Degradación in vivo de la materia seca y materia orgánica del Teocinte (Zea nicaraguensis Iltis y Benz)

Este estudio se realizó con el objetivo de determinar la degradabilidad in vivo del Teocinte (Zea nicaraguensis Iltis & Benz). El ensayo de degradabilidad fue realizado en la galera experimental de la Facultad de Ciencia Animal adscrita a la Universidad Nacional Agraria. En este ensayo se utilizaron dos vacas de raza Reyna, secas y vacías, con fistulas ruminales. Los tiempos de incubación (0, 0.5, 1, 3, 6, 9, 12, 24, 48, 72, 96 horas) fueron considerados los tratamientos (11) por 4 réplicas por cada tiempo. Las variables evaluadas fueron: degradación de materia seca (DMS) y degradación de materia orgánica (DMO). Para la determinación de las variables, se utilizó el modelo exponencial descrito por Orskov and McDonald (1979). Como resultados finales de la Degradabilidad de la MS se obtuvo una fracción soluble de 2.44% (a), fracción insoluble pero potencialmente degradable del 23.35% (b), una tasa de degradación de la fracción potencialmente degradada estimada del 2.61%* horas (c) y la fracción no degradable [100-(2.44 + 23.35)] del 74.05%. En la Degradabilidad de la MO la fracción soluble fue de 2.43% (a), la fracción insoluble pero potencialmente degradable de 23.35% (b), una tasa de degradación de la fracción potencialmente degradada estimada de 2.61%* horas y la fracción no degradable [100-(2.43 + 23.35)] de 74.22%. El Teocinte en la edad de corte utilizada en este experimento no tiene valores de degradabilidad compatibles con sistemas de producción animal de alta intensidad por lo que se sugiere seguir investigando en su potencialidad.

In vivo regulation of the Vi antigen in Salmonella and induction of immune responses with an in vivo-inducible promoter.

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo-inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.

A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo.

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+)), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+) colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+) and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+) resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(-) infected animals. C5.507 Vi(+) infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-). The modulating effect associated with Vi was not observed in MyD88(-/-) and was reduced in TLR4(-/-) mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.

Pravastatin inhibits cell proliferation and increased MAT1A expression in hepatocarcinoma cells and in vivo models

Background: Statins may have therapeutic effects on hepatocarcinoma (HCC). This type of disorder is the most common malignant primary tumour in the liver. Our objective was to determine whether pravastatin had a therapeutic effect in vitro and in vivo models. Method: We design in vitro and in vivo model. In vitro we used PLC and determine cell proliferation. In vivo, we used and animal model to determined, PCNA and MAT1A expression and transaminases levels. Results: We found that pravastatin decreases cell proliferation in vitro (cell proliferation in pravastatin group was 82%, in sorafenib group 51% and in combined group 40%) and in vivo (in pravastatin group 80%, in sorafenib group 76.4% and in combined group 72.72%). The MAT1A levels, was significantly higher in Pravastatin group (D 62%, P 94%, S 71%, P + S 91%). The transaminases levels, decreased significantly in Pravastatin group (GOT and GPT levels D 619.5 U/L; 271 U/L) (P 117.5 U/L; 43.5 U/L) (S 147 U/L; 59 U/L) (P + S 142 U/L; 59 U/L). Conclusion: The combination of pravastatin + sorafenib were more effective than Sorafenib alone.

Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

227 págs.

In Vitro and In Vivo Evaluation of a Folate-Targeted Copolymeric Submicrohydrogel Based on N-Isopropylacrylamide as 5-Fluorouracil Delivery System

Folate-targeted poly[(p-nitrophenyl acrylate)-co-(N-isopropylacrylamide)] nanohydrogel (F-SubMG) was loaded with 5-fluorouracil (5-FU) to obtain low (16.3 +/- 1.9 mu g 5-FU/mg F-SubMG) and high (46.8 +/- 3.8 mu g 5-FU/mg F-SubMG) load 5-FU-loaded F-SubMGs. The complete in vitro drug release took place in 8 h. The cytotoxicity of unloaded F-SubMGs in MCF7 and HeLa cells was low; although it increased for high F-SubMG concentration. The administration of 10 mu M 5-FU by 5-FU-loaded F-SubMGs was effective on both cellular types. Cell uptake of F-SubMGs took place in both cell types, but it was higher in HeLa cells because they are folate receptor positive. After subcutaneous administration (28 mg 5-FU/kg b.w.) in Wistar rats, F-SubMGs were detected at the site of injection under the skin. Histological studies indicated that the F-SubMGs were surrounded by connective tissue, without any signs of rejections, even 60 days after injection. Pharmacokinetic study showed an increase in MRT (mean residence time) of 5-FU when the drug was administered by drug-loaded F-SubMGs.

Anatomical and developmental patterns of interleukin-2 gene expression in the mouse: analysis of IL-2 expressing cells and partial characterization of interactions involved in mediating IL-2 gene expression in vivo

Interleukin-2 (IL-2) is an important mediator in the vertebrate immune system. IL-2 is a potent growth factor that mature T lymphocytes use as a proliferation signal and the production of IL-2 is crucial for the clonal expansion of antigen-specific T cells in the primary immune response. IL-2 driven proliferation is dependent on the interaction of the lymphokine with its cognate multichain receptor. IL-2 expression is induced only upon stimulation and transcriptional activation of the IL-2 gene relies extensively on the coordinate interaction of numerous inducible and constitutive trans-acting factors. Over the past several years, thousands of papers have been published regarding molecular and cellular aspects of IL-2 gene expression and IL-2 function. The vast majority of these reports describe work that has been carried out in vitro. However, considerably less is known about control of IL-2 gene expression and IL-2 function in vivo.

To gain new insight into the regulation of IL-2 gene expression in vivo, anatomical and developmental patterns of IL-2 gene expression in the mouse were established by employing in situ hybridization and immunohistochemical staining methodologies to tissue sections generated from normal mice and mutant animals in which T -cell development was perturbed. Results from these studies revealed several interesting aspects of IL-2 gene expression, such as (1) induction of IL-2 gene expression and protein synthesis in the thymus, the primary site of T-cell development in the body, (2) cell-type specificity of IL-2 gene expression in vivo, (3) participation of IL-2 in the extrathymic expansion of mature T cells in particular tissues, independent of an acute immune response to foreign antigen, (4) involvement of IL-2 in maintaining immunologic balance in the mucosal immune system, and (5) potential function of IL-2 in early events associated with hematopoiesis.

Extensive analysis of IL-2 mRNA accumulation and protein production in the murine thymus at various stages of development established the existence of two classes of intrathymic IL-2 producing cells. One class of intrathymic IL-2 producers was found exclusively in the fetal thymus. Cells belonging to this subset were restricted to the outermost region of the thymus. IL-2 expression in the fetal thymus was highly transient; a dramatic peak ofiL-2 mRNA accumulation was identified at day 14.5 of gestation and maximal IL-2 protein production was observed 12 hours later, after which both IL-2 mRNA and protein levels rapidly decreased. Significantly, the presence of IL-2 expressing cells in the day 14-15 fetal thymus was not contingent on the generation of T-cell receptor (TcR) positive cells. The second class of IL-2 producing cells was also detectable in the fetal thymus (cells found in this class represented a minority subset of IL-2 producers in the fetal thymus) but persist in the thymus during later stages of development and after birth. Intrathymic IL-2 producers in postnatal animals were located in the subcapsular region and cortex, indicating that these cells reside in the same areas where immature T cells are consigned. The frequency of IL-2 expressing cells in the postnatal thymus was extremely low, indicating that induction of IL-2 expression and protein synthesis are indicative of a rare activation event. Unlike the fetal class of intrathymic IL-2 producers, the presence of IL-2 producing cells in the postnatal thymus was dependent on to the generation of TcR+ cells. Subsequent examination of intrathymic IL-2 production in mutant postnatal mice unable to produce either αβ or γδ T cells showed that postnatal IL-2 producers in the thymus belong to both αβ and γδ lineages. Additionally, further studies indicated that IL-2 synthesis by immature αβ -T cells depends on the expression of bonafide TcR αβ-heterodimers. Taken altogether, IL-2 production in the postnatal thymus relies on the generation of αβ or γδ-TcR^+ cells and induction of IL-2 protein synthesis can be linked to an activation event mediated via the TcR.

With regard to tissue specificity of IL-2 gene expression in vivo, analysis of whole body sections obtained from normal neonatal mouse pups by in situ hybridization demonstrated that IL-2 mRNA^+ cells were found in both lymphoid and nonlymphoid tissues with which T cells are associated, such as the thymus (as described above), dermis and gut. Tissues devoid of IL-2 mRNA^+ cells included brain, heart, lung, liver, stomach, spine, spinal cord, kidney, and bladder. Additional analysis of isolated tissues taken from older animals revealed that IL-2 expression was undetectable in bone marrow and in nonactivated spleen and lymph nodes. Thus, it appears that extrathymic IL-2 expressing cells in nonimmunologically challenged animals are relegated to particular epidermal and epithelial tissues in which characterized subsets of T cells reside and thatinduction of IL-2 gene expression associated with these tissues may be a result of T-cell activation therein.

Based on the neonatal in situ hybridization results, a detailed investigation into possible induction of IL-2 expression resulting in IL-2 protein synthesis in the skin and gut revealed that IL-2 expression is induced in the epidermis and intestine and IL-2 protein is available to drive cell proliferation of resident cells and/or participate in immune function in these tissues. Pertaining to IL-2 expression in the skin, maximal IL-2 mRNA accumulation and protein production were observed when resident Vγ_3^+ T-cell populations were expanding. At this age, both IL-2 mRNA^+ cells and IL-2 protein production were intimately associated with hair follicles. Likewise, at this age a significant number of CD3ε^+ cells were also found in association with follicles. The colocalization of IL-2 expression and CD3ε^+ cells suggests that IL-2 expression is induced when T cells are in contact with hair follicles. In contrast, neither IL-2 mRNA nor IL-2 protein were readily detected once T-cell density in the skin reached steady-state proportions. At this point, T cells were no longer found associated with hair follicles but were evenly distributed throughout the epidermis. In addition, IL-2 expression in the skin was contingent upon the presence of mature T cells therein and induction of IL-2 protein synthesis in the skin did not depend on the expression of a specific TcR on resident T cells. These newly disclosed properties of IL-2 expression in the skin indicate that IL-2 may play an additional role in controlling mature T-cell proliferation by participating in the extrathymic expansion of T cells, particularly those associated with the epidermis.

Finally, regarding IL-2 expression and protein synthesis in the gut, IL-2 producing cells were found associated with the lamina propria of neonatal animals and gut-associated IL-2 production persisted throughout life. In older animals, the frequency of IL-2 producing cells in the small intestine was not identical to that in the large intestine and this difference may reflect regional specialization of the mucosal immune system in response to enteric antigen. Similar to other instances of IL-2 gene expression in vivo, a failure to generate mature T cells also led to an abrogation of IL-2 protein production in the gut. The presence of IL-2 producing cells in the neonatal gut suggested that these cells may be generated during fetal development. Examination of the fetal gut to determine the distribution of IL-2 producing cells therein indicated that there was a tenfold increase in the number of gut-associated IL-2 producers at day 20 of gestation compared to that observed four days earlier and there was little difference between the frequency of IL-2 producing cells in prenatal versus neonatal gut. The origin of these fetally-derived IL-2 producing cells is unclear. Prior to the immigration of IL-2 inducible cells to the fetal gut and/or induction of IL-2 expression therein, IL-2 protein was observed in the fetal liver and fetal omentum, as well as the fetal thymus. Considering that induction of IL-2 protein synthesis may be an indication of future functional capability, detection of IL-2 producing cells in the fetal liver and fetal omentum raises the possibility that IL-2 producing cells in the fetal gut may be extrathymic in origin and IL-2 producing cells in these fetal tissues may not belong solely to the T lineage. Overall, these results provide increased understanding of the nature of IL-2 producing cells in the gut and how the absence of IL-2 production therein and in fetal hematopoietic tissues can result in the acute pathology observed in IL-2 deficient animals.

An in vivo approach to tRNA identity

A leucine-inserting tRNA has been transformed into a serine-inserting tRNA by changing 12 nucleotides. Only 8 of the 12 changes are required to effect the conversion of the leucine tRNA to serine tRNA identity. The 8 essential changes reside in basepair 11-24 in the D stem, basepairs 3-70, 2-71 and nucleotides 72 and 73, all of the acceptor stem.

Functional amber suppressor tRNA genes were generated for 14 species of tRNA in E. coli, and their amino acid specificities determined. The suppressors can be classified into three groups, based upon their specificities. Class I suppressors, tRNA^(Ala2)_(CUA), tRNA^(GlyU)_(CUA), tRNA^(HisA)_(CUA), tRNA^(Lys)_(CUA), and tRNA^(ProH)_(CUA), inserted the predicted amino acid. The Class II suppressors, tRNA^(GluA)_(CUA) , tRNA^(GlyT)_(CUA), and tRNA^(Ile1)_(CUA) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase (AAS). The Class III suppressors, tRNA^(Arg)_(CUA), tRNA^(AspM)_(CUA), tRNA^(Ile2)_(CUA), tRNA^(Thr2)_(CUA), tRNA^(Met(m))_(CUA) and tRNA^(Val)_(CUA) inserted predominantly lysine.

Flexible microimplants for in vivo sensing

The work in this thesis develops two types of microimplants for the application of cardiovascular in vivo biomedical sensing, one for short-term diagnosis and the other for long-term monitoring.

Despite advances in diagnosis and therapy, atherosclerotic cardiovascular disease remains the leading cause of morbidity and mortality in the Western world. Predicting metabolically active atherosclerotic plaques has remained an unmet clinical need. A stretchable impedance sensor manifested as a pair of quasi-concentric microelectrodes was developed to detect unstable intravascular. By integrating the impedance sensor with a cardiac catheter, high-resolution Electrochemical Impedance Spectroscopy (EIS) measurements can be conducted during cardiac catheterization. An inflatable silicone balloon is added to the sensor to secure a well-controlled contact with the plaque under test in vivo. By deploying the device to the explants of NZW rabbit aorta and live animals, distinct EIS measurements were observed for unstable atherosclerotic plaques that harbored active lipids and inflammatory cells.

On the other hand, zebrafish (Danio rerio) is an emerging genetic model for heart regenerative medicine. In humans, myocardial infarction results in the irreversible loss of cardiomyocytes. Zebrafish hearts can fully regenerate after two months with 20% ventricular resection. Long-term electrocardiogram (ECG) recording can characterize the heart regeneration in a functional dimension. A flexible microelectrode membrane was developed to be percutaneously implanted onto a zebrafish heart and record epicardial ECG signals from specific regions on it. Region-specific aberrant cardiac signals were obtained from injured and regenerated hearts. Following that, in order to achieve continuous and wireless recording from non-sedated and non-restricted small animal models, a wireless ECG recording system was designed for the microelectrode membrane, prototyped on a printed circuit board and demonstrated on a one-day-old neonatal mouse. Furthermore, a flexible and compact parylene C printed circuit membrane was used as the integration platform for the wireless ECG recording electronics. A substantially miniature wireless ECG recording system was achieved.