The neuroactive steroid pregnanolone glutamate (Pg glu), a synthetic analogue of the naturally occurring pregnanolone sulfate (3alpha5betaS), has neuroprotective properties and a minimum of adverse effects. The subject of my thesis is the influence of selected structural modifications of the molecule Pg glu on biological effects. The first modification involves an increase of lipophilicity, the second involves the attachment of a positively charged group to C3. All these neuroactive steroids are use-dependent inhibitors of NMDA receptors. The first aim of this thesis was to determine the neuroprotective effectiveness of the neuroactive steroids chosen. The second aim was to explore the influence of selected neuroactive steroids on motor coordination, reflexes, anxiety and locomotor activity, as well as the effect of their high doses. The third aim was to create a battery of behavioural tests for screening the biological effects of analogues of Pg glu in laboratory rodents. The neuroprotective effects were evaluated in a model of excitotoxic damage of hippocampus in the rat on the basis of its behavioural consequences. The neuroprotective efficacy of androstane glutamate (And glu) and Pg glu was demonstrated. In the case of positively charged molecules, neuroprotective efficacy was not demonstrated....

Normotensive hydrocephalus (NPH) is a neurodegenerative disease that occurs mainly in patients of high age. The disorder causes accumulation of cerebrospinal fluid (CSF), which leads to enlargement of ventricles and pressure exerted on cerebral structures. Clinical manifestations (gait disorders, development of dementia, incontinence) can be easily confused with symptoms of other neurodegenerative diseases; unlike other such disorders, however, NPH can be treated by surgery, if diagnosed in time. Patients are indicated for the procedure by a lumbar drainage test. There are currently no reliable laboratory biomarkers known that could be the basis of NPH diagnostics. In the past, steroids proved to be linked to neuronal activity in neurodegenerative diseases with the help of specific diagnostic markers. An instrumental method has been developed for the purposes of this thesis and it was used to gauge the level of certain steroids in CSF in a sample group of NPH patients and a control sample group of healthy individuals. A significant difference has been found in levels of aldosterone and cortisone. Aldosterone was higher in NPH sufferers, while cortisone levels were higher in the control group. It is crucial to differentiate patients with NPH from patients with similar clinical manifestations during...

Cationic steroid antimicrobials (CSAs or ceragenins) are a novel class of synthetic, cholic acid-based mimics of endogenous antimicrobial peptides. These small molecule compounds display broad bactericidal activity against gram-negative and gram-positive bacteria, potent ability against fungal pathogens, and cidal effects against drug resistant and multidrug resistant microbes. Implantable medical devices provide an abiotic surface upon which bacteria and fungi can accumulate--thereby leading to localized or systemic infection. We proposed that CSA antibiotics can be incorporated into medical device surface coatings which can be optimized for the active release or elution of the CSA compounds over time to prevent device-associated infections. This report will discuss the progress of developing and testing coating systems for 3 such devices: cardiac implantable electronic devices (CIED), silicone nasal splints, and breast tissue expanders. In the case of CIEDs, an envelope material containing CSA was created using bioresorbable polymers. We found that this envelope elutes CSA antibiotics and kills all surrounding bacteria or fungi in both planktonic and biofilm forms within 1 hour of exposure. We also developed a nasal splint coating which is directly adhered to the surface of the silicone splint. This coating system demonstrated more than 8 days of protective ability (full microbicidal activity to the detection limit) against Candida albicans, and reduced microbial growth of P. aeruginosa, Candida auris, and MRSA for approximately 6 days. Lastly, in the case of tissue expanders, we developed a layered coating which displays fully-reductive antimicrobial activity against MRSA for 8 days with reintroduction of bacteria every 24 hours. Additionally, this work will discuss our investigations into the secondary properties of ceragenin compounds. On the basis of studies which have demonstrated the pro-osteogenic properties of CSA, we probed the mechanism of this effect. We studied the potential effects of ceragenins on the proliferation, differentiation, and migration of bone-derived mesenchymal stem cells (MSCs). We have determined the absence of any positive proliferative effects of ceragenins on these cells; however, we have demonstrated the significant migration-promoting chemoattractant properties of CSA. In the case of CSA-13, we have observed up to a 400% increase in migration compared to the control. Also, we demonstrated that the P2X7 receptor is strongly implicated in the cellular mechanism of this effect. Our studies of the differentiation-promoting properties of CSA on MSCs have been largely inconclusive, but further investigations are proposed in this report. Lastly, this work includes a report on our investigations into the potential synergistic interactions between CSA-131/CSA-44 with amphotericin B or caspofungin, two commonly used antifungal agents.

The PDZ and LIM domain 5 (PDLIM5) gene may play a role in cancer, bipolar disorder, major depression, alcohol dependence and schizophrenia; however, little is known about the interaction effect of steroid and PDLIM5 gene on cancer. This study examined 47 single-nucleotide polymorphisms (SNPs) within the PDLIM5 gene in the Marshfield sample with 716 cancer patients (any diagnosed cancer, excluding minor skin cancer) and 2848 noncancer controls. Multiple logistic regression model in PLINK software was used to examine the association of each SNP with cancer. Bayesian logistic regression in PROC GENMOD in SAS statistical software, ver. 9.4 was used to detect gene–steroid interactions influencing cancer. Single marker analysis using PLINK identified 12 SNPs associated with cancer (P < 0.05); especially, SNP rs6532496 revealed the strongest association with cancer (P = 6.84 × 10−3); while the next best signal was rs951613 (P = 7.46 × 10−3). Classic logistic regression in PROC GENMOD showed that both rs6532496 and rs951613 revealed strong gene–steroid interaction effects (OR = 2.18, 95% CI = 1.31−3.63 with P = 2.9 × 10−3 for rs6532496 and OR = 2.07, 95% CI = 1.24 −3.45 with P = 5.43 × 10−3 for rs951613, respectively). Results from Bayesian logistic regression showed stronger interaction effects (OR = 2.26, 95% CI = 1.2 −3.38 for rs6532496 and OR = 2.14, 95% CI = 1.14 −3.2 for rs951613, respectively). All the 12 SNPs associated with cancer revealed significant gene–steroid interaction effects (P < 0.05); whereas 13 SNPs showed gene–steroid interaction effects without main effect on cancer. SNP rs4634230 revealed the strongest gene–steroid interaction effect (OR = 2.49, 95% CI = 1.5 −4.13 with P = 4.0 × 10−4 based on the classic logistic regression and OR = 2.59, 95% CI = 1.4 −3.97 from Bayesian logistic regression; respectively). This study provides evidence of common genetic variants within the PDLIM5 gene and interactions between PLDIM5 gene polymorphisms and steroid use influencing cancer.

Liquid chromatography, coupled with tandem mass spectrometry, presents a powerful tool for the quantification of the sex steroid hormones 17-β estradiol, progesterone and testosterone from biological matrices. The importance of accurate quantification with these hormones, even at endogenous levels, has evolved with our understanding of the role these regulators play in human development, fertility and disease risk and manifestation. Routine monitoring of these analytes can be accomplished by immunoassay techniques, which face limitations on specificity and sensitivity, or using gas chromatography–mass spectrometry. LC–MS/MS is growing in capability and acceptance for clinically relevant quantification of sex steroid hormones in biological matrices and is able to overcome many of the limitations of immunoassays. Analyte specificity has improved through the use of novel derivatizing agents, and sensitivity has been refined through the use of high-resolution chromatography and mass spectrometric technology. This review highlights these innovations, among others, in LC–MS/MS steroid hormone analysis captured in the literature over the last decade.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells , while the steroid 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by l,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against a putative 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake (P2D3, each in Cells exposed to hormone alone again showed an increased accumulation of 32P from T=5-10 min, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with 100 nM phorbol-13-myristate (PMA) increased 32P uptake to 143% of controls, while cells pretreated with 50 μM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity at T=1-3 min (P2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.

Cortisol, a hormone produced by a hormone produced by the adrenal gland, is responsible for many regulatory functions in the body. Cortisol release is mediated by adrenocorticotrophic hormone, or ACTH, through the hypothalamus-pituitary-adrenal or HPA axis. This HPA axis is the major release pathway used during acute stress, during which the levels of ACTH parallel those of cortisol. However, in states of chronic stress, the level of ACTH drops dramatically, while cortisol remains high. This study focuses on the pathway of cortisol release during these chronic stress states, specifically examining the role of IL-6 with respect to STATs and the Jak/STAT pathway. It has been shown that IL-6 increases cortisol levels, and that IL-6 utilizes the Jak/STAT pathway. Also, the steroidogenic acute regulatory (StAR) promoter contains multiple STAT binding sites. Thus, STATs could be mediating the effects of IL-6 in the chronic release of cortisol by inducing expression of StAR. Experiments were performed to identify whether IL-6 has a direct effect on StAR promoter activity, StAR mRNA and StAR protein levels. Electromobility Shift Assays (EMSA) were performed to show that STATs bind to the full STAT site within the StAR promoter region. Various experiments were also carried out in the presence of IL-6 alone or, congruently with either a Jak (AG490) or STAT3 (Piceatannol) inhibitor, to show the effects of STATs and the Jak/STAT pathway on StAR. Luciferase assays were performed in order to observe the effects on induction of the StAR promoter. RT-PCR and western blots were also performed to observe the effect of Jak/STAT inhibition on both StAR mRNA levels and StAR protein levels. These experiments showed a marked decrease in the IL-6-stimulated StAR promoter activity, mRNA and protein expression when treated with wither Jak or STAT inhibitor. Therefore, IL-6 regulates expression of StAR through utilization of the Jak/STAT pathway; which phosphorylates and subsequently dimerizes STAT, allowing STAT to translocate to the nucleus and bind to the StAR promoter, thus increasing StAR expression and thereby inducing synthesis of cortisol.

Cationic steroid antimicrobial agents (CSAs) are a family of bile acid derivatives. These compounds are amphiphilic and mimic endogenous antimicrobial peptides. The antimicrobial activities of CSA-13 have been investigated and due to portent bactericidal activities and low toxicity, a large amount of CSA-13 is demanded for clinic trails and other antimicrobial applications. During our studies, we optimized the synthetic route of CSA-13, so that it can be prepared at the kilogram, even in tons scale. We investigated three routes and one of them is suitable for industry, because only recrystallization is needed in the synthesis. Natural killer T cells (NKT cells) are a kind of lymphocyte that bridge the adaptive immune system with the innate immune system. Once stimulated by glycolipids, NKT cells influence immune responses. To search for better glycolipid ligands, scientists have isolated many natural products to get inspiration. Thrautochysides A-C was isolated from a group of marine protists. These compounds have an interesting structure on their sphingosine lipid chains. We finished the iii synthesis of thraustochyside B, and made substantial progress toward the synthesis of thraustochyside A. Toll like receptors (TLRs) are integral components of the innate immune system. They recognize antigens and induce dendritic cells to give immune responses. TLR1, TLR2 and TLR6 recognize lipopetides, and these TLRs function as heterodimers. TLR1/TLR2 dimer recognition gives inflammatory responses, and TLR2/TLR6 dimer recognition gives immunomodulatory responses. We used modeling of TLRs to find a compound, which can fill the lipid binding pockets of the TLR2 and TLR6 dimer. In our study, we found the peptide chain of the antigen Pam2CSK4 can be replaced by a water soluble polyamine, which confirmed the function of the peptide to increase the water solubility.

Nanotechnology applications have emerged enormously in recent times. Of particular interest is that area that overlaps the areas of nanotechnology, biology and medicine: nanomedicine. One advantage of nanomedicines is it that it can be used as an enabling technology by pharmaceutical researchers and industry to overcome issues associated with the low bioavailability of hydrophobic drugs. In the first part of the current study, nanosuspensions of two of hydrophobic steroid drugs: hydrocortisone and prednisolone were produced. Nanosuspensions were prepared using a bottom-up approach: the anti-solvent precipitation method using microfluidic reactors. Surface modification was carried out on these nanosuspensions using cationic surfactants to obtain nanoparticles with different levels of surface positive charge as indicated by ¿-potential values. Dynamic light scattering (DLS) and transmission electron microscope (TEM) techniques were used to characterize the prepared nanoparticles. Powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC) were also used to characterize hydrocortisone nanoparticles. In the second part, cellular uptake of both coated and uncoated nanoparticles by HaCaT keratinocytes cell line was examined and indicated by quantifying the anti- inflammatory effect of nanoparticles on the LPS-induced inflammation. Also, TEM was employed to evaluate the cellular uptake of hydrocortisone nanoparticles. Results showed higher ant-inflammatory effect of coated nanoparticles over uncoated nanoparticles. Furthermore, the anti-inflammatory effect of coated nanoparticles was correlated to the degree of positive surface charge. / Libyan government

The uptake and binding of tritiated cortisol, corticosterone, and dexamethasone in cytosols isolated from various regions of non-adrenalectomized (intact) and adrenalectomized sheep brains and pituitaries were studied. [3H]-cortisol and [3H]-corticosterone were bound preferentially by cytosols from the hippocampal and septal regions of the brain, whereas, [3H]-dexamethasone was preferentially bound by cytosol from the pituitary. Cytosols isolated from the brains of adrenalectomized sheep bound significantly more [3H]-corticosterone than did cytosol from the brains of intact animals. Pre-incubation of cytosols from the hippocampus of adrenalectomized animals with non-radioactive testosterone or estradiol did not significantly block the subsequent binding of [3H]-cortisol. Results of pre-incubations with non-radioactive progesterone were non-reproducable with a large standard deviation among replicates. These findings provide evidence that the sites of feedback inhibition are the same in the sheep as described for other mammalian systems, and that in the sheep as in other mammals the principle site of localization of cortisol and corticosterone is in limbic structures of the brain, whereas, the principle site of uptake of dexamethasone is in the pituitary.