Coiled coil protein 1, a novel gene downstream of FGF2 expressed in the developing brain

Fibroblast Growth Factor 2 (FGF2) plays an important role in modulating cell proliferation and differentiation in the developing central nervous system. It has been shown that FGF2 affects the transcription of several genes. In this study, a previously uncharacterised gene downstream of FGF2, named 'coiled coil protein 1' (ccpl), was identified and characterised. The expression at the tissue levels, particularly, in the brain, and cellular localization of ccp1 was investigated. In addition, functional analysis was carried out by overexpression and RNAi approaches. Ccp1 was identified by previous microarray analysis of primary cortical neuron culture derived from mouse embryonic brains stimulated by FGF2. Microarray data showed that ccp1 was up-regulated by 2.3-fold upon FGF2 stimulation. The study in this thesis showed that FGF2 up-regulated ccp1 both at the transcript and protein levels. Ccp1 cDNA is 1697 nucleotides long and the protein size is predicted to be 180 amino acids. Bioinformatic analysis revealed ccp1 homologues in various species, including human, rat, zebrafish, and drosophila. The gene is located in mouse chromosome 1,1B and its human homolog in chromosome 2q21.2. Next, in order to gain insight into the physiological role of ccp1 in mouse forebrains, its expression pattern was further analysed using in situ RNA hybridisation. Ccp1 was highly expressed early in the brain development in a ventrolateral area of the cortex and in the specific layer of the dorsal cortex. The pattern of ccp1 expression reflects some of the aspects of the tangential and radial migration that occurs at the early stages of brain development. A number of conserved domains are contained within the amino acid sequence of ccp1, including two coiled coil regions, a leucine zipper and two nuclear exporting signals (NES). These domains are related to specific protein functions and they may provide information in determining a role of ccp1 in the cell and in animal development. Analysis of enhanced green fluorescent protein (EGFP)-tagged ccp1 expressed in cells demonstrated that ccp1 was localised to the cytoplasm in a punctate pattern. Finally, ccp1 function was investigated using a retroviral overexpression system and RNAi in vitro. Ccp1 overexpression induced cell proliferation in the absence and presence of serum. Silencing of ccp1 expression by RNAi decreased cell proliferation, which provided further evidence for the role of ccp1 in this process. Incorporation of the S phase marker, 5-Bromo 2-Deoxyuridine (BrdU), has indicated that ccp1 enhances G1-S transition. To address the signalling mechanism by which ccp1 induces proliferation, involvement of 'Mitogen-Activated Protein Kinase' (MAPK) pathway was investigated. The addition of the 'MAPK or ERK Kinase' (MEK) inhibitor, U0126, blocked cell proliferation in cells overexpressing ccp1 in the presence of serum. The data suggested that MAPK pathway may be at least partially responsible for the ccp1-induced cell proliferation. The data presented in this thesis form the bases for functional study of ccp1 in vivo in the future, and provides novel insights into the mechanism of cell proliferation by growth factors

Role of state and trait attachment dimensions on involvement in a close relationship

The aim of the study was to investigate the association between trait and state attachment features and involvement in a couple-relationship. Eighty-four participants of different nationalities completed Childhood Trauma Questionnaire (CTQ), Toronto Alexithymia Scale (TAS-20), Attachment Style Questionnaire (ASQ), Experiences in Close Relationships-Revised (ECR-R), State Adult Attachment Measure (SAAM) and a personal questionnaire focused on involvement and amount of time spent in a couple-relationship. Results of the study showed that trait attachment features predicted involvement in a close relationship and the presence of a couple relationship predicted attachment state dimensions. Correlation analyses showed that the involvement in couple relationship was associated to CTQ Physical Neglect and SAAM Anxiety while participants without a partner had higher scores on CTQ Emotional Abuse, ECR- R Avoidance and SAAM Avoidance. Regression analyses showed that trait attachment features predicted time spent in a close relationship, while time spent in relationship predicted state attachment dimensions. Moreover, regression analysis showed that SAAM Security was predicted by ECR-R scales only in the sample of participants involved in a couple relationship

The Dopaminergic System in Peripheral Blood Lymphocytes: From Physiology to Pharmacology and Potential Applications to Neuropsychiatric Disorders

Besides its action on the nervous system, dopamine (DA) plays a role on neural-immune interactions. Here we review the current evidence on the dopaminergic system in human peripheral blood lymphocytes (PBL). PBL synthesize DA through the tyrosine-hydroxylase/DOPA-decarboxylase pathway, and express DA receptors and DA transporter (DAT) on their plasma membrane. Stimulation of DA receptors on PBL membrane contributes to modulate the development and initiation of immune responses under physiological conditions and in immune system pathologies such as autoimmunity or immunodeficiency. The characterization of DA system in PBL gave rise to a further line of research investigating the feasibility of PBL as a cellular model for studying DA derangement in neuropsychiatric disorders. Several reports showed changes of the expression of DAT and/or DA receptors in PBL from patients suffering from several neuropsychiatric disorders, in particular parkinsonian syndromes, schizophrenia and drug-or alcohol-abuse. Despite some methodological and theoretical limitations, these findings suggest that PBL may prove a cellular tool with which to identify the derangement of DA transmission in neuropsychiatric diseases, as well as to monitor the effects of pharmacological treatments

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein ​CrkL, a major substrate of the oncogenic tyrosine kinase ​BCR-​ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <104 cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-​CrkL and the protein tyrosine phosphatase ​PTPRC/​CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents

Prodromal non-motor symptoms of Parkinson’s disease

The motor symptoms of Parkinson’s disease (PD), bradykinesia, muscular rigidity, and tremor depend upon degeneration of the dopaminergic neurons in the substantia nigra pars compacta. Recent neuropathological studies show that the Lewy bodies, the intraneuronal landmark of PD, accumulate in several neuronal cell types in the brain. An ascending gradient of pathological involvement, from the medulla oblongata to neocortical areas has been reported. Thus the original view of PD as a disease characterized by selective damage of the dopaminergic neurons in the mesencephalon should be updated into the concept of a severe multisystemic neurodegenerative disorder. Additionally, the neuropathological alterations outside the substantia nigra are soundly correlated with the non-motor symptoms of PD. As a result of these findings, interest is growing in the identification of prodromal non-motor symptoms of PD. Indeed, data from the literature suggest that autonomic disturbances, olfactory dysfunctions, depression and sleep disorders (in particular REM-sleep behavior disorder) may represent prodromal non-motor symptoms of PD. Several tests are available to detect most of these symptoms. Thus, the identification of prodromal non-motor symptoms may contribute to the precocious diagnosis of PD, and might be useful in the future to test the efficacy of neuroprotective agents