Molecular analysis of Sigma-1 receptor modulation of the dopamine transporter

Sigma (σ) receptors are well established as a non-opioid, non-phencyclidine, and haloperidol-sensitive receptor family with its own binding profile and a characteristic distribution in the central nervous system (CNS) as well as in endocrine, immune, and some peripheral tissues. Two σ receptors subtypes, termed σ1 and σ2, have been pharmacologically characterized, but, to date, only the σ1 has also been cloned. Activation of σ1 receptors alter several neurotransmitter systems and dopamine (DA) neurotrasmission has been often shown to constitute an important target of σ receptors in different experimental models; however the exact role of σ1 receptor in dopaminergic neurotransmission remains unclear. The DA transporter (DAT) modulates the spatial and temporal aspects of dopaminergic synaptic transmission and interprer the primary mechanism by wich dopaminergic neurons terminate the signal transmission. For this reason present studies have been focused in understanding whether, in cell models, the human subtype of σ1 (hσ1) receptor is able to directly modulate the human DA transporter (hDAT). In the first part of this thesis, HEK-293 and SH-SY5Y cells were permanently transfected with the hσ1 receptor. Subsequently, they were transfected with another plasmid for transiently expressing the hDAT. The hDAT activity was estimated using the described [3H]DA uptake assay and the effects of σ ligands were evaluated by measuring the uptaken [3H]DA after treating the cells with known σ agonists and antagonists. Results illustrated in this thesis demonstrate that activation of overexpressed hσ1 receptors by (+)-pentazocine, the σ1 agonist prototype, determines an increase of 40% of the extracellular [3H]DA uptake, in comparison to non-treated controls and the σ1 antagonists BD-1047 and NE-100 prevent the positive effect of (+)-pentazocine on DA reuptake DA is likely to be considered a neurotoxic molecule. In fact, when levels of intracellular DA abnormally invrease, vescicles can’t sequester the DA which is metabolized by MAO (A and B) and COMT with consequent overproduction of oxygen reactive species and toxic catabolites. Stress induced by these molecules leads cells to death. Thus, for the second part of this thesis, experiments have been performed in order to investigate functional alterations caused by the (+)-pentazocine-mediated increase of DA uptake; particularly it has been investigated if the increase of intracellular [DA] could affect cells viability. Results obtained from this study demonstrate that (+)-pentazocine alone increases DA cell toxicity in a concentration-dependent manner only in cells co-expressing hσ1 and hDAT and σ1 antagonists are able to revert the (+)-pentazocine-induced increase of cell susceptibility to DA toxicity. In the last part of this thesis, the functional cross-talking between hσ1 receptor and hDAT has been further investigated using confocal microscopy. From the acquired data it could be suggested that, following exposure to (+)-pentazocine, the hσ1 receptors massively translocate towards the plasma membrane and colocalize with the hDATs. However, any physical interaction between the two proteins remains to be proved. In conclusion, the presented study shows for the first time that, in cell models, hσ1 receptors directly modulate the hDAT activity. Facilitation of DA uptake induced by (+)-pentazocine is reflected on the increased cell susceptibility to DA toxicity; these effects are prevented by σ1 selective antagonists. Since numerous compounds, including several drugs of abuse, bind to σ1 receptors and activating them could facilitate the damage of dopaminergic neurons, the reported protective effect showed by σ1 antagonists would represent the pharmacological basis to test these compounds in experimental models of dopaminergic neurodegenerative diseases (i.e. Parkinson’s Disease).

Molecular approach to Neurodegenerative Disorders: Role of Nociceptin/Orphanin FQ - NOP System in Parkinson and Epigenetic Mechanism in Alzheimer's Disease

With life expectancies increasing around the world, populations are getting age and neurodegenerative diseases have become a global issue. For this reason we have focused our attention on the two most important neurodegenerative diseases: Parkinson’s and Alzheimer’s. Parkinson’s disease is a chronic progressive neurodegenerative movement disorder of multi-factorial origin. Environmental toxins as well as agricultural chemicals have been associated with PD. Has been observed that N/OFQ contributes to both neurotoxicity and symptoms associated with PD and that pronociceptin gene expression is up-regulated in rat SN of 6-OHDA and MPP induced experimental parkinsonism. First, we investigated the role of N/OFQ-NOP system in the pathogenesis of PD in an animal model developed using PQ and/or MB. Then we studied Alzheimer's disease. This disorder is defined as a progressive neurologic disease of the brain leading to the irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. Effective biomarker tests could prevent such devastating damage occurring. We utilized the peripheral blood cells of AD discordant monozygotic twin in the search of peripheral markers which could reflect the pathology within the brain, and also support the hypothesis that PBMC might be a useful model of epigenetic gene regulation in the brain. We investigated the mRNA levels in several genes involve in AD pathogenesis, as well DNA methylation by MSP Real-Time PCR. Finally by Western Blotting we assess the immunoreactivity levels for histone modifications. Our results support the idea that epigenetic changes assessed in PBMCs can also be useful in neurodegenerative disorders, like AD and PD, enabling identification of new biomarkers in order to develop early diagnostic programs.

Velhas drogas, novas terapêuticas: investigação da utilização de antagonistas de adrenoceptores α1 e de bloqueadores de canais de cálcio no retardo da ejaculação

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência dos fatores de crescimento semelhantes à insulina (IGF-l e ll), seus receptores (IGFR-l e II), proteínas ligantes (IGFBP-2 e 4) e PAPP-A na aquisição de tolerância ao estresse térmico de embriões bovinos (Nelore vs Holandês) produzidos in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

New molecular approaches to control rhabdomyosarcoma onset and metastasis in preclinical systems

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. The aim of this study was to identify molecular events involved in rhabdomyosarcoma onset for the development of new therapeutic approaches against specific molecular targets. BALB-p53neu mice develop pelvic rhabdomyosarcoma and combines the activation of HER-2/neu oncogene with the inactivation of an allele of p53 oncosuppressor gene. Gene expression profiling led to the identification of genes potentially involved in rhabdomyosarcoma genesis and therefore of candidate targets. The pattern of expression of p53, HER-2/neu, CDKN2A/p19ARF and IGF-2 suggested that these alterations might be involved in gender-, site- and strain-specific development of rhabdomyosarcoma. Other genes such as CDKN1A/p21 might be involved. The role of IGF-2, CDKN2A/p19ARF and CDKN1A/p21 in tumor growth was investigated with siRNA in murine rhabdomyosarcoma cells. Silencing of p19ARF and p21 induced inhibition of growth and of migration ability, indicating a possible pro-tumor and pro-metastatic role in rhabdomyosarcoma in absence of p53. In addition the autocrine IGF-2/IGF-1R loop found in early phases of cancer progression strengthens its key role in sustaining rhabdomyosarcoma growth. As rhabdomyosarcoma displays defective myogenic differentiation, a therapeutic approach aimed at enhancing myogenic differentiation of rhabdomyosarcoma cells. Forced expression of myogenin was able to restore myogenic differentiation, significantly reduced cell motility and impaired tumor growth and metastatic spread. IL-4 treatment increased rhabdomyosarcoma cell growth, decreased myogenin expression and promoted migration of cells lacking myogenin. Another approach was based on small kinase inhibitors. Agents specifically targeting members of the HER family (Lapatinib), of the IGF system (NVP-AEW541) or downstream signal transducers (NVP-BEZ235) were investigated in vitro in human rhabdomyosarcoma cell lines as therapeutic anti-tumor and anti-metastatic tools. The major effects were obtained with NVP-BEZ235 treatment that was able to strongly inhibit cell growth in vitro and showed anti-metastatic effects in vivo.

Cellular and Molecular Biomarkers for Risk Assessment of Colorectal cancer

The detection of Colorectal Cancer (CRC), at early stages, is one of the proven strategies resulting in a higher cure rate. In recent years, several studies have appeared identifying potential cancer markers in serum, plasma and stool in an attempt to improve actual screening procedures. Thus, the aim of the study was (1) Evaluate MN frequency, (2) Evaluate plasma ultrafiltrate capacity to induce MN formation, (3) Evaluate SEPT9 and NOTCH3 promoter methylation profile in peripheral blood lymphocytes from subjects resulted positive to fecal occult blood test and examined by colonoscopy. MN frequency was significantly higher in subjects with histological diagnosis of CRC and adenoma than control (p ≤ 0.001 and p ≤ 0.01, respectively). About, CF-MN analysis, a statistically significant difference was observed between CRC and control (p ≤ 0.05). On the other hand, SEPT9 and NOTCH3 promoter methylation status was significantly lower in CRC subjects than controls; additionally, NOTCH3 promoter methylation status was significantly lower in CRC subjects than adenoma subjects (p ≤ 0.01). The results obtained allow conclude that MN frequency varies according CRC pathologic status and, together with other variables, is a valid biomarker for adenoma and CRC risk. Additionally, the plasma of patients affected with CRC not only serve as a biomarker for oxidative stress but also as biomarker of genetic damage correlated with the carcinogenic process that verifies in colon-rectum. SEPT9 and NOTCH3 promoter methylation status, at peripheral blood level, varies according hystopathological changes observed in colon-rectum, suggesting that promoter methylation profile of these genes could be a reliable biomarker for CRC risk.

Neurodegenerative diseases: molecular mechanisms and new strategies for neuroprotection

The term neurodegeneration defines numerous conditions that modify neuron’s normal functions in the human brain where is possible to observe a progressive and consistent neuronal loss. The mechanisms involved in neurodegenerative chronic and acute diseases evolution are not completely understood yet, however they share common characteristics such as misfolded proteins, oxidative stress, inflammation, excitotoxicity, and neuronal loss. Many studies have shown the frequency to develop neurodegenerative chronic diseases several years after an acute brain injury. In addition, many patients show, after a traumatic brain injury, motor and cognitive manifestations that are close to which are observed in neurodegenerative chronic patients. For this reason it is evident how is fundamental the concept of neuroprotection as a way to modulate the neurodegenerative processes evolution. Neuroinflammation, oxidative stress and the apoptotic process may be functional targets where operate to this end. Taking into account these considerations, the aim of the present study is to identify potential common pathogenetic pathways in neurodegenerative diseases using an integrated approach of preclinical studies. The goal is to delineate therapeutic strategies for the prevention of neuroinflammation, neurodegeneration and dysfunctions associated to Parkinson’s disease (PD) and cerebral ischemia. In the present study we used a murine model of PD treated with an isothiocyanate, 6-MSITC, able to quench ROS formation, restore the antioxidant GSH system, slow down the apoptotic neuronal death and counteract motor dysfunction induced by 6-OHDA. In the second study we utilized a transgenic mouse model knockout for CD36 receptor to investigate the inflammation involvement in a long term study of MCAo, which shows a better outcome after the damage induced. In conclusion, results in this study allow underlying the connection among these pathologies, and the importance of a neuroprotective strategy able to restore neurons activity where current drugs therapies have shown palliative but not healing abilities.

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes : (Part 10) The infrared and Raman characteristics of phthalocyanine in heteroleptic bis(phthalocyaninato) rare earth complexes with decreased molecular symmetry

The infrared (IR) spectroscopic data for a series of eleven heteroleptic bis(phthalocyaninato) rare earth complexes MIII(Pc)[Pc(α-OC5H11)4] (M = Sm–Lu, Y) [H2Pc = unsubstituted phthalocyanine, H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected with 2 cm−1 resolution. Raman spectroscopic properties in the range of 500–1800 cm−1 for these double-decker molecules have also been comparatively studied using laser excitation sources emitting at 632.8 and 785 nm. Both the IR and Raman spectra for M(Pc)[Pc(α-OC5H11)4] are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues due to the decreased molecular symmetry of these double-decker compounds, namely C4. For this series, the IR Pc√− marker band appears as an intense absorption at 1309–1317 cm−1, attributed to the pyrrole stretching. With laser excitation at 632.8 nm, Raman vibrations derived from isoindole ring and aza stretchings in the range of 1300–1600 cm−1 are selectively intensified. In contrast, when excited with laser radiation of 785 nm, the ring radial vibrations of isoindole moieties and dihedral plane deformations between 500 and 1000 cm−1 for M(Pc)[Pc(α-OC5H11)4] intensify to become the strongest scatterings. Both techniques reveal that the frequencies of pyrrole stretching, isoindole breathing, isoindole stretchings, aza stretchings and coupling of pyrrole and aza stretchings depend on the rare earth ionic size, shifting to higher energy along with the lanthanide contraction due to the increased ring-ring interaction across the series. The assignments of the vibrational bands for these compounds have been made and discussed in relation to other unsubstituted and substituted bis(phthalocyaninato) rare earth analogues, such as M(Pc)2 and M(OOPc)2 [H2OOPc = 2,3,9,10,16,17,23,24-octakis(octyloxy)phthalocyanine].

Time-Dependent Density Functional Molecular Orbital and Excited State Calculations on Bis(porphyrinyl)butadiynes in the Monocationic, Neutral, Monoanionic, and Dianionic Oxidation States

We report a theoretical study of the multiple oxidation states (1+, 0, 1−, and 2−) of a meso,meso-linked diporphyrin, namely bis[10,15,20-triphenylporphyrinatozinc(II)-5-yl]butadiyne (4), using Time-Dependent Density Functional Theory (TDDFT). The origin of electronic transitions of singlet excited states is discussed in comparison to experimental spectra for the corresponding oxidation states of the close analogue bis{10,15,20-tris[3‘,5‘-di-tert-butylphenyl]porphyrinatozinc(II)-5-yl}butadiyne (3). The latter were measured in previous work under in situ spectroelectrochemical conditions. Excitation energies and orbital compositions of the excited states were obtained for these large delocalized aromatic radicals, which are unique examples of organic mixed-valence systems. The radical cations and anions of butadiyne-bridged diporphyrins such as 3 display characteristic electronic absorption bands in the near-IR region, which have been successfully predicted with use of these computational methods. The radicals are clearly of the “fully delocalized” or Class III type. The key spectral features of the neutral and dianionic states were also reproduced, although due to the large size of these molecules, quantitative agreement of energies with observations is not as good in the blue end of the visible region. The TDDFT calculations are largely in accord with a previous empirical model for the spectra, which was based simplistically on one-electron transitions among the eight key frontier orbitals of the C4 (1,4-butadiyne) linked diporphyrins.

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

Comparison of molecular markers to detect fresh sewage in environmental waters

Human-specific Bacteroides HF183 (HS-HF183), human-specific Enterococci faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., Escherichia coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% hostspecificity. All the human-specific markers showed >97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10_5, 10_4 and 10_3 respectively.HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10_4 whilst EVs were detected up to dilution 10_5. The ability of the HS-HF183 marker to detect freshsewagewas3–4 orders ofmagnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices was similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.