Mastitis is often bacterial in origin. Lipoteichoic acid (LTA) and lipopolysaccharide (LPS), endotoxins from gram-positive and gram-negative bacteria, respectively, are potent inducers of mammary gland inflammation. Inflammation can alter expression of transporters responsible for transport of substrates important in synthesis of milk constituents and cellular metabolic energy. Since, gram-positive and gram-negative bacterial infections cause a different clinical course of mastitis, I investigated whether LTA and LPS differentially alter proton-coupled (MCT1) and sodium-coupled monocarboxylate transporter (SMCT1, SMCT2) expression and functional outcomes of altered expression. Human mammary epithelial cells (MCF-12A) were incubated with 1 microgram/mL LPS or LTA for 6, 12 and 24 hours and mRNA expression of TNF-alpha, IL-1β, IL-6, MCT1, SMCT1, and SMCT2 were measured using Quantitative RT-PCR. LPS decreased SMCT1, but increased SMCT2 expression after 6 h, while LTA increased MCT1 expression at 6 h, followed by gradual decrease in expression until 24 h. To know whether such differential changes in transporter expression by LPS and LTA could cause changes in cellular energy production, I quantified creatine (Cr) and high-energy phosphate substrates (CrP, ATP, ADP, AMP) and oxygen consumption rates using HPLC and Hansatech oxygen electrode, respectively. At 12 h, LPS increased concentrations of Cr, CrP, ATP and ADP, whereas LTA caused changes in CrP and ADP concentrations relative to control. Both LPS and LTA decreased oxygen consumption rates after 12 h. Furthermore, to know whether changes in transporter expression lead to differences in substrate availability, I performed uptake studies for carnitine using radiolabelled tritium L-carnitine. LPS and LTA challenge did not affect the affinity, but caused a 2-3-fold increase in maximal activity (Vmax) of carnitine transport. Although increases in Vmax were not significant, the increase in Vmax after 12 h exposure by LPS and LTA corresponds to changes in mRNA expression of the OCTN2 transporter (previously reported in the laboratory). In conclusion, LPS and LTA differentially alter mRNA expression of transporters, which leads to changes in cellular energy levels and oxygen consumption rates and possibly to changes in the functional activity of transporters. Whether such differences contribute to the different clinical course of mastitis warrants further investigation.

Background: Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model. Methods: Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O-2, 60 min), hypoxia (Hx, 6% O-2, 60 min) or H/R (6% O-2, 60 min followed by 21% O-2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean +/- SD and analyzed via ANOVA followed by the post hoc Bonferroni t test. Results: We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in brain microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation. Conclusions: Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Experiments are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression. Such studies will determine utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.

Type 1 diabetes (T1D) results from a loss of functional insulin-producing pancreatic beta cells. The etiology of T1D is poorly understood, but the detection of infiltrating inflammatory cells in the pancreas and circulating autoantibodies has led to the common notion that an autoimmune process plays a central role in the pathogenesis of the disease. The aim of this doctoral thesis was to assess various aspects of the immunopathology of type 1 diabetes. To this purpose, studies have been conducted on pancreatic material from the Network for Pancreatic Organ Donors with Diabetes (nPOD) collection, the Nordic Network for Islet Transplantation, and the Diabetes Virus Detection (DiViD) study. Paper I is a study on pancreatic tissue from organ donors with varying duration of T1D as well as non-diabetic donors and subjects with other types of diabetes, in which persistent expression of glucose transporters was shown on the beta cell membrane despite several years of T1D. Glucose transporter 1 was also confirmed as the predominant glucose transporter on human pancreatic islets. In paper II, we report on signs of inflammation in the exocrine but not in the endocrine pancreas in non-diabetic organ donors with diabetes-related autoantibodies, suggesting that diabetes-associated autoantibodies can occur in response to unspecific pancreatic lesions. Paper III aimed to characterize the T cell-infiltration of pancreatic islets in material from recent-onset T1D patients. Insulitis was shown in all subjects, but with distinct differences in expression analysis of T- and B cell activation to cell-mediated allorejected kidney transplant. Also Paper IV was conducted on material from recent-onset cases and showed increased islet glucagon content, in combination with a reduced number of islets but sustained mean islet size. Together, these results provide expansion of our knowledge of the immunopathology in T1D, and will hopefully assist in bringing us towards a deeper understanding of T1D aetiology and eventually an effective cure.

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Fluoroquinolones (FQ) are broad-spectrum charged antimicrobials exhibiting excellent tissue/fluid permeation. Thus, FQ disposition depends essentially on active transport and facilitative diffusion. Although most early transporter studies investigating renal elimination of FQs have focused on apical efflux of FQs from renal proximal tubule cell (RPTC) into urine, their basolateral uptake mechanism(s) from blood into RPTC (i.e., first step to tubular secretion) has not yet been explored in detail. Renally expressed SLC22 members: organic anion (OATs) and cation (OCTs) transporters are known to transport such small organic ionic substrates (molecular weight ~400 Da). Hence it is of interest to explore the role of these basolateral transporters in renal elimination of FQs, and to further quantitatively assess their impact in clinically observed FQ drug-drug interactions (DDI). An initial systematic review of clinical literature for FQs (n=18) demonstrated substantial differences among their renal clearance (CLren~46-fold) and unbound renal clearance (CLrenu~20-fold), and suggested that tubular secretion and reabsorption could be major determinants of FQ half-life, efficacy, and DDIs. FQs (n=13) identified from the above review were investigated by in-vitro transport studies using stably transfected cell lines, for potential interactions with organic cation [human (h) OCT1, hOCT2 and hOCT3] and anion [mouse (m) and hOAT3, hOAT1; and hOAT4] transporters. Further, kinetic inhibition studies were conducted to determine inhibition potency (Ki/IC50 values) for those FQs exhibiting significant OCT/OAT inhibition in preliminary interaction experiments. Gatifloxacin, moxifloxacin, prulifloxacin, and sparfloxacin were determined to be competitive inhibitors of hOCT1 with Ki = 250±18, 161±19, 136±33, and 94±8 μM, respectively. Moxifloxacin competitively inhibited hOCT3-mediated uptake, Ki = 1,598±146 μM. Enoxacin, fleroxacin, levofloxacin, lomefloxacin, moxifloxacin, prulifloxacin, and sparfloxacin exhibited competitive inhibition for mOat3 with Ki = 396±15, 817±31, 515±22, 539±27, 1356±114, 299±35, 205±12 μM, respectively. Fleroxacin and pefloxacin were found to inhibit hOAT1 with IC50 = 2228±84 and 1819±144 respectively. Despite expression in enterocytes, hepatocytes, and RPTC, hOCT3 does not appear to contribute significantly to FQ disposition. However, due to hepatic and potential RPTC expression, hOCT1 could play an important role in elimination of these antimicrobials. Among renally expressed OATs in humans, hOAT1 and hOAT3 are likely to be involved in FQ elimination.

The obligate human pathogen Neisseria gonorrhoeae successfully overcomes host strategies to limit essential nutrients, termed “nutritional immunity” by expression of TonB-dependent transporters (TdTs): outer membrane receptors that facilitate nutrient transport in an energy-dependent manner. N. gonorrhoeae encodes eight TdTs, five of which facilitate utilization of iron or iron-chelates from host derived proteins including transferrin, lactoferrin and hemoglobin, in addition to siderophores from neighboring bacteria. The transferrin utilization system was previously shown to be critical for establishing infection in human males; demonstrating the possible contributions of TdTs to gonococcal pathogenesis. As such, studies describing the biological function and contribution to pathogenesis of the remaining three uncharacterized TdTs (TdfG, TdfH and TdfJ) are needed. In this study we report that neither TdfG, TdfH nor TdfJ are heme receptors as gonococcal heme utilization occurs passively, independent of energy derived from the TonB system. We also report that TdfH and TdfJ are zinc (Zn) regulated and identify virulence associated regulators that modulate expression of these TdTs, which is in some cases strain-specific. We report that both TdfH and TdfJ contribute to Zn acquisition in N. gonorrhoeae and we characterize TdfH as a calprotectin receptor. Calprotectin, an immune effector protein highly expressed in neutrophils, has antimicrobial activity due to its ability to sequester Zn and Mn. We present evidence that TdfH confers resistance to calprotectin and that TdfH facilitates gonococcal calprotectin binding and Zn accumulation in the presence or absence of calprotectin. Finally, we demonstrate that TdfH expression enhances N. gonorrhoeae NET survival. These studies identify for the first time a novel gonococcal defense strategy to host-mediated nutritional immunity, in which N. gonorrhoeae, via the TdT TdfH, utilizes calprotectin as a Zn source neutralizing its antimicrobial activity. These studies have yielded novel insights into the function and regulation of TdfG, TdfH and TdfJ in N. gonorrhoeae and have laid the framework for future investigation of TdT-mediated Zn acquisition and its role in bacterial pathogenesis.

Oxidative stress (OS) is a frequent complication of various disease conditions such as Alzheimer’s and Parkinson’s disease, atherosclerosis, preeclampsia, rheumatoid arthritis, diabetes including gestational diabetes, etc. OS is defined as an imbalance between the production of reactive species and the ability of an organism to detoxify the reactive intermediates and repair the damage. As a result of OS, the excess of reactive species such as oxygen superoxide (O2-), hydroxyl radical (OH), peroxynitrite (ONOO−), 4-hydroxynonenal (4HNE), etc., have a tendency to react with nearby proteins/nucleic acids/lipids changing their functionality or inactivating them completely. The organism has many ways to protect itself from the harmful effects of oxidants. One strategy employs antioxidants introduced to the body with food. The purpose of this thesis was to investigate the effect of reactive species on the active transport mediated by ABC efflux transporters as well as exploring the possibility of using antioxidants not as interceptors of reactive species but rather as inhibitors of metabolic enzymes and transporters. The BCRP/ABCG2 efflux transporter was selected for the investigation of the effect of reactive anion, ONOO−, generated during OS and the product of OS, 4HNE, formed after a series of chain reactions involving ROS. Experiments conducted with Sf9 membrane vesicles overexpressing BCRP/ABCG2 revealed that both species are capable of inactivating this ABC transporter with IC50 being 31 ± 2.7 μM and 92 ± 1.4 μM for ONOO− and 4HNE, respectively. In presence of 4HNE, Vmax decreased 4-fold and Km remained unchanged, suggesting a noncompetitive inhibition mechanism. However, with addition of 4HNE, positive cooperativity was also observed. With ONOO−, the situation was different: both Vmax and Km changed consistent with mixed type inhibition. Overall, OS-mediated BCRP/ABCG2 inactivation occurred at biologically relevant concentrations of the reactive species. Antioxidants are substances that are known to reduce the amount of ROS/RNS accumulated during OS, but this research considered the use of antioxidants not only as interceptors of ROS/RNS but rather as inhibitors of metabolic enzymes. The effect of the dietary antioxidant, quercetin (Qc), on the metabolism of 2-methoxyestradiol (2Me-E2), a promising potential anticancer agent was investigated. Qc possesses five hydroxyl groups, several of which are targets for UDP-glucuronosyltransferases (UGTs). Thus, the simultaneous presence of Qc and 2Me-E2 could result in decreased glucuronidation of 2Me-E2. Using the LS180 intestinal human colon adenocarcinoma cell line, glucuronidation of 2Me-E2 resulted in formation of only one major glucuronide, 2-Methoxyestradiol-3-glucuronide (2Me-E2-3G). Qc effectively reduced its formation (IC50 = 7.8 ± 0.26 μM) to a minimum level. The decrease in the activity of UGTs increased the intracellular concentration of parent 2Me-E2. Additional increase in cellular concentration of 2Me-E2 was achieved when LS180 cells were pre-incubated with Qc prior the addition of 2Me-E2. Transwell experiments with MDCKII – BCRP cells revealed that BCRP/ABCG2 did not appear to transport 2Me-E2. All in all, the present study showed that OS has a negative impact on active transport mediated by ABC transporters. This, in turn, can affect drug disposition and protection of endogenous organs and tissues. Antioxidants are one of the mechanisms that can effectively reduce the negative impact caused by oxidative species. Nevertheless, this research revealed that they can also be an effective tool to reduce the excessive metabolism of therapeutic drugs. Thus, Qc was found to be a dietary antioxidant that could reduce metabolism of 2Me-E2 and increase it intracellular concentration.

The ankyrins comprise a family of proteins serving as components of the membrane cytoskeleton, and participate in a diverse set of associations with multiple binding partners including the cytoplasmic domains of transporters, ion channels, some classes of receptors, and cell adhesion proteins. Moreover, evidence is accumulating that ankyrin participates in defining functionally distinct subcellular regions. The complex functional and structural roles of ankyrins indicate they are likely to play essential roles in the pathology of traumatic axonal injury. The current study examined changes in ankyrin-G expression following a moderate central fluid percussion injury administered to adult rats. At 1d, 3d, and 7d postinjury (or following a sham control injury), protein levels of ankyrin-G in the corpus callosum and cerebral cortex were assessed using Western Blot analysis. Three immunopositive bands were identified in both brain regions as 220,212, and 75 kD forms of ankyrin-G. Time-dependent changes in ankyrin-G were observed in the corpus callosum. At 1d injury-induced elevations were observed in the callosal 220 kD (+147% relative to sham levels) and in the 212 kD (+73%) forms of ankyrin-G, but in both cases the expression decreased to control levels by 3d and 7d. In contrast, the 75 kD form showed moderate increases at 1d postinjury, but was significantly below control levels at 3d (-54%) and at 7d (-41%). Ankyrin-G expression in the cerebral cortex was only slightly affected by the injury, with a significant decrease in the `220 kD form occurring between 1d and 3d. These data suggest that the 220 and 212 kD changes probably represent postinjury proteolytic fragments derived from intact ankyrin-G isoforms of 480 andor 270 kD, while the 75 kD effects are likely breakdown products of intact 190 kD ankyrin-G. These results were discussed as they relate to prior findings of differential vulnerabilities of callosal myelinated and unmyelinated axons to injury. In this context, the 220,212 kD changes may reflect pathology within myelinated axons, and alterations to the 75 kD form may reflect more persistent pathology affecting unmyelinated callosal fibers.

Intracellular pH affects nearly all biochemical processes in yeast, the processes regulating the cytosolic pH includes function of many transport proteins. In this work, the impact of selected sodium transporters on cytosolic pH has been studied in two yeast species: Saccharomyces cerevisiae and Zygosaccharomyces rouxii including wild-type and mutants with affected sodium transport. Measurements of cytosolic pH and buffering capacity have been performed using fluorescent protein probe pHluorin - a pH sensitive derivate of green fluorescence protein. Several procedures for calibration of pHluorin fluorescence response have been compared and the importance of a proper correction of the calibration curve has been demonstrated. It has been shown that cytosolic pH is influenced by the function of Nha1 transport protein in S. cerevisiae as well as in Z. rouxii but not by Sod2-22 transporter in Z. rouxii. It has been demonstrated that the buffering capacity of cytosol decrease in the presence of glucose in all strains studied.

Estrogen signalling pathway plays crucial role in carcinogenesis of breast cancer. Estrogen receptor (ER) is a prototypical hormone receptor that upon binding its ligand, estradiol, translocates into the nucleus and turns on target genes related to cellular proliferation and survival. Although estrogen signalling physiologically supports normal breast tissue development, deregulations of this pathway contribute to development of breast tumours that are estrogen receptor dependent. One of the main obstacles in breast cancer treatment is acquired resistance to common anticancer drugs also known as multidrug resistance (MDR). The switch between chemotherapy responsive to chemotherapy resistant cell phenotype is usually accompanied by increased expression of ABC transporters, special membrane proteins responsible for export of various kinds of commonly used anticancer drugs from the intracellular to extracellular space and is also linked to the existence of cancer stem cells (CSCs). ABC transporters can not only export chemotherapeutic drugs but may modulate tumour microenvironment through the transport of endogenous intracellular substrates such as leukotrienes (LTs), sphingolipids and prostaglandins PGs). This function may also play important role in carcinogenesis. The aim of the thesis was to...