1,720,968 research outputs found
Caracterización de los genes que codifican para las proteínas de estrés térmico Hsp60 y Hsp70 de Piscirickettsia salmonis y su uso potencial para la formulación de una vacuna
No full textLa industria salmonera en Chile se ha convertido en una importante
actividad económica contribuyendo con el crecimiento y desarrollo del país.
Sin embargo, las enfermedades de peces que afectan el desarrollo de esta
actividad han generado grandes pérdidas económicas. Uno de los
patógenos responsables es Piscirickettsia salmonis, causante del Síndrome
Rickettsial Salmónideo (SRS). Esta enfermedad afecta a gran parte de los
cultivos intensivos del país y es por esto que se han desarrollado técnicas de
diagnóstico y se ha tratado de controlar la enfermedad mediante el uso de
antibióticos y vacunas. No obstante, no se ha tenido éxito.
Las proteínas de estrés térmico han sido consideradas como
antígenos principales en la respuesta inmune y se han utilizado como
vacunas en diversos ensayos con alto grado de éxito. Por esta razón, el
objetivo de esta tesis fue identificar, clonar, caracterizar y expresar los genes
de las proteínas de estrés térmico 60 (hsp60) y 70 (hsp70) de P. salmonis
con el objeto de desarrollar una potencial vacuna contra el SRS. Ambos
genes fueron clonados en el vector pGEM-T, los vectores de expresión
eucarionte pUK21-A2, pCMV-Bios y el vector de expresión procarionte
pET32a(+). Los genes fueron secuenciados y expresados en E. coli Origami,
logrando obtener las proteínas recombinantes Hsp60 y Hsp70, que fueron
posteriormente purificadas y utilizadas para elaborar una vacuna
recombinante, la cual fue ensayada en ratones Balb/c, generándose una
respuesta inmune anti-Hsp60 y anti-Hsp70.
Por otro lado, se inmunizaron ratones Balb/c con los genes hsp60 y
hsp70 clonados en el vector pUK21-A2. La respuesta inmune generada por
esta vacuna fue medida por ELISA, detectándose una respuesta inmune
sólo en ratones vacunados con el vector pUK21-A2 + hsp70. Además,
ambas proteínas recombinantes fueron utilizadas para evaluar la presencia
de anticuerpos anti-Hsp60 o anti-Hsp70 en ratones inmunizados con la
vacuna de ADN o con la vacuna recombinante mediante"Western blot". De
esta manera, se detectaron anticuerpos que reconocen tanto a Hsp60 como
a Hsp70 en sueros de ratones inmunizados con las vacunas de proteína
recombinante y también fue detectada la presencia de anticuerpos en el
suero de ratones inmunizados con el vector de expresión pUK21-A2 +
hsp70, lo cual indica que este gen clonado en el vector eucariótico se
expresa in vivo. Finalmente, a través de anticuerpos monoclonales antiHsp60 y anti-Hsp70, desarrollados en BiosChile 1.G.S.A, se demostró la
existencia de estas proteínas en extractos de P. salmonis indicando que
estos genes efectivamente son expresados por el patógeno.The Salmon industry has become in a important economic activity wich
contributes to the growth and the development of Chile. However, the
diseases that affect the development of this activity have caused catastrophic
economic loses. One of the pathogens responsible for this is Piscirickettsia
salmonis which causes the Septicemial Rickettsial Syndrome (SRS). This
disease affects most salmon cultures in Chile and for this reason diagnostic
techniques have been developed. Antibiotics and vaccines has failed in the
attempt to control the diseases.
The heat shock proteins are considered the principal antigens to obtain an
immune response and these have been used as vaccines in different of
models with a high degree of success. The objective of this work was the
identification, cloning, characterization and the expression of the heat shock
protein genes hsp60 and hsp70 of P. salmonis, order to develop a vaccine
against the SRS. Both genes were succesfully cloned in the pGEM-T vector,
the pUK21-A2 and pCMV-Bios eukaryotic expression vectors and in the
PET32a(+) procariontes expression vector. The genes were sequenced and
expressed in E. coli Origami, obtaining the recombinant proteins Hsp60 and
Hsp70. These proteins were purified tested as recombinant vaccines in
mouse Balb/c where an immune response was generated against Hsp60 and
Hsp70.
In another set of studies Balb/c mice were immunizated using the hsp60 and
hsp70 genes, cloned in the pUK21-A2 eukaryotic expression vector. The
immune response generated by this DNA vaccine was determinate by ELISA
which showed an immune response only in the mice vaccinated with pUK21-
A2 + hsp70 vector. Western blotting detected the presence of Hsp60 and
Hsp70 antibodies in serum of mice which had been immunized with the
recombinant vaccines as well as in serum of mice vaccinated with pUK21-A2
+ hsp70 eukaryotic expression vector, demonstrating that this gene cloning in
the eukaryotic vector are expressed in vivo. Finally, using monoclonal
antibodies against Hsp60 and Hsp70, we demonstrated the presences of
these P. salmonis proteins witch indicates that these genes were indeed
expressed by this pathogen
Genetische Analyse der Thymusentwicklung im Zebrabärbling (Danio rerio)
No full textThe zebrafish model has been proposed as a genetic and immunological tool to understand the processes of thymopoiesis and lymphopoiesis. In this work using forward genetic analysis, a collection of three mutants IU191, IP109 and IP045, which exhibited a defect in thymus development were characterized and the mutations were mapped by linkage analysis using SSLP markers for subsequent positional cloning.
The positional cloning revealed that IU191 mutant embryos harbor a mutation (G to A transition) in the splice donor sequence of exon 11 of the gene encoding cleavage stimulation factor subunit-77 kDa (cstf3). The analysis of the mutants revealed that the mutation affects the normal splicing of intron 11, such that exon 11 is followed by the full intron 11 sequence in the mature mRNA. Moreover, by using splice-site morpholino in wild-type embryos, it was possible to reproduce the mutant phenotype and the same splicing defect. The inclusion of the intron 11 generates a premature stop codon, 21 bp into the intron sequence, possibly causing the generation of a truncated CstF protein. These mutant embryos exhibited a defect in the Meckel’s cartilage as determined by Alcian blue staining. However, no subsequent craniofacial defects were observed during further development of these mutants. Furthermore, the pharyngeal arches form normally and foxn1 expression is also normal in the thymic anlage.
Detailed characterization of these mutants by WISH using T-cell markers (rag1, ikaros, ccr9 and tcrβ) showed a severe reduction of thymopoiesis. However, the hematopoietic program was not affected in the mutant, since the expression of gata1 was not affected and l-palstin was affected especially in the thymus. Finally, preliminary results suggest that knock-down of p53 rescues rag1 expression in IU191 embryos.
In the IP109 mutant, a mutation (T to A transversion) in the proto-oncogene cmyb was found. This mutation results in an isoleucine to asparagine substitution in the R3 repeat of the DNA binding domain. These mutants lack thymopoiesis since T-cell markers (rag1, ikaros, ccr9, gata3 and tcrβ) are absent of the thymic anlage. However, the primitive hematopoiesis is not affected, given that these mutants have only a slightly reduction on gata1, cmyb and l-plastin. No tcrβ transcripts could be obtained by RT-PCR from IP109 mutants, indicating that V(D)J recombination is absent in these mutants. Finally, in order to prove that the phenotype was caused by the mutation in cmyb, a rescue by BAC (with the sequence of the wild-type cmyb) injection was attempted. Only 12.5% instead of the expected 25% injected fish did not exhibit rag1 expression in the thymus. Moreover, 9 fish that were homozygous for the mutant allele at flanking markers showed normal rag1 expression in the thymus, suggesting that the BAC injection rescued the wild-type rag1 expression in the thymus.
Finally the mutant IP045 was characterized with T-cell markers (rag1, ikaros, and tcrβ) and the macrophage marker l-plastin. This mutant exhibited a severe reduction in thymopoiesis. So far, the mutation that affect this line was linked to the chromosome 8 and the critical interval (6.26 cM) was defined by flanking markers.
Zebrafish ha sido considerado como un modelo genético e inmunológico ideal para la identifiación de genes involucrados tanto en el desarrollo del timo como en la diferenciación de las células linfoides. En el presente trabajo, se analizó una colección de tres mutantes (IU191, IP109 y IP045), a través de “forward genetics”, los cuales excibían defectos en el desarrollo del timo. La mutación responsable fue identificada usando marcadores moleculares (SSLP), y los mutantes fueron caracterizados mediante diferentes técnias moleculares. El clonamiento posicional de la mutación responsable del fenotipo IU191, reveló un cambio de base (G to A) en la última base nucleótidica del exón 11 (donor splicing) del gen que codifica para la subunidad 77 kDa del “cleavage stimulation factor” (cstf3) . Esta mutación impide el “splicing” normal de este exón, provocando la inserción de la secuencia intrónica. Esta al ser incluida, provoca la aparición de un “stop codon” después de 21 pares de bases, posiblemente abortando la producción de una proteína normal. Además, a través de la injección de morholinos en embriones control (silvestres), fue posible reproducir el fenotipo mutante. Estos mutantes también presentan una deformación del cartílago de Meckel. Sin embargo, otras estructuras cranio faciales no presentan ningún defecto en comparación a los individuos controles. Es mas, la formación de los arcos branquiales y la cavidad tímica (formada por células epiteliales del timo) es normal, de acuerdo a la expresión de foxn1.La caracterización detallada de estos mutantes mediante hibridación “in situ”, usando sondas específicas para linfocitos T (rag1, ikaros, ccr9 and tcrβ) demostraron que en ellos existe una severa reducción en la timopoiesis. No obstante, el análisis del programa hematopoietico, de acuerdo a la expresión de gata1, demostró que al menos durante la primera fase hematopoiética estos mutantes no presentan ninguna anomalía. De hecho, la expresión de l-plastin en estos mutantes se ve muy afectada en el timo pero no en otras regiones de estos embriones. Finalmente, resultados preliminares sugieren que al abolir la función de p53 es posible recobrar la expresión de rag1 en los mutantes IU191. El analysis de los embriones mutantes IP109, reveló una transversión T to A en el proto-oncogen cmyb. La mutación encontrada produce una sustitución aminoácidica (isoleucina a asparagina) en una zona altamente conservada del dominio de unión al ADN R3. Estos mutantes carecen the timopoiesis, ya que todos las sondas específicas para células T evaludas (rag1, ikaros, ccr9, gata3 and tcrβ) no se manifiestan en la cavidad tímica. Sin embargo, la primera hematopoiesis o hematopoiesis primitiva sólo se ve parcialmente afectada, de acuerdo a la expresión de gata1, cmyb y l-plastin. Del mismo modo, en los embriones mutantes no fue posible detectar la expresión de de tcrβ, ya sea mediante hibridación “in situ” o por RT-PCR. Este último resultado indicaría que la recombinación V(D)J no ocurriría en estos mutantes. Finalmente, con el fin de demostrar que el fenotipo observado era causado por una mutación en el gen que codifica para el proto-oncogen CMyb, se realizó un rescate de la expresión de rag1 en el timo mediante injeccciones de BAC ADN. Los resultados indican que en 12.5% de los embriones injectados (en vez del 25% esperados) no se obtuvo ninguna expresión de rag1 en el timo. De hecho 9 embriones homozygotos mutantes, de acuerdo con la patrón de amplificación de los marcadores ligados a la mutación, recobraron la expresión de rag1 en el timo. Estos resultados suguieren que la injección de BAC ADN pudo recobrar la expression de rag1 en el timo, demostrando así que cmyb es responsable del fenotipo observado. Finalmente, en este trabajo se caracterizó mediante hibridación “in situ” los embriones mutantes IP045. Con este fin se utilizaron sondas específicas para células T (rag1, ikaros, and tcrβ) y macrófagos (l-plastin). En general, estos mutantes muestran una severa reducción en la timopoiesis. Hasta el momento, la mutación ha sido ligada al cromosoma 8 y el inervalo crítico ha sido definido por maracadores específicos a 6.26 cM
Efecto de la Inclusión de Harina de Plasma Bovino en la Dieta de Individuos Hembras Triploides de Trucha Arcoiris (Oncorhynchus myskiss, Walbaun), Sobre la Respuesta Productiva en su Primera Etapa de Desarrollo
No full textTesis (Ingeniero en Acuicultura)Se estudió el efecto de la inclusión, en distintos porcentajes (2% y 5%), de harina
de plasma bovino en reemplazo de la harina de pescado en la dieta, sobre la respuesta
productiva de hembras triploides de trucha arcoiris (Oncorhynchus my kiss, Walbaum), en
su primera etapa de desarrollo.
Este ensayo se realizó desde los 0,267 gr. por 180 días, evaluando la respuesta
productiva a través de las variables, peso vivo, ganancia de peso, tasa de crecimiento
específico, consumo de alimento, eficiencia de conversión alimenticia, longitudes totales,
índice de condición, mortalidad y conveniencia económica del uso de harina de pl asma
bovino en reemplazo de harina de pescado.
Se encontraron diferencias estadísticamente significativas (P < 0,05) en las
ganancias de peso, longitud totales y las eficiencias de conversión alimenticias entre los
grupos de peces en experimentación; siendo siempre los peces alimentados con 5% de
harina de plasma bovino (tratamiento 3) los que presentaron los mejores valores para cada
variable, seguidos de los peces alimentados con inclusión de 2% de harina de plasma
bovino (tratamiento 2) y finalmente de los alimentados con el tratamiento 1 o control (sin
inclusión de harina de plasma bovino).
Para las variables tasa de crecimiento específico, índice de condición, consumo de
alimento y mortalidad, no se observaron diferencias estadísticamente significativas
(P > 0,05) entre los tres tratamientos.
El análisis de costos evidenció diferencias estadísticamente significativas
(P < 0,05) entre los peces alimentados con el tratamiento 3 con respecto a los peces
pertenecientes al tratamiento control y los del tratamiento 2; siendo los peces
alimentados con el tratamiento 2, los que lograron los menores costos de producción del
kg. de trucha bruto.lt was studied the effect of the inclusion of different percentages (2% and 5%) of
plasma bovine meal, in diet of triploid female rainbow trout (Oncorhynchus my kiss,
Walbaum) on productive responses in fry phase.
The experiment was start in October 1997 and !asted for 180 days. The initial
mean weight of rainbow trout was O, 267 gr ..
In these trial, the productive responses was evaluated through the variables body
weight, gaining weight, total growth, specific growth rate, feed consumption, feeding
efficiency, condition index, mortality and cost analysis of the bovine plasma meal as a
partial substitute for fish meal.
Statistically significant differences (P < 0,05), were found in the fishes of the tri al ,
for the body weight, gaining weight, total growth and feeding efficiency. The results
show that the fishes fed with an experimental feed containing 5% plasma bovine meal
(treatment 3) as a partial substitute of fishmeal, have the best values for each variable.
The fishes fed with an experimental feed containing 2% plasma bovine meal (treatment 2)
in the diet, have the second best valuables and finally the control diet or treatment 1
(fishes fed with an experimental feed not containing plasma bovine mea!).
The specific growth rate, feed consumption, condition index and mortality shows
no statistically significant differences (P > 0.05) in the fishes of the experimental
treatments.
The cost analysis shows statistically significant differences (P < 0,05) between the
fishes of treatment 3 in compare with fishes of treatment 2 and treatment l . At last fishes
fed with treatment 2 show the lowest gross cost for kg. production
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
- …
