The Processing of Amyloid Precursor Protein in the Central Nervous System of Humans and Rhesus Macaques

Alzheimer\u27s disease: AD) is the most common dementia, currently afflicting 30 million people worldwide, with prevalence steadily increasing. Over 99% of AD cases are classified as sporadic, in which the major risk factors are age: greater than 65 years) and the ApoE-ε4 allele: in a gene dose dependent manner). The minority of AD cases: less than 1%) are caused by autosomal dominant inheritance of a genetic mutation in one of three genes: Amyloid Precursor Protein: APP), Presenilin-1: PSEN1), or Presenilin-2: PSEN2). Mutation carriers will generally notice cognitive decline starting at a relatively young age, anytime between their 30s-50s, and eventually succumb to this early-onset AD. Early-onset AD may also be the result of an extra copy of the APP gene, as manifested in Down Syndrome: DS) patients, who universally develop AD. Alzheimer\u27s disease pathophysiology appears to revolve around APP. This transmembrane protein may be processed through one of two pathways. If APP is processed down the amyloidogenic: β-secretase) pathway, soluble APPβ: sAPPβ) will be released first, and with a subsequent cleavage, β-amyloid: Aβ), will be released into the brain where it could sequester into amyloid plaques, which are a major hallmark of AD. The other processing pathway is caused by cleavage of APP by α-secretase. This results in the release of soluble APPα: sAPPα), which precludes the release of Aβ, thus making this pathway non-amyloidogenic. Several studies have focused on measuring the products of APP cleavages in an effort to elucidate the pathophysiology of AD. These studies give a static view of APP because they measure absolute amounts of protein in single samples. However, proteins are constantly being produced, cleared, and aggregated which may result in protein concentrations fluctuating over time. Recently it was reported that Aβ exhibits a diurnal pattern, the definitive cause of which is still unknown. The general goal of this thesis is to determine the interplay of the α- and β;-secretase APP processing pathways in both physiological and pathophysiological settings in humans, to establish the human metabolism rates of total sAPP, sAPPα, and sAPPβ, and to determine whether drug intervention of one of the two APP pathways, would have an effect the other pathway. The aims of this thesis are: 1) To determine the extent of interdependence of the α-secretase and the β-secretase APP processing pathways in humans and whether β diurnal patterns were being driven by APP. Human cerebrospinal fluid: CSF) was collected hourly for 36 h, and concentrations of sAPPα, sAPPβ, Aβ40, and Aβ42 were measured by four metabolite-specific ELISAs. Parameters associated with diurnal patterns were compared among metabolites. Further, correlation analyses were used to determine if any correlation among metabolites at a single time-point was evident. Samples from Alzheimer\u27s patients, age-matched controls, and young healthy controls were analyzed. 2) To determine the physiological and pathophysiological metabolism of sAPPα and sAPPβ in humans. Total sAPP was isolated by immunoprecipitation from CSF of participants undergoing stable isotope labeling kinetics studies. Isolated sAPP underwent proteolytic cleavage by trypsin and turnover rates of total sAPP were determined by LC-MS and compared to turnover rates of Aβ. Isolated total sAPP that underwent proteolytic cleavage by Arginine-C produced some preliminary results that could lead to eventual determination of sAPPα or sAPPβ specific turnover rates in humans. 3) To determine the physiological metabolism of sAPPα and sAPPβ in rhesus macaques in comparison to Aβ metabolism and to determine if drug intervention of the β-secretase pathway by a BACE1 inhibitor would affect the α-secretase pathway. Soluble APPβ, sAPPα and Aβ were isolated from rhesus macaque CSF by serial immunoprecipitation. Monkeys were undergoing stable isotope labeling kinetics studies. Isolated APP metabolites underwent proteolytic cleavage by LysN, and then the turnover rates of these metabolites were measured by LC-MS to determine if the BACE1 inhibitor was hitting its target, and if it was having any effect of the α-secretase pathway

Optimization of conditions for organic acid extraction from edible plant material as applied to radish sprouts

In recent years, there has been growing interest in the influence of sprouts on health. Fruit and vegetables are the main sources of organic acids for humans; however, little is yet known about organic acids in sprouts. In this study, the selection of the optimal parameters for extraction of organic acids from fresh, edible sprouts is reported. Two extraction techniques: microwave-assisted (MAE) and ultrasound- assisted were compared. The experimental conditions were optimized in terms of ext raction time, temperature, and composition of extraction solution. To determine the influence of time and temperature of extraction or sample cooling, solvents used for extraction, on the analytical signal in isotachophoretic separation, the methods of experimental planning fractional factorial design: 3^{k-1} were used (three factor, three-level design). The optimal conditions for extraction of organic acids from radish sprouts were MAE, 90 °C; 18 min; and 0.01 M NaOH as a solvent

Determination of selenium as a biomarker of thyroid cancer by HG-AFS method

A simple and versatile procedure has been developed for the determination of selenium in biological samples for clinical purposes. The procedure consists of microwave sample digestion and the determination of selenium using atomic fluorescence spectrometry with a hydride generation system (HG-AFS). The method allows the determination of selenium in a range of 0.5–100.0 μg L–1 with a detection limit not higher than 0.2 μg L–1 and with good repeatability not exceeding 1%. It was applied for determination of selenium in women’s plasma samples (n = 90) with a suspicion of a thyroid cancer and the control group of women (n = 87). Most of the obtained results were in the range 30.0–60.0 μg L–1 and either did not match the physiological level of selenium in human plasma or indicate moderate selenium deficiency. In the further examinations the thyroid tissues taken from 30 patients were analyzed. In most of the cases the selenium concentration was found to be lower (0.14–1.67 μg g–1 wet weight) than its physiological level in a healthy tissue. On this basis the hypothesis has been drawn that selenium can be considered as an additional marker of the thyroid cancer disease

Influence of brassica sprouts on short chain fatty acids concentration in stools of rats with thyroid dysfunction

The aim of the study was to investigate the influence of kohlrabi and broccoli sprouts added to the diet on the concentrations of short-chain fatty acids in feces samples of rats with thyroid dysfunction. Two models of hypothyroidism were used. The first one was based on the diet with iodine deficiency and the second by sulfadimethoxine addition to the animal drinking water. In most cases, the concentrations of acetic acid, propanoic acid and butanoic acid in stool samples obtained from rats with thyroid dysfunction were insignificantly lower than found in the control group. Moreover, almost in all cases the diet enriched with brassica sprouts insignificantly increased the concentration of fatty acids in investigated material. A significant positive correlation between concentrations of butanoic and acetic acids was found in different groups of rats. In conclusion, the addition of broccoli or kohlrabi sprouts in ratís diet may slightly alleviate the decline of concentrations of some fatty acids in the stool of animals with thyroid dysfunction. Long term effects of this phenomenon should be evaluated in future studies

Developed and validated capillary isotachophoresis method for the rapid determining organic acids in childrens saliva

One of the current challenges facing researchers is the search for alternative biological material, as opposed to routinely and invasively collected (such as blood), as the analysis of the former would provide information about the state of human health, allowing for the diagnosis of diseases in their early stages. With the search for disease biomarkers in alternative materials, the development of newer analytical solutions has been observed. This study aims to develop a reliable analytical method using the capillary isotachophoresis technique for the determination of organic acids in children’s saliva, the presence/elevation of which can be used in the future for diagnostic purposes. Organic acids such as formic, lactic, acetic, propionic, and butyric acid, were determined in the saliva of healthy children without carious lesions. The limit of quantification determined in the validation process was found to vary from 0.05 to 1.56 mg/L, the recoveries at the two levels were determined to vary between 90% and 110% for level I, while for level II the corresponding values of 75% and 106% were found; the presentation, expressed as relative standard deviation values (RSD), did not exceed 5%. The parameters determined while validating the results method indicated that the obtained are reliable. The Red–Green–Blue (RGB) additive color model was used for the evaluation of the method. This comparative analysis allowed us to define the color of the method, which expresses whether it meets the given assumptions and requirements. According to the RGB model, the isotachophoresis method developed requires less reagent input, shorter sample preparation times, and results with lower energy consumption. Thus, the subject procedure may provide an alternative, routine tool for determining organic acids in human saliva, to be applied in the diagnosing of diseases of various etiological origins

SNF1-Related Protein Kinases SnRK2.4 and SnRK2.10 Modulate ROS Homeostasis in Plant Response to Salt Stress

In response to salinity and various other environmental stresses, plants accumulate reactive oxygen species (ROS). The ROS produced at very early stages of the stress response act as signaling molecules activating defense mechanisms, whereas those produced at later stages in an uncontrolled way are detrimental to plant cells by damaging lipids, DNA, and proteins. Multiple systems are involved in ROS generation and also in ROS scavenging. Their level and activity are tightly controlled to ensure ROS homeostasis and protect the plant against the negative effects of the environment. The signaling pathways responsible for maintaining ROS homeostasis in abiotic stress conditions remain largely unknown. Here, we show that in Arabidopsis thaliana, two abscisic acid- (ABA)-non-activated SNF1-releted protein kinases 2 (SnRK2) kinases, SnRK2.4 and SnRK2.10, are involved in the regulation of ROS homeostasis in response to salinity. They regulate the expression of several genes responsible for ROS generation at early stages of the stress response as well as those responsible for their removal. Moreover, the SnRK2.4 regulate catalase levels and its activity and the level of ascorbate in seedlings exposed to salt stress