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More than 70,000 people worldwide suffer from cystic fibrosis, a genetic disease characterized by chronic accumulation of mucus in patients’ lungs provoking bacterial infections, and leading to respiratory failure. An employed age-old treatment to prevent the symptoms of the disease is inhalation of hypertonic saline solution, NaCl at concentrations higher than in the human body (~150 mM). This procedure clears the mucus in the lungs, bringing relief to the patient. However, the biophysical mechanisms underlying this process are not entirely clear. We undertook a new experimental approach to understand the effects of sprayed saline solutions on model lung surfactants towards understanding the mechanisms of the treatment. The surface of lungs contains mainly 1,2-Dipalmitol-sn-glycero-3-phosphocoline (DPPC). As previously assumed by others, we considered that monolayer of DPPC at the air-water interface serves as model system for the lungs surface; we employed a Langmuir-Blodgett (LB) trough and PM-IRRAS to measure surface-specific infrared spectra of the surfactant monolayers and effects on the interfacial tensions. We investigated spraying hyper-saline solutions onto surfactant monolayers at the airwater interface in two parts: (i) validation of our methodology and techniques with stearic acid and (ii) experiments with DPPC monolayers at the air-water interface. Remarkably, when micro-droplets of NaCl were sprayed to the monolayer of stearic acid, we observed enhanced organization of the surfactant, interpreted from the intensities of the CH2 peaks in the surface-specific IR spectra. However, our results with DPPC monolayers didn’t show an effect with the salt added as aerosol, possibly indicating that the experimental methodology proposed is not adequate for the phenomena studied. In parallel, we mimicked respiratory mucous by preparing salt solutions containing 1% (wt%) agar and measured effects on their viscosities. Interestingly, we found that NaCl was much more effective than NaI and NaClO4. This thesis reports structural dynamics of monolayers of stearic acid and DPPC at the airwater interfaces and those of aqueous solutions towards understanding mechanisms underlying the most commonly employed treatment for cystic fibrosis. Our methodology has never been reported before; but requires further modifications to gain deeper insights into the effects of salt sprays on model lung systems.

This problem was concerned with determining the total lipid content and individual lipid composition of the larvae of a local chironomid, Glyptotendipes barbipes (Staeger).

This investigation was concerned with characterizing effects of this tumor line on lipid metabolism in DBA/lJ mice and serum protein levels and cellular changes in DBA/lJ and Swiss white mice. Total lipids, lipid phosphorus, neutral lipids, and changes in fatty acids were determined in liver, spleen, skin, and tumor of DBA/lJ mice bearing the lymphosarcoma at various days after injection of tumor cells.

AIM To evaluate magnitude/direction of changes in peripheral lipid profiles in patients undergoing direct acting therapy for hepatitis C by genotype. METHODS Mono-infected patients with hepatitis C were treated with guideline-based DAAs at a university-based liver clinic. Patient characteristics and laboratory values were collected before and after the treatment period. Baseline demographics included age, ethnicity, hypertension, diabetes, hyperlipidemia, treatment regimen, and fibrosis stage. Total cholesterol (TCHOL), high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides (TG), and liver function tests were measured prior to treatment and ETR. Changes in lipid and liver function were evaluated by subgroups with respect to genotype. Mean differences were calculated for each lipid profile and liver function component (direction/magnitude). The mean differences in lipid profiles were then compared between genotypes for differences in direction/magnitude. Lipid profile and liver function changes were evaluated with Levene's test and student's t test. Mean differences in lipid profiles were compared between genotypes using ANOVA, post hoc analysis via the Bonferroni correction or Dunnett T3. RESULTS Three hundred and seventy five patients enrolled with 321 (85.6%) achieving sustained-viral response at 12 wk. 72.3% were genotype 1 (GT1), 18.1% genotype 2 (GT2), 9.7% genotype 3 (GT3). Baseline demographics were similar. Significant change in lipid profiles were seen with GT1 and GT3 (Delta GT1, p and Delta GT3, p), with TCHOL increasing (+ 5.3, P = 0.005 and + 16.1, P < 0.001), HDL increasing (+ 12.5, P < 0.001 and + 7.9, P = 0.038), LDL increasing (+ 7.4, P = 0.058 and + 12.5, P < 0.001), and TG decreasing (-5.9, P = 0.044 and -9.80 P = 0.067). Among genotypes (Delta GT1 v.Delta GT2 v.Delta GT3, ANOVA), significant mean differences were seen with TCHOL (+ 5.3 v. + 0.1 v. + 16.1, P = 0.017) and HDL (+ 12.3 v. + 2 v. + 7.9, P = 0.040). Post-hoc, GT3 was associated with a greater increase in TCHOL than GT1 and GT2 (P = 0.028 and P = 0.019). CONCLUSION Successful DAA therapy results in increases in TCHOL, LDL, and HDL and decrease in TG, particularly in GT1/ GT3. Changes are most pronounced in GT3.

This diploma thesis deals with hyphenation of liquid chromatography with mass spectrometric detection based on microfluidic chip. Firstly, a miniaturized ion source for atmospheric-pressure chemical ionization (APCI), and atmospheric-pressure photoionization (APPI) was constructed. The main component of this source was a glass microfluidic chip. Geometry and the working conditions of the chip were optimized. Since both ion sources work under the same conditions, possible advantages resulting from APCI/APPI combination were investigated. Furthermore, the performance characteristics of the sources were evaluated, and compared to the conventional high flow-rate sources. The best performing source, APCI, was then hyphenated with HPLC using low flow-rate. A method for separation of fatty acids methyl esters using Supelco 37 standard was developed. The separation conditions were as follows: C18 reversed stationary phase, and acetonitrile containing 0.1 % formic acids was used as the mobile phase. A temperature gradient was used in order to enhance the speed of the separation. The limits of detection and quantitation of for selected analytes using the chip micro-APCI were calculated, and compared to the ones obtained using a commercially available micro-APCI source. The method was used for separation of...

Protein folding and stability are essential for protein function. Changes in these characteristics can lead to altered physiological states and to the development of certain pathologies. While extensive research has focused on the stability of soluble proteins, membrane protein stability has received much less attention. Understanding the stability of membrane proteins can provide insight into folding mechanisms and the etiology of various pathologies. The purpose of this project is to prepare molecular tools to perform comparative studies of homologous membrane proteins that are found in various environments. To this end, thermophilic (Pyrococcus abyssi), mesophilic (Escherichia coli), and psychrophilic (Exiguobacterium 255-15) transmembrane Zn2+ transporting ATPases were cloned, expressed, and functionally characterized to correlate thermostability with optimal functional temperatures. In addition, the lipid environments and composition (rigid or fluid lipids) may also be involved in determining the stability of membrane proteins. Toward exploring the role of extremophilic lipids, Archaeoglobus fulgidus and Thermotoga maritima were grown and lipids were extracted. Availability of these molecular tools will enable physical-chemical studies toward understanding the structural factors that determine functional stability.

Phospholipids are the major components of cell membranes and have a wide variety of structures, shapes and properties. Different ratios of phospholipid species confer different properties to membranes and contribute to the normal function of organelles. We have previously shown that acute phosphatidylcholine (PC) biosynthesis inhibition leads to a severe form of lipid imbalance that disrupts ER morphology and structure. Furthermore, our previous studies also revealed a mechanism for ER proteostasis under conditions of lipid-imbalance-induced ER stress in yeast, whereby unfolded ER proteins are removed by lipid droplets (LDs) and targeted to the vacuole for degradation by microlipophagy. Here, we find that LDs also contribute to ER proteostasis during chemically induced ER stress. Furthermore, we find that ER stress results in an increase in ubiquitinated proteins in LDs as well as recruitment of cytosolic and ER heat shock proteins, as well as ER proteins to LDs. ER stress-induced microlipophagy does not require core ATG genes and can occur in the absence of lipid ordered microdomains (Lo) in the vacuolar membrane. Instead, we find that the ESCRT machinery is up-regulated and localizes to the vacuolar membrane in response to ER stress induced microlipophagy and that ESCRT I, II and III complexes are required for microlipophagy in response to each of these stressors. Similar to yeast, we find that lipid imbalance in skeletal muscle from CHKB CMD, new autosomal recessive CMD (Congenital Muscular Dystrophy) caused by a mutation of choline kinase beta (CHKB), results in abnormal SR/ER morphology. CHKB is the first enzyme in the de novo PC biosynthesis pathway and causes phospholipid imbalance in cell membranes similar to that observed in yeast. Besides the disruption of SR morphology, we also detect a dysfunction of the Ryanodine Receptor (RyR), the Ca2+ channels responsible for initiating muscle contraction. Specifically, we observe abnormal RyR morphology and increased association of RyRs with lipid droplets (LD) in muscle fibers from a CHKB CMD patient. Finally, we detect ER stress and pronounced UPR activation in rmd mice, a mouse model of CHKB CMD. Given these results, we propose that inhibition of PC biosynthesis leads to phospholipid imbalance in the SR, which in turn, causes RyR leakage and ER stress which lead to mitochondrial dysfunction and dystrophy in CHKB CMD.