia. Inicialment, STV va ser associat a distints símptomes de decoloració i anomalies en la maduració del fruit. Però la presència freqüent de virus aguts en les plantes infectades amb STV i la detecció d'aqueste en plantes asimptomàtiques, posen en dubte la seua patogenicitat i l'impacte que pot tindre en el cultiu. En aquesta tesi doctoral es va realitzar la posada al punt de la RT-LAMP i la RT- qPCR per a la detecció específica i sensible de STV. La RT-LAMP va permetre reduir costos i simplificar el procediment, sent útil per a la detecció del virus en camp. La RT-qPCR és una tècnica molt sensible que ens va permetre detectar i quantificar STV en distints tipus de teixits, incloent-hi llavors individuals. El virus s'acumulava principalment en arrels i fulles, i en les llavors es trobava tant en la coberta com en l'embrió. Es va comprovar que les taxes de transmissió per llavor, la incidència en camp i en vivers era molt elevada, major en les varietats comercials que en les locals. Els estudis filogenètics realitzats van mostrar que el virus tenia una baixa diversitat genètica amb una forta pressió de selecció negativa. No hi havia una correlació entre distància genètica del virus i origen geogràfic, degut per una ràpida dispersió a traves de llavors infectades i/o a la forta pressió de selecció negativa. En aquest treball es van obtindre evidències de què STV en condicions d'infecció simple no induïa símptomes en la planta, no alterava la producció, ni afectava paràmetres fisiològics com a conductivitat estomacal, fotosíntesi i pes en condicions d'estrés salí. Tampoc, es van observar cap presència de partícules virals ni canvis a nivell tissular ni cel·lular. No obstant això, STV era capaç de modificar l'expressió d'alguns miRNAs amb importants funcions. Es van detectar molt poca quantitat de vsiRNAs derivats del STV, podria deure's a la supressió del mecanisme de silenciamient gènic per acció d'un supressor. Els assajos d'expressió transitòria en plantes de N. benthamiana 16C va determinar que la p42 de STV no va tindre capacitat supressora de silenciamient gènic. Per finalitzar, vam estudiar l'efecte que podia tindre STV en infeccions mixtes amb altres virus aguts com CMV i PepMV. Els resultats obtinguts d'un assaig amb diferents combinacions d'infeccions van mostrar interaccions complexes entre els virus que implicaven variacions en la severitat de símptomes, en els nivells d'acumulació viral i en les poblacions de siRNA. STV i CMV establien una interacció sinèrgica que produïa l'avanç i l'increment dels símptomes, i un augment de l'acumulació de CMV. D'altra banda, la presència de STV en plantes infectades amb PepMV també produïa un avanç dels símptomes, però no hi havia variacions en l'acumulació de PepMV. En el grup de plantes co-infectades amb CMV i PepMV es va observar un efecte antagònic que dificultava la replicació de CMV, alterant-se els símptomes de la planta. STV era capaç de trencar aquest efecte antagònic restaurant la concentració de CMV i modificant els símptomes. Els anàlisis de siRNAs van permetre identificar un total de 78 miRNAs, 47 corresponien a miRNAs novells, que s'expressaven de forma diferent als grups de plantes infectades amb els diversos virus, respecte a les plantes control sense infectar. Aquests miRNAs estaven implicats en la regulació d'importants funcions i tant el seu nombre com el seu nivell d'expressió variaven. També es van identificar vsiRNAs d'origen viral i es va observar que la seua proporció variava depenent de la combinació viral. La quantitat de vsiRNAs de STV s'incrementava notablement amb la presència d'altres virus. Les freqüències de vsiRNA en els genomes virals no eren uniformes, no obstant això, els pat / [EN] Southern tomato virus (STV) is a persistent virus (genus Amalgavirus, family Amalgaviridae) which was detected in several countries such Spain and Italy. STV was associated with symptoms of discoloration and maturation of tomato fruit. However, STV was frequently detected in mixed infections with acute viruses and in some asymptomatic tomato plants. For these reasons, it is not clear the STV pathogenic role and its real impact on tomato crops. In this PhD we improved the specific and sensitive detection of the virus by using the RT-LAMP and RT-qPCR. RT-LAMP is very useful for field STV detection since it is a simple and cheap technique. The high RT-qPCR sensitivity enabled this technique to detect and quantify STV from different plant tissues even individual sees. The highest STV concentrations were found in tomato leaves and roots. In the seeds, STV could be detected in both coat and embryo. The virus transmission by seed and the STV incidence in fields and seedlings was very high, being higher in commercial tomato varieties than in local ones. Phylogenetic analysis from different STV isolates showed a low genetic diversity with a high negative selection pressure. Moreover, there was no correlation between genetic diversity and geographic region. This could be explained by a quick dispersion of infected seeds and/or by the high negative selection pressure. It was shown that STV did not induce any apparent plant symptom and did not affect the plant production in single infection conditions. Also, physiological parameters related to stomatic conductivity, photosynthesis, and plant weight were no affected by STV infection in saline stress conditions. Optic and transmission electron microscopy did not reveal viral particles or structural changes in STV tomato tissues. However, the population analysis of miRNAs showed that STV was able to modify the expression of some miRNAs which modulated important plant functions. Low vsiRNAs were detected in STV tomato infected plants. It could be produced by the action of a suppressor which could suppress the gene silencing pathway in the plants. A transient expression assay of p42 in N. benthamiana 16C plants did not show suppressor activity of this STV protein. Finally, we studied the effect of STV in mixed infections with other acute viruses such as CMV and PepMV. The virus mixes infection assay in tomato plants showed complex interactions between viruses that modify the symptoms severity, the viral accumulation and the siRNA population. STV and CMV established a synergistic interaction in co-infected tomato plants producing the advancement of the symptoms and an increase in its severity. STV and CMV co-infection increased the CMV accumulation in the early stages of infection. On the other hand, the presence of STV in plants co-infected with PepMV also produced an advance of symptoms, but with no variation in the PepMV accumulation. In the group of plants co-infected with CMV and PepMV, it was observed an antagonistic effect that delayed the CMV accumulation, altering the symptoms of the plant with respect to the simple infections. STV was able to break this antagonistic effect by increasing the CMV viral concentration and changing the symptomatology. The siRNAs analysis allowed to identify a total of 78 miRNAs, 47 corresponding to novel miRNAs, that were expressed differentially in the plants infected respect to no infected plants. These miRNAs were involved in the regulation of important functions and their number and their level of expression varied depending on the virus combination. vsiRNAs of the different viruses were also identified and it was observed that rates varied depending on the virus combination. The number of vsiRNAs in STV single infected tomato plants was very small, but it increased with the presence of the other viruses. The frequencies of vsiRNAs in the viral genomes were not uniform and these frequencies were not influenced by other viruses in mixed infections. / Elvira González, L. (2021). Detección y caracterización del virus meridional del tomate (STV) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/165207

Cette étude porte sur l'identification et la dispersion des bioaérosols générés sur les plates-formes de compostage et plus précisément lors du retournement des andains en cours de fermentation. L'analyse des bioaérosols émis sur cinq plates-formes par des inventaires moléculaires (ADNr 16S et ADNr 18S) a permis de montrer la dominance de deux phyla bactériens Firmicutes et Actinobacteria et du phylum fongique Ascomycota. En comparant la structure de la population microbienne des cinq bioaérosols, une signature a été identifiée. Dix phylotypes microbiens sont communs à au moins quatre bioaérosols. Deux sont présents dans les cinq bioaérosols : NA07 appartenant à l'espèce Saccharopolyspora rectivirgula et représentant 7% des séquences bactériennes totales et EQ07 affiliée à Thermomyces (49% des séquences fongiques). Un second phylotype bactérien, NC38, affilié à la famille des Thermoactinomycetaceae a été sélectionné du fait q u'il n'ait été identifié que dans le compost. Des systèmes de PCRq ont été développés pour quantifier ces trois indicateurs potentiels. Ces derniers ont été validés expérimentalement par des mesures sur sites industriels. La dispersion des bioaérosols a ensuite été caractérisée en utilisant plusieurs méthodologies, dont les trois indicateurs conçus et une technique d'empreinte moléculaire, la SSCP. Lors d'une activité de retournement, la concentration des indicateurs est supérieure à leur niveau basal dans l'air (bruit de fond). Les indicateurs présentent des profils de dispersion différents d'où l'intérêt de les coupler afin d'obtenir une meilleure vision de la dispersion des bioaérosols de compostage. / The aim of this work was to analyze the diversity and the dispersal of composting bioaerosol emitted during the turning of compost windrows in thermophilic phase on composting platforms. The study of the microbial diversity of aerosols emitted on five composting plants was realized by 16S and 18S rDNA molecular inventories. Two bacterial phyla Firmicutes and Actinobacteria and one fungal phylum Ascomycota dominated. A common microbial signature emerged from the five composting bioaerosols: ten microbial phylotypes (seven bacterial and three fungal) were common to at least four bioaerosols. Two have been identified in five bioaerosols: NA07 belonging to the species Saccharopolyspora rectivirgula, and representing 7% of total number of bacterial sequences, and EQ05,affiliated to Thermomyces (49% of total number of fungal sequences). A second bacterial phylotype, NC38, affiliated to the Thermoactinomycetaceae family, was selected because it was id entified only in the environmental source compost'. qPCR systems were then designed for each phylotype. Measurements performed on industrial composting sites validated the use of these microorganisms as indicators of composting bioaerosols. The dispersal of composting bioaerosols was then characterized using the three indicators developed and a fingerprint technique, the SSCP.