Role of G protein coupled inwardly rectifier K+ channels (GIRK) on the neurobiology depression

353 págs.

Tissue engineering active biological machines : bio-inspired design, directed self-assembly, and characterization of muscular pumps simulating the embryonic heart

Biological machines are active devices that are comprised of cells and other biological components. These functional devices are best suited for physiological environments that support cellular function and survival. Biological machines have the potential to revolutionize the engineering of biomedical devices intended for implantation, where the human body can provide the required physiological environment. For engineering such cell-based machines, bio-inspired design can serve as a guiding platform as it provides functionally proven designs that are attainable by living cells. In the present work, a systematic approach was used to tissue engineer one such machine by exclusively using biological building blocks and by employing a bio-inspired design. Valveless impedance pumps were constructed based on the working principles of the embryonic vertebrate heart and by using cells and tissue derived from rats. The function of these tissue-engineered muscular pumps was characterized by exploring their spatiotemporal and flow behavior in order to better understand the capabilities and limitations of cells when used as the engines of biological machines.

Developing peptide based capture agents for diagnostics and therapeutics

Iterative in situ click chemistry (IISCC) is a robust general technology for development of high throughput, inexpensive protein detection agents. In IISCC, the target protein acts as a template and catalyst, and assembles its own ligand from modular blocks of peptides. This process of ligand discovery is iterated to add peptide arms to develop a multivalent ligand with increased affinity and selectivity. The peptide based protein capture agents (PCC) should ideally have the same degree of selectivity and specificity as a monoclonal antibody, along with improved chemical stability. We had previously reported developing a PCC agent against bovine carbonic anhydrase II (bCAII) that could replace a polyclonal antibody. To further enhance the affinity or specificity of the PCC agent, I explore branching the peptide arms to develop branched PCC agents against bCAII. The developed branched capture agents have two to three fold higher affinities for the target protein. In the second part of my thesis, I describe the epitope targeting strategy, a strategy for directing the development of a peptide ligand against specific region or fragment of the protein. The strategy is successfully demonstrated by developing PCC agents with low nanomolar binding affinities that target the C-terminal hydrophobic motif of Akt2 kinase. One of the developed triligands inhibits the kinase activity of Akt. This suggests that, if targeted against the right epitope, the PCC agents can also influence the functional properties of the protein. The exquisite control of the epitope targeting strategy is further demonstrated by developing a cyclic ligand against Akt2. The cyclic ligand acts as an inhibitor by itself, without any iteration of the ligand discovery process. The epitope targeting strategy is a cornerstone of the IISCC technology and opens up new opportunities, leading to the development of protein detection agents and of modulators of protein functions.

A cocktail of thermally stable, chemically synthesized capture agents for the efficient detection of anti-gp41 antibodies from human sera and techniques

This thesis reports on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60°C.

Development of protein-catalyzed capture (PCC) agents with application to the specific targeting of the E17K Point mutation of Akt1

This thesis describes the expansion and improvement of the iterative in situ click chemistry OBOC peptide library screening technology. Previous work provided a proof-of-concept demonstration that this technique was advantageous for the production of protein-catalyzed capture (PCC) agents that could be used as drop-in replacements for antibodies in a variety of applications. Chapter 2 describes the technology development that was undertaken to optimize this screening process and make it readily available for a wide variety of targets. This optimization is what has allowed for the explosive growth of the PCC agent project over the past few years.

These technology improvements were applied to the discovery of PCC agents specific for single amino acid point mutations in proteins, which have many applications in cancer detection and treatment. Chapter 3 describes the use of a general all-chemical epitope-targeting strategy that can focus PCC agent development directly to a site of interest on a protein surface. This technique utilizes a chemically-synthesized chunk of the protein, called an epitope, substituted with a click handle in combination with the OBOC in situ click chemistry libraries in order to focus ligand development at a site of interest. Specifically, Chapter 3 discusses the use of this technique in developing a PCC agent specific for the E17K mutation of Akt1. Chapter 4 details the expansion of this ligand into a mutation-specific inhibitor, with applications in therapeutics.

Análise da composição da matriz extracelular das camadas urotelial e muscular da bexiga de mulheres em diferentes idades

A complacência da bexiga depende de músculos lisos, fibras colágenas, fibras elásiticas e suas relações. O objetivo deste trabalho é determinar a composição da matriz extracelular em amostras de bexigas normais através de análise bioquímica de colágeno e glicosaminoglicanos em amostras obtidas de mulheres em diferentes grupos de idade, analisando separadamente as camadas urotelial e muscular. Avaliamos 17 amostras de bexiga divididas em três grupos: infância (N=5), menacme (N=6) e pós-menopausa (N=6). As bexigas foram analisadas para concentração de GAG total e colágeno e para análise qualitativa de GAG por eletroforese em gel de agarose. Na camada muscular, não houve diferença entre os grupos tanto para GAG quanto para colágeno. Na camada urotelial, a análise da concentração de colágeno não mostrou diferença entre os grupos, mas a concentração de GAG no grupo da pós-menopausa (0.21 0.12 μg de ácido hexurônico/mg de tecido seco) apresentou diferença em relação aos grupos do menacme (1.78 1.62 μg de ácido hexurônico/mg de tecido seco) e da infância ( 2.29 1.32 μg de ácido hexurônico/mg de tecido seco).Nosso trabalho concluiu que a concentração de GAG está substancialmente diminuída na camada urotelial da bexiga de mulheres na pós-menopausa.

Imidazolium-Based Organic Structure Directing Agents for the Synthesis of Microporous Materials

The central theme of this thesis is the use of imidazolium-based organic structure directing agents (OSDAs) in microporous materials synthesis. Imidazoliums are advantageous OSDAs as they are relatively inexpensive and simple to prepare, show robust stability under microporous material synthesis conditions, have led to a wide range of products, and have many permutations in structure that can be explored. The work I present involves the use of mono-, di-, and triquaternary imidazolium-based OSDAs in a wide variety of microporous material syntheses. Much of this work was motivated by successful computational predictions (Chapter 2) that led me to continue to explore these types of OSDAs. Some of the important discoveries with these OSDAs include the following: 1) Experimental evaluation and confirmation of a computational method that predicted a new OSDA for pure-silica STW, a desired framework containing helical pores that was previously very difficult to synthesize. 2) Discovery of a number of new imidazolium OSDAs to synthesize zeolite RTH, a zeolite desired for both the methanol-to-olefins reaction as well as NOX reduction in exhaust gases. This discovery enables the use of RTH for many additional investigations as the previous OSDA used to make this framework was difficult to synthesize, such that no large scale preparations would be practical. 3) The synthesis of pure-silica RTH by topotactic condensation from a layered precursor (denoted CIT-10), that can also be pillared to make a new framework material with an expanded pore system, denoted CIT-11, that can be calcined to form a new microporous material, denoted CIT-12. CIT-10 is also interesting since it is the first layered material to contain 8 membered rings through the layers, making it potentially useful in separations if delamination methods can be developed. 4) The synthesis of a new microporous material, denoted CIT-7 (framework code CSV) that contains a 2-dimensional system of 8 and 10 membered rings with a large cage at channel intersections. This material is especially important since it can be synthesized as a pure-silica framework under low-water, fluoride-mediated synthesis conditions, and as an aluminosilicate material under hydroxide mediated conditions. 5) The synthesis of high-silica heulandite (HEU) by topotactic condensation as well as direct synthesis, demonstrating new, more hydrothermally stable compositions of a previously known framework. 6) The synthesis of germanosilicate and aluminophosphate LTA using a triquaternary OSDA. All of these materials show the diverse range of products that can be formed from OSDAs that can be prepared by straightforward syntheses and have made many of these materials accessible for the first time under facile zeolite synthesis conditions.

Perinatal hipoxic ischemic injury in the auditory pathway and the effect of several neuroprotective agents

192 p.

Análisis nutricional y su relación con el estrés psico-físico y el daño muscular en jugadoras profesionales de voleibol a lo largo de una temporada

261 p.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of alpha-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg(-1), 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle-regulated by both diet and CB1 receptor activity-through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.