A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Modulation of the expression of the transcription factor Max in rat retinal ganglion cells by a recombinant adeno-associated viral vector

Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.

Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

Sentence Compression with Supervised Machine Learning

The subject of the thesis is automatic sentence compression with machine learning, so that the compressed sentences remain both grammatical and retain their essential meaning. There are multiple possible uses for the compression of natural language sentences. In this thesis the focus is generation of television program subtitles, which often are compressed version of the original script of the program. The main part of the thesis consists of machine learning experiments for automatic sentence compression using different approaches to the problem. The machine learning methods used for this work are linear-chain conditional random fields and support vector machines. Also we take a look which automatic text analysis methods provide useful features for the task. The data used for machine learning is supplied by Lingsoft Inc. and consists of subtitles in both compressed an uncompressed form. The models are compared to a baseline system and comparisons are made both automatically and also using human evaluation, because of the potentially subjective nature of the output. The best result is achieved using a CRF - sequence classification using a rich feature set. All text analysis methods help classification and most useful method is morphological analysis. Tutkielman aihe on suomenkielisten lauseiden automaattinen tiivistäminen koneellisesti, niin että lyhennetyt lauseet säilyttävät olennaisen informaationsa ja pysyvät kieliopillisina. Luonnollisen kielen lauseiden tiivistämiselle on monta käyttötarkoitusta, mutta tässä tutkielmassa aihetta lähestytään television ohjelmien tekstittämisen kautta, johon käytännössä kuuluu alkuperäisen tekstin lyhentäminen televisioruudulle paremmin sopivaksi. Tutkielmassa kokeillaan erilaisia koneoppimismenetelmiä tekstin automaatiseen lyhentämiseen ja tarkastellaan miten hyvin erilaiset luonnollisen kielen analyysimenetelmät tuottavat informaatiota, joka auttaa näitä menetelmiä lyhentämään lauseita. Lisäksi tarkastellaan minkälainen lähestymistapa tuottaa parhaan lopputuloksen. Käytetyt koneoppimismenetelmät ovat tukivektorikone ja lineaarisen sekvenssin mallinen CRF. Koneoppimisen tukena käytetään tekstityksiä niiden eri käsittelyvaiheissa, jotka on saatu Lingsoft OY:ltä. Luotuja malleja vertaillaan Lopulta mallien lopputuloksia evaluoidaan automaattisesti ja koska teksti lopputuksena on jossain määrin subjektiivinen myös ihmisarviointiin perustuen. Vertailukohtana toimii kirjallisuudesta poimittu menetelmä. Tutkielman tuloksena paras lopputulos saadaan aikaan käyttäen CRF sekvenssi-luokittelijaa laajalla piirrejoukolla. Kaikki kokeillut teksin analyysimenetelmät auttavat luokittelussa, joista tärkeimmän panoksen antaa morfologinen analyysi.

Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

A tour from the City of New-York, to Detroit, in the Michigan Territory, made between the 2d of May and the 22d of September, 1818 : the tour extends from New-York, by Albany, Schenectady, and Utica to Sacket's Harbor, and thence throught Lake Ontario, to St. Lawrence river, and down that stream to Hamilton village. Thence along both banks of the St. Lawrence, from Hamilton to the Thousand Islands; thence to Sacket's Harbor by water; from that place by the route of great Sodus, Geneva, Cananaigua and Batavia, to Buffalo; and from thence to Black Rock, Fort Erie, the Falls of Niagara, Queenston, Lewiston, and the memorable fields of Bridgewater and Chippewa after viewing the interesting place of Niagara, the author traversed the south shore of Lake Erie to the City of Detroit, and visited in the latter range, Dunkirk, Erie, Cleveland, Sandusky, and other places of less note. The tour is accompanied with a map upon which the route will be designated; a particular map of the Falls andRiver of Niagara, and the environs of the City of Detroit /

Errata slip inserted.

Sketches of the Battle Fields of Ontario from the War of 1812, July 15, 1876.

Accompanying caption from the Canadian Illustrated News, July 15, 1876: “We publish today a page of sketches consisting of the following battle fields in Ontario :--Lundy’s Lane where, without doubt, the hardest fought battle of 1812-15 took place, and in which more troops were engaged than in any other engagement of that war : the battle field of Stony Creek where the Canadians and Indians made a night attack on the Americans and achieved a victory over a greatly superior force and obliged the Americans to retreat back to the shelter of Old Fort George which was the scene of many engagements during the war. Beaver Dam battle field is just in the suburbs of the thriving village of Thorold, and the monument covers the remains of several soldiers whose bodies were unearthed during the building of the new Welland Canal at that place.”

Newspaper Clipping - Fields' homestead

A newspaper clipping describing a fire at the Fields' homestead near Queenston. The caption describes the house as where "Sir Isaac Brock slept the night before he was killed".

Indenture - Land sold by Hart Smith of Lincoln County to James Fields of Newark in Oxford County

Indenture regarding land sold by Hart Smith of Lincoln County to James Fields of Newark in Oxford County, First Concession, Lot no. 3. October 11, 1797.

Respuesta funcional de Toxorhynchites theobaldi (DYAR y KNAB) sobre larvas de Aedes aegypti (LINN.) vector del dengue

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Control del vector del dengue Aedes aegypti (l) con la participación de estudiantes de primaria como educadores de salud en la cuidad de Guadalupe Nuevo Léon

Tesis (Maestria en Ciencias con Especialidad en Entomología Médica) UANL

Comparación de la eficiencia de rociados domiciliares en ultra bajo volumen (ULV) y termonebulización para el control de poblaciones adultas del vector del dengue Aedes aegypti (L.) en una colonia de Guadalupe, Nuevo León, México

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Bionomia de Aedes aegypti L., vector del dengue en el sur del estado de Jalisco, México

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) UANL

Bionomía del vector de la malaria Anopheles vestitipennis Dyar & Knab (Díptera: Culicidae) en la cuenca del Río Polochic, Alta Verapaz, Guatemala, C.A. [por] Jaime Abraham Juárez Sandoval

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) U.A.N.L.