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Cancer is the second most common cause of death in the world. One of the objectives of this thesis is to biologically evaluate a series of anti-cancer polymeric aromatic/heterocyclic bis-sulfonamides and pyridinium sulfonamides which were synthesized from three established aminosulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor pharmacophores. Testing of these novel inhibitors and their precursors against a panel of membrane-bound CA isoforms, including tumor-overexpressed CA IX and XII and cytosolic isozymes, identified nanomolar-potent inhibitors against both classes and several compounds with medium isoform selectivity. In the case of pyridinium sulfonamides we used complexes of the inhibitors with cyclodextrins or sulfocalixarene to enhance aqueous solubility for biological testing. The ability of CA inhibitors to kill tumor cells overexpressing CA IX and XII was tested under normoxic and hypoxic conditions, using 2D and 3D in vitro cellular models. The study identified a nanomolar potent PEGylated bis-sulfonamide CA inhibitor (25), as well as cyclodextrin and sulfocalixarenes complexes, which were able to significantly reduce the viability of colon HT-29, breast MDA-MB231, and ovarian SKOV-3 cancer cell lines, thus revealing the potential of polymer conjugates in CA inhibition and cancer treatment. As a different disease state yet still a concern, cognitive dysfunction markedly impacts patients with a host of psychiatric conditions including attention deficit hyperactivity disorder, autism spectrum disorder, drug addiction, schizophrenia, depression, bipolar disorder, obsessive-compulsive disorder, and of course, Parkinson’s and Alzheimer’s diseases and other types of dementia. Another objective of this thesis was to profile several series of bis-imidazoles for physicochemical, in-vitro and in-vivo properties as potential memory and learning enhancers (nootropics). Biological testing on eight isozymes of carbonic anhydrase (CA) present in the human brain revealed compounds with nanomolar potency against at least one membrane bound, cytosolic or mitochondrial CA isozymes, combined with good physicochemical properties. We also identified lead compounds with the ability to rescue experimental animals from drug-induced memory deficits, using an optimized Novel Object Recognition Task (NORT) procedure. / Pharmaceutical Sciences

The fragile X mental retardation protein (FMRP) is an important regulator of protein translation, and a lack of FMRP expression leads to a cognitive disorder known as fragile X syndrome (FXS). Clinical symptoms characterizing FXS include learning impairments and heightened anxiety in response to stressful situations. The Fmr1-/y mouse has previously been shown to have deficits in context discrimination and novel object recognition tasks, which primarily rely on the dentate gyrus (DG) region of the hippocampal formation, but not in the Morris water maze (MWM) or the elevated plus-maze tasks, which primarily depend on the Cornu Ammonis (CA1) region. Furthermore, previous research has demonstrated N-methyl-D-aspartate receptor (NMDAR)-associated synaptic plasticity impairments in the DG but not in the CA1. However, the impact of acute stress on synaptic plasticity in the Fmr1-/y hippocampus has not been examined. The current study sought to extend previous behavioural investigations in the Fmr1-/y mouse, as well as examine the impact of stress on activation of the hypothalamic-pituitary-adrenal (HPA)-axis and on hippocampal synaptic plasticity. To further characterize hippocampus-dependent behaviour in this mouse model, the DG-dependent metric change spatial processing and CA1-dependent temporal order discrimination tasks were evaluated. The results reported here support previous findings and demonstrate that Fmr1-/y mice have performance deficits in the DG-dependent task but not in the CA1-dependent task, suggesting that previously reported subregional differences in NMDAR-associated synaptic plasticity deficits in the hippocampus of the Fmr1-/y mouse model may also manifest as selective behavioural deficits in hippocampus-dependent tasks. In addition, following acute stress, mice lacking FMRP showed a faster elevation of the glucocorticoid corticosterone and a more immediate impairment in long-term potentiation (LTP) in the DG. Stress-induced LTP impairments were rescued by administering the glucocorticoid receptor (GR) antagonist RU38486. Administration of RU38486 also enhanced LTP in Fmr1-/y mice in the absence of acute stress to wild-type levels, and this enhancement was blocked by application of the NMDAR antagonist 2-amino-5-phosphonopentanoic acid. These results suggest that a loss of FMRP results in enhanced GR signalling that may adversely affect NMDAR-dependent synaptic plasticity in the DG. Finally, synaptic plasticity alterations reported in this work were found to be specific to the DG and were unidirectional, i.e., restricted to LTP, as NMDAR- and metabotropic glutamate receptor (mGluR)-LTD were both unaffected by acute stress in the DG or the CA1 regions. This study offers new insights into synaptic plasticity impairments in the Fmr1-/y mouse model, and suggests stress and GRs as important contributors to learning and memory deficits in FXS. / Graduate

Spontaneous activity in neuronal networks in vitro is common and has been well documented. However, alteration of spontaneous activity in such networks via conditioning electrical stimulation has received much less experimental attention. Two different patterns of electrical stimulation were used to enhance or depress the level of spontaneous activity in spinal cord cultures. High-frequency stimulation (HFS), a method routinely shown to increase the efficacy of synaptic transmission, was employed to augment spontaneous activity. Low-frequency stimulation (LFS), the technique often applied to depress synaptic efficacy, was employed to decrease spontaneous activity. In addition, LFS was used to reverse the effect of HFS on spontaneous activity. Likewise, HFS was applied to counter the effect of LFS. Because these networks were grown on multi-microelectrode plates (MMEPs), this allowed the simultaneous stimulation of any combination of the 64 electrodes in the array. Thus, the possible differences in response to single versus multi-electrode stimulation were also addressed. Finally, test-pulses were delivered before and after the conditioning stimulation on the same stimulation electrode(s) in order to assess the change in mean evoked action potentials (MEAPs). Dissociated spinal tissue from embryonic mice was allowed to mature into self-organized networks that exhibited spontaneous bursting activity after two weeks of incubation. Spontaneous activity was monitored from up to 14 recording channels simultaneously. Although uniform responses to stimulation across all recording electrodes were rarely observed, a large majority of the recording channels had similar responses. Spontaneous activity was increased in 52% of 89 HFS trials, whereas activity was decreased in 35% of 75 LFS trials. The duration of most of these increases was less than 5 minutes. When there were substantial and long-term (> 15 min) changes in spontaneous activity, the opposing stimulation pattern successfully reversed the effect of the previous stimulation. The percent change in MEAPs following conditioning stimulation suggested that synaptic modification had taken place in 75% of all test-pulse stimulation trials.

Stroke is a common and multifactorial disease influenced by genetic and environmental risk factors. It is a highly heterogeneous entity consisting of two main types, ischemic (80%) and hemorrhagic (20%) stroke. The most common form of hemorrhagic stroke is intracerebral hemorrhage (ICH). Ischemic stroke mainly results from thrombotic or embolic events, while ICH is caused by the rupture of an artery in the brain. The mean age of first-ever stroke is 75 years (73 vs. 78 years, for men and women, respectively) and the age-specific stroke incidence is higher for men as compared to women, suggesting that hormonal factors confer protection. A large body of experimental and observational studies shows that estrogens exert beneficial effects in the cardiovascular system. However, large, recent, clinical randomized trials have failed to demonstrate a lower risk of stroke with hormone replacement therapy (HRT) in elderly postmenopausal women. It is possible that HRT may only protect a subgroup of women. Here, genetic predisposition might be involved. Stroke incidence is 50% higher in northern compared to southern Sweden, suggesting a genetic predisposition in this population. This relatively homogeneous population displays founder effects, making it well suited for genetic studies. Since 1985, the MONICA and VIP projects have conducted large-scale cardiovascular health surveys in this population. Information about conventional stroke risk determinants and also DNA have been collected, and two prospective, nested case-referent cohorts (113 cases and 226 controls; 275 cases and 549 controls) have been sampled. To investigate whether genes of the estrogen signaling system may be important in stroke development, we performed genetic association studies, including specific functional single nucleotide polymorphisms in the genes for estrogen receptor alpha (ERα, ESR1), and its target genes osteoprotegerin (OPG, TNFRS11B) and interleukin-6 (IL-6, IL6). We found a significant association between the common c.454-397T/T genotype in ESR1 and ICH, remaining after adjustments for conventional stroke risk factors. The c.454-397T/T genotype also associated with increased systolic (SBP) and diastolic blood pressure (DBP). The combination of c.454- 397T/T and either hypertension, increased SBP, or increased DBP boosted this association substantially and significant synergistic effects on ICH risk between this genotype and increased blood pressure were demonstrated. In a second study, we found a similar association between the common OPG-1181C/C genotype and ICH. Cognitive impairments, including spatial memory and learning deficiencies, are common after stroke. Estrogens improve cognitive functions, including memory and learning processes, in postmenopausal women and ovariectomized rodents. Post-ischemic housing of rats in an enriched environment (EE) improves recovery of spatial memory and learning impairments. Both estrogen and EE induce neuroplasticity in the hippocampus. We hypothesized that 17β- estradiol combined with EE would accelerate recovery after experimental focal brain ischemia in ovariectomized rats and that such improvements could be related to expression of nerve growth factor-induced gene A (NGFI-A) in the hippocampus. Five to six weeks after middle cerebral artery occlusion, 17β-estradiol–treated rats housed in an EE showed significant improvements in cognitive function (i.e., shorter latency and path in the Morris water maze task) and significantly higher NGFI-A mRNA expression in bilateral cornu ammonis 1 (CA1) and ipsilateral dentate gyrus (DG) compared to placebo-treated animals in EE. In conclusion, we present evidence for the association between polymorphic variants in the ESR1 and TNFRS11B genes and ICH and show that 17β-estradiol in combination with EE accelerates cognitive functions in a rat stroke model, putatively through upregulation of NGFI-A in hippocampal subregions. These findings may contribute to an increased understanding of the underlying genetic etiology of ICH and may be informative for the primary prevention of this disease. They also provide hope for 17β-estradiol combined with early environmental enrichment as a novel therapeutic option following ischemic stroke.

Genetic deletion or pharmacological blockade of the CB1 receptor has been reported to impair extinction learning in aversive conditioning (i.e., conditioned fear and Morris water maze) paradigms, but not in operant procedures in which food reinforcement is earned. It is difficult to discern whether the differential effects caused by CB1 receptor disruption on extinction result from the hedonics (i.e., aversive vs. appetitive) or is related to the required responses associated with these disparate tasks. In order to evaluate whether the hedonics is the determining factor, we used either aversive (i.e., escape from bright lights and air turbulence) or appetitive (i.e., to gain access to water) motivators in the Barnes maze task, a model in which mice are required to enter a hidden goal box. Administration of the CB1 receptor antagonist, rimonabant, disrupted extinction learning under aversive conditions, but not under appetitive conditions. This is the first study to show a differential effect of rimonabant on extinction in a task that required identical motor behaviors, but only differed in hedonic nature of the reinforcer. In addition, genetic ablation of CB1 receptor signaling impaired acquisition of the task under both aversive and appetitive conditioning procedures. Conversely, enhancing endocannabinoid signaling, via genetic deletion of the FAAH enzyme, accelerated acquisition of the task under aversive, but not appetitive, conditioning procedures. Accordingly, these data strongly support the hypothesis that the endogenous cannabinoid system plays a necessary role in the extinction of aversively motivated behaviors, but is expendable in appetitively motivated behaviors. While these findings underscore concerns over potential side effects associated with CB1 receptor antagonists, they also suggest that stimulating the endogenous cannabinoid system may be a promising pharmacological approach to treat maladaptive behaviors that arise from stress or trauma.

No description available.

Previous research on the relationship between spatial memory and adult hippocampal neurogenesis has been controversial. In the present study, neurogenesis was compared between two natural populations of the same species that differ in their reliance on spatial memory to cache and retrieve stored food. Western red squirrels store food in a single site whereas eastern red squirrels store food in multiple sites. Neurogenesis was assessed using endogenous markers of the number of proliferating cells (Ki-67) and the number of immature neurons (DCX), and neuronal recruitment was determined by measuring the area of the dentate gyrus of the hippocampus. The number of proliferating cells, immature neurons and neuronal recruitment were enhanced in the eastern compared to the western red squirrels, reflecting the food storing strategies used by the squirrels. This suggests that there is a positive correlation between adult hippocampal neurogenesis and spatial learning and memory.

Previous research on the relationship between spatial memory and adult hippocampal neurogenesis has been controversial. In the present study, neurogenesis was compared between two natural populations of the same species that differ in their reliance on spatial memory to cache and retrieve stored food. Western red squirrels store food in a single site whereas eastern red squirrels store food in multiple sites. Neurogenesis was assessed using endogenous markers of the number of proliferating cells (Ki-67) and the number of immature neurons (DCX), and neuronal recruitment was determined by measuring the area of the dentate gyrus of the hippocampus. The number of proliferating cells, immature neurons and neuronal recruitment were enhanced in the eastern compared to the western red squirrels, reflecting the food storing strategies used by the squirrels. This suggests that there is a positive correlation between adult hippocampal neurogenesis and spatial learning and memory.

The aim of the present study was to examine retention of spatial reference memory after 6 (Experiment I) and 15days (Experiment II) of binge-like drinking and during alcohol withdrawal in young adult Wistar rats. Prior to alcohol treatment, rats received Morris Water Maze (MWM) training. Afterwards, rats were intragastrically administered ethanol at the dose increasing from 4.5g-to-12g/kg. Intubation control groups (n=7 and n=10, respectively) received infusions of a sucrose solution without ethanol. Subsequently, all subjects were given a single probe trial in the MWM to test memory retention. In both experiments, there were three alcohol groups: A0 group (n=7) tested 4h after the last alcohol administration for acute effects of ethanol / A24 group (n=7) tested 24h after alcohol cessation, when acute ethanol effects disappear but withdrawal symptoms does not develop yet / A72 group (n=7) tested 72h after the last ethanol infusion for withdrawal effects. Finally, potential molecular changes in hippocampus were examined using Fourier Transform Infra-Red (FT-IR) spectroscopy. The blood alcohol concentration was 605.67&plusmn / 36mg/dl. In Experiment I, due to the low overall level of performance in the memory retention task the behavioral effects of ethanol could not be evaluated and no significant between&ndash / group differences were observed in Experiment II. In Experiment I, no significant changes in the molecular make-up of the hippocampus were noted. Conversely, in Experiment II, significant changes in protein, lipid, and nucleic acid profiles related to ethanol intake and withdrawal were found. They are linked to both development of tolerance to ethanol and adverse withdrawal effects.