On crack path stability in a layered material

Crack paths in an elastic layer on top of a substrate are considered. Crack growth is initiated from an edge crack in the layer. The plane of the initially straight crack forms an angle to the free surface. The load consists of a pair of forces applied at the crack mouth and parallel to the interface. Crack paths are calculated using a boundary element method. Crack growth is assumed to proceed along a path for which the mode II stress intensity factor vanishes. The inclination and the length of the initial crack are varied. The effect of two different substrates on the crack path evolution is demonstrated. A crack path initially leading perpendicularly to the interface is shown to be directionally unstable for a rigid substrate. Irrespective of its initial angle, the crack does not reach the interface, but reaches the free surface if the layer is infinitely long. At finite layer length the crack reaches the upper free surface if the initial crack inclination to the surface is small enough. For an inextendable flexible substrate, on the other hand, the crack reaches the interface if its initial inclination is large enough. For the flexible substrate an unstable path parallel with the sides of an infinitely long layer is identified. The results are compared with experimental results and discussed in view of characterisation of directionally unstable crack paths. The energy release rate for an inclined edge crack is determined analytically.

Sidewall flow stability in the MHD channel flows

The velocity distribution between two sidewalls is; M-shaped for the MHD channel flows with rectangular cross section and thin conducting walls in a strong transverse magnetic field. Assume that the dimensionless numbers R(m) much less than 1, M, N much greater than 1, and sigma

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.

Forward-looking Pairwise Stability in Networks with Externalities

We consider cooperation situations where players have network relations. Networks evolve according to a stationary transition probability matrix and at each moment in time players receive payoffs from a stationary allocation rule. Players discount the future by a common factor. The pair formed by an allocation rule and a transition probability matrix is called a forward-looking network formation scheme if, first, the probability that a link is created is positive if the discounted, expected gains to its two participants are positive, and if, second, the probability that a link is eliminated is positive if the discounted, expected gains to at least one of its two participants are positive. The main result is the existence, for all discount factors and all value functions, of a forward-looking network formation scheme. Furthermore, we can always nd a forward-looking network formation scheme such that (i) the allocation rule is component balanced and (ii) the transition probabilities increase in the di erence in payo s for the corresponding players responsible for the transition. We use this dynamic solution concept to explore the tension between e ciency and stability.

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.

Temporal and spacial control of RNA stability in the early embryo of Drosophila melanogaster

Early embryogenesis in metazoa is controlled by maternally synthesized products. Among these products, the mature egg is loaded with transcripts representing approximately two thirds of the genome. A subset of this maternal RNA pool is degraded prior to the transition to zygotic control of development. This transfer of control of development from maternal to zygotic products is referred to as the midblastula transition (or MBT). It is believed that the degradation of maternal transcripts is required to terminate maternal control of development and to allow zygotic control of development to begin. Until now this process of maternal transcript degradation and the subsequent timing of the MBT has been poorly understood. I have demonstrated that in the early embryo there are two independent RNA degradation pathways, either of which is sufficient for transcript elimination. However, only the concerted action of both pathways leads to elimination of transcripts with the correct timing, at the MBT. The first pathway is maternally encoded, is triggered by egg activation, and is targeted to specific classes of mRNAs through cis-acting elements in the 3' untranslated region (UTR}. The second pathway is activated 2 hr after fertilization and functions together with the maternal pathway to ensure that transcripts are degraded by the MBT. In addition, some transcripts fail to degrade at select subcellular locations adding an element of spatial control to RNA degradation. The spatial control of RNA degradation is achieved by protecting, or masking, transcripts from the degradation machinery. The RNA degradation and protection events are regulated by distinct cis-elements in the 3' untranslated region (UTR). These results provide the first systematic dissection of this highly conserved process in development and demonstrate that RNA degradation is a novel mechanism used for both temporal and spatial control of development.

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.

In vivo analysis of interactions between trans-acting factors and their target genes

The investigations presented in this thesis use various in vivo techniques to understand how trans-acting factors control gene expression. The first part addresses the transcriptional regulation of muscle creatine kinase (MCK). MCK expression is activated during the course of development and is found only in differentiated muscle. Several in vivo footprints are observed at the enhancer of this gene, but all of these interactions are limited to cell types that express MCK. This is interesting because two of the footprints appear to represent muscle specific use of general transcription factors, while the other two correspond to sites that can bind the myogenic regulator, MyoD1, in vitro. MyoD1 and these general factors are present in myoblasts, but can bind to the enhancer only in myocytes. This suggests that either the factors themselves are post-translationally modified (phosphorylation or protein:protein interactions), or the accessibility of the enhancer to the factors is limited (changes in chromatin structure). The in vivo footprinting study of MCK was performed with a new ligation mediated, single-sided PCR (polymerase chain reaction) technique that I have developed.

The second half of the thesis concerns the regulation of mouse metallothionein (MT). Metallothioneins are a family of highly conserved housekeeping genes whose expression can be induced by heavy metals, steroids, and other stresses. By adapting a primer extension method of genomic sequencing to in vivo footprinting, I've observed both metal inducible and noninducible interactions at the promoter of MT-I. From these results I've been able to limit the possible mechanisms by which metal responsive trans-acting factors induce transcription. These interpretations correlate with a second line of experiments involving the stable titration of positive acting factors necessary for induction of MT. I've amplified the promoter of MT to 10^2-10^3 copies per cell by fusing the 5' and 3' ends of the MT gene to the coding region of DHFR and selecting cells for methotrexate resistance. In these cells, there is a metal-specific titration effect, and although it acts at the level of transcription, it appears to be independent of direct DNA binding factors.