In order for organisms to co-exist with nonliving matter, envelop to protect their delicate internal functions must be present. There are other reasons for this boundary to exist, e.g. to limit the volume occupied by the organism and to compartmentalize the contents of the organism so that certain critical concentrations may be easily maintained. Such envelopes are, of course, the cellular membranes. Membranes differ greatly between species, as well as within species. Given the complexity to which organisms have evolved, membranes have developed with a myriad of functions and components. It is easy to see differences between plant cellular membranes and animal cell membranes as well as to see differences between mitochondrial and nuclear membranes within a single cell.

No / This work explored the role of skin appendages (shunt route) in passive and iontophoretic drug and liposome penetration. The technique used an epidermis and stratum corneum sandwich from the same skin donor with the additional stratum corneum forming the top layer of the sandwich. Penetration was monitored during occluded passive and iontophoretic (0.5 mA cm-2) delivery of mannitol and estradiol solutions, and ultradeformable liposomes containing estradiol. The shunt route had a significant role during passive penetration of mannitol (hydrophilic compound), but was negligible during penetration of estradiol (lipophilic drug) and liposomes. In iontophoresis, the shunt route significantly contributed to the overall flux of all preparations, being highest for mannitol. However, shunts were not the only pathway for iontophoretic drug delivery and evidence was observed for the creation of new aqueous pathways via disorganization of the intercellular lipid domain of stratum corneum. The skin sandwich technique should prove valuable for general studies on routes of skin penetration.

The primary purpose of this study was to investigate the effect of acute high-intensity interval exercise (HIIE) vs. continuous moderate-intensity exercise (CME) on serum CTRP9 and brachial FMD responses in obese and normal-weight subjects. Sixteen participants (9 obese and 7 normal-weight) completed HIIE and CME in a randomized fashion. Our results showed a significant time effect for CTRP9 immediately following acute HIIE and CME in both groups. Furthermore, both significant treatment by time and group by time interactions for FMD were observed following both exercise protocols, with greater CME-induced FMD response in obese subjects than normal-weight subjects. Additionally, a positive correlation in percent change (baseline to peak) between CTRP9 and FMD was observed following acute CME. These findings support acute CME for improvement of endothelial function in obesity. Furthermore, the novel results from this study provide a foundation for additional examination of the mechanisms of exercise-mediated CTRP9 on endothelial function. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Solute diffusion is critical to maintenance of cellular function and matrix integrity in articular cartilage. Nutrient deficiency due to transport limitations is thought to be one of the causes of the pathological degeneration of the cartilage tissue. Thus, a study of diffusion within cartilage will lead to a better understanding of the causes of cartilage degeneration. To accurately estimate diffusion coefficients in cartilage and other hydrated medium, we developed a finite-element based method, the Direct Diffusion Simulation Parameter Estimation method (DDSPE), to be used for quantitative determination of solute diffusivities from Fluorescence Recovery After Photobleaching data. Analyses of simulated and experimental FRAP data demonstrated that this method was more accurate than existing analytical methods, including having a low sensitivity to variations in the spot radius. Subsequently, the roles of extracellular matrix (ECM) composition and tissue orientation in solute diffusion within immature bovine cartilage were explored. Diffusivities were measured through the cartilage depth and in two different orientations (radial and transverse). Diffusivities were then correlated with ECM components. Matrix water content was found to be the best predictor of solute diffusion rates in immature cartilage. Although no specific experiments were done to measure the effect of structure, our results suggested that matrix structure did indeed modulate transport. Diffusional anisotropy, defined as the ratio of the diffusivities in both orientations, was observed to be significant in all the immature cartilage zones. As a consequence, the differences in solute diffusion between immature and mature bovine cartilage were investigated. Diffusion rates and diffusional anisotropy decreased in the mature cartilage superficial zone. The decrease in diffusivities observed in mature cartilage suggests that there may be a reduction in nutrient and growth factor supply to the cells. Nevertheless, healthy adult cartilage can still maintain its normal function even with a reduction in solute diffusion rates as nutrient diffusion distances are shorter in mature cartilage. However, any disruption in the mechanical or biological environment could cause an imbalance in tissue homeostasis, which when combined with decreased diffusivities, could trigger matrix degeneration. Thus, decreased diffusivity may be a necessary but not a sufficient prerequisite of matrix degeneration.

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Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

The thesis research was designed to study the characteristics of two important physiological processes in smooth muscle: Ca2+ transport mediated by the plasmalemmal Ca2+-ATPase and muscarinic receptor-G protein interactions. In resting smooth muscle, several Ca2+ extrusion or sequestration processes offset the passive inward leak of Ca2+. Although biochemical evidence suggests that the plasmalemmal Ca2+ pump plays a key role in this process, the precise role of this enzyme could not be proven until a reliable estimate of the inward Ca2+ leak was measured. Recent studies using dispersed smooth muscle cells from the toad stomach provided an estimate of the basal transmembrane Ca2+ flux rate; thus, we examined the transport capacity of the plasmalemmal Ca2+pump in this tissue. Gastric smooth muscle tissue was disrupted by homogenization and nitrogen cavitation. Membranes enriched 20 fold for plasma membrane markers were obtained using differential centrifugation and purification by flotation on discontinuous sucrose gradients. The membrane vesicles exhibited an ATP-dependent 45Ca uptake that was insensitive to azide or oxalate but sensitive to stimulation by calmodulin or inhibition by orthovanadate and the calmodulin antagonists trifluoperazine (TFP) or calmidazolium (CMZ). 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by agents that stimulate Ca2+ release from the sarcoplasmic rettculum (caffeine, inositol trisphosphate, GTP). However, both CMZ and TFP evoked a Ca2+ release that was comparable to that observed in the presence of Ca2+ ionophore, suggesting that these compounds have profound effects on membrane Ca2+permeability. 45Ca transport exhibited a high affinity for Ca2+ (KD 0.2 μM) and a high transport capacity, producing a > 12,000-fold gradient for Ca2+and a transmembrane flux rate at least 3-fold greater than that observed in resting smooth muscle cells. As a first step toward understanding the biochemical basis for the diversity of muscarinic cholinergic actions on smooth muscle, we examined the distribution of muscarinic receptor subtypes and coupling to guantne nucleotide-binding (G) proteins in airway and gastric smooth muscle. Receptor subtypes were classified in membranes prepared from bovine trachea and toad stomach based on the relative abilities of the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3) to displace the binding of nonselective antagonist [3H]QNB (quinuclidinyl benzilate). Based on the binding profiles for these antagonists, it was concluded that both smooth muscle types contain a mixture of M2 and M3 subtypes. In trachea the majority of receptors (86%) were M2, whereas in stomach the majority of receptors (88%) were M3. The displacement of [3H]QNB binding by the agonist oxotremorine indicated a mixed population of high affinity (KD = 4 nM) and low affinity (KD = 2-4 μM) binding sites. The addition of GTPγS abolished all high affinity agonist binding, suggesting that coupling of the receptors to G proteins may confer high affinity. Reaction of membranes with pertussis toxin in the presence of [32P]NAD caused the [32P]-labelling of a ~ 41 kD protein in both gastric and tracheal smooth musc1e. Pretreatment of the membranes with pertussis toxin and NAD completely abolished high affinity agonist binding in gastric smooth muscle, but produced little if any decrease in high affinity agonist binding in trachea. We conclude that, although muscarinic receptor activation leads to the elevation of intracellular Ca2+ and to contraction of both airway and gastric smooth muscle, the dissimilar distributions of receptor subtypes and distinct patterns of coupling to G proteins may indicate that each smooth muscle type uses different receptor-G protein interactions to regulate intracellular signalling pathways.

Plasma membrane transport proteins play a crucial role in all cells by conferring to the cell surface a selective permeability to a wide range of ions and small molecules. The activity of these transporters is often regulated by controlling their amount at the plasma membrane, via intracellular trafficking. The recent boom in the numbers of crystallized transporters shows that many of them that belong to different functional families with little sequence similarity adopt the same structural fold implying a conserved transport mechanism. These proteins belong to the APC (Amino acid-Polyamine-organoCation) superfamily and their fold is typified by the bacterial leucine transporter LeuT. This LeuT fold is characterized by inverted structural repeats of 5 transmembrane domains that harbor the central substrate-binding site and a pseudo-symmetry axis parallel to the membrane. The yeast Saccharomyces cerevisiae possesses about 16 amino acid permeases (yAAPs) that belong to the APC superfamily and that display various substrate specificity ranges and affinities. Topological, mutational analysis and in silico data indicate that yAAPS adopt the LeuT fold.<p><p>In this work we combined computational modeling and yeast genetics to study substrate binding by yAAPs and the endocytosis of these transporters in response to substrate transport. In the first part of this work, we analyzed the selective recognition of arginine by the yeast specific arginine permease, Can1. We constructed three-dimensional models of Can1 using as a template the recently resolved structure of AdiC, the bacterial arginine:agmatine antiporter, which is also a member of the APC superfamily. By comparison of the binding pockets of Can1 and Lyp1, the yeast specific lysine permease, we identified key residues that are involved in the recognition of the main and side chains of arginine. We first showed that the network of interactions of arginine in Can1 is similar to that of AdiC, and that the selective recognition of arginine is mediated by two residues: Asn 176 and Thr 456. Substituting these residues by their corresponding residues in Lyp1 converted Can1 into a specific lysine permease. In the second part of this work, we studied the regulation of two permeases, Can1 and the yeast general amino acid permease, Gap1. In the presence of their substrates, Gap1 and Can1 undergo ubiquitin-dependent endocytosis and targeting to the vacuolar lumen for degradation. We showed that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. By permease structural modeling, mutagenesis, and kinetic parameter analysis, we showed that Gap1 and Can1 need to switch to an intermediary conformational state and persist a minimal time in this state after binding the substrate to trigger their endocytosis. This down-regulation depends on the Rsp5 ubiquitin ligase and involves the recruitment of arrestin-like adaptors, resulting in the ubiquitylation and endocytosis of the permease.<p><p>Our work shows the importance of the structural analysis of yAAPs to get further insight into the different aspects of their function and regulation. We validate the use of a bacterial APC transporter, AdiC, to construct three-dimensional models of yAAPs that can be used to guide experimental analyses and to provide a molecular framework for data interpretation. Our results contribute to a better understating of the recognition mode of amino acids by their permeases, and the regulation of this transport in response to substrate binding. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished