The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

This thesis aims to present a color segmentation approach for traffic sign recognition based on LVQ neural networks. The RGB images were converted into HSV color space, and segmented using LVQ depending on the hue and saturation values of each pixel in the HSV color space. LVQ neural network was used to segment red, blue and yellow colors on the road and traffic signs to detect and recognize them. LVQ was effectively applied to 536 sampled images taken from different countries in different conditions with 89% accuracy and the execution time of each image among 31 images was calculated in between 0.726sec to 0.844sec. The method was tested in different environmental conditions and LVQ showed its capacity to reasonably segment color despite remarkable illumination differences. The results showed high robustness.

The unfolded protein response (UPR) is induced by a variety of external and internal stimuli, including accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses such as Herpes Simplex Virus type 1 (HSV-1) induce host cells to produce viral proteins many of which undergo glycosylation and other modifications in the ER, causing stress to the ER and consequently UPR activation. I have tested the hypothesis that HSV-1 has evolved strategies to regulate the UPR in order to suppress aspects of the UPR that might interfere with viral replication and to promote pathways that aid its own survival and replication. The purpose of this study was to test the hypothesis that HSV-1 selectively modulates the three pathways (PERK, ATF6, and IRE-1) of the UPR in epithelial and neuronal cells and to examine the similarities and the differences between these two types of cells in their responses to ER stress. Vero and ONS-76 cells were used as models of epithelial and neuronal cells respectively and qRT PCR technique was used for analyzing RNA levels of transcripts of spliced Xbp1, HERP, CHOP and BIP, selected target genes for three pathways of the UPR. HSV-1 DNA synthesis and infectious virus production in infected cells showed that compared to the permissive Vero cells, ONS-76 cells seemed to be semi-permissive to HSV-1 infection with limited viral DNA synthesis and infectious virus production. The kinetics of transcript and protein synthesis for genes representing immediate early, early and late classes of viral genes was also monitored. Expression of the immediate early gene, ICP0, was similar in both cell types but the expression of the early gene, TK and late genes VP16 and VP 5 was different. My work reveals that HSV-1 infection in cells of epithelial and neuronal origins results in activation of the UPR, but through cell type selective regulation of the three signal transduction pathways of the UPR (PERK, ATF6, and IRE-1). While HSV-1 infection resulted in upregulation of Spliced Xbp1 and its target gene HERP (IRE1 pathway) and downregulation of BIP (ATF6 pathway) in both cell types, CHOP (PERK pathway) was upregulated only in ONS cells. My results suggest that some aspects of the UPR are regulated differently in cells representing the sites for HSV-1 lytic and latent infections. This may indicate the need for increasing the capacity for protein folding and degradation (Xbp1 and ATF6-induced) in both cells but a requirement for suppressing apoptosis (PERK-induced) only in epithelial cells. As well, I show that HSV-1 infection not only selectively activates the UPR pathways in different cell types, but also inactivates some components of the UPR pathways activated by the drug thapsigargin.

Knockdown of the M5 acetylcholine muscarinic receptor in the ventral tegmental area (VTA) reduces brain-stimulation reward sensitivity in rats. Knockout (KO) of the M5 receptor in mice reduces morphine-induced dopamine efflux, locomotion, conditionedplace- preference, and mating-induced 30-110 kHz ultrasonic vocalizations (USVs). The GFP-labeled M5 receptor gene was transfected using a Herpes simplex virus either into the VTA or 0.2-0.7 mm posterior in the medial tegmentum (MT) of male M5 KO mice. HSV-M5-GFP transfection in VTA fully restored mating-induced USVs and augmented morphine-induced locomotion and stereotypy consistent with activation of DA neurons by M5 receptors. HSV-M5-GFP transfection sites in the MT inhibited USVs and morphine-induced locomotion presumably through inhibition of DA neurons. Putative transfection of M5 in GABA neurons of the rostromedial tegmental nucleus (RMTg) or 5HT neurons of the median raphe (mR) may explain this inhibition. Therefore, HSV-M5- GFP transfection in the VTA enhances DA-mediated behaviours while MT transfections inhibits these behaviours.

No description available.

Knockdown of the M5 acetylcholine muscarinic receptor in the ventral tegmental area (VTA) reduces brain-stimulation reward sensitivity in rats. Knockout (KO) of the M5 receptor in mice reduces morphine-induced dopamine efflux, locomotion, conditionedplace- preference, and mating-induced 30-110 kHz ultrasonic vocalizations (USVs). The GFP-labeled M5 receptor gene was transfected using a Herpes simplex virus either into the VTA or 0.2-0.7 mm posterior in the medial tegmentum (MT) of male M5 KO mice. HSV-M5-GFP transfection in VTA fully restored mating-induced USVs and augmented morphine-induced locomotion and stereotypy consistent with activation of DA neurons by M5 receptors. HSV-M5-GFP transfection sites in the MT inhibited USVs and morphine-induced locomotion presumably through inhibition of DA neurons. Putative transfection of M5 in GABA neurons of the rostromedial tegmental nucleus (RMTg) or 5HT neurons of the median raphe (mR) may explain this inhibition. Therefore, HSV-M5- GFP transfection in the VTA enhances DA-mediated behaviours while MT transfections inhibits these behaviours.

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.

Submitted by Marta Toyoda (1144061@mackenzie.br) on 2018-05-02T21:51:39Z No. of bitstreams: 2 MARCELLO GUARIENTO BERTACCHI.pdf: 10727812 bytes, checksum: cdcb205e08ced81eddded18a3138a873 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Paola Damato (repositorio@mackenzie.br) on 2018-05-04T15:59:12Z (GMT) No. of bitstreams: 2 MARCELLO GUARIENTO BERTACCHI.pdf: 10727812 bytes, checksum: cdcb205e08ced81eddded18a3138a873 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-04T15:59:12Z (GMT). No. of bitstreams: 2 MARCELLO GUARIENTO BERTACCHI.pdf: 10727812 bytes, checksum: cdcb205e08ced81eddded18a3138a873 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Initially, facial feature recognition was only used intuitively, which means that one individual recognized another by certain characteristics relevant for their identification. Time passed, and with technological advancement, other methods were created for this purpose. However, the addition of artificial characteristics could have a negative influence in the process of facial recognition. Hence the choice of the cosmetic application field, with the purpose of exploring in more details both the effects in recognition as well as the process of detection of facial makeup. For this purpose, the color model CMYK was chosen due to its satisfactory performance in skin detection. The objective of this work is to emphasize the feasibility of applying the color model CMYK in Computational Vision procedures and Image Analysis, in comparisson to another model widely used, which is the HSV. For the makeup classification process, it was chosen a variant of Artificial Neural Networks known as Neural Network Convolutional, which is based on the visual cortex of cats. First, it was proved the negative influence of makeup in face recognition, through the LBP descriptor. In sequence, six neural networks were trained to detect makeup, achieving an accuracy of 97 percentage points on the eye region, 95 points percent on the face and 80 percentage points on the lips, in CMYK’s model, and 91 percentage points on the eye region, 92 points percent on the face and 73 percentage points on the lips, in HSV’s model. Consequently, CMYK was proven to be a color space that deserves attention in the fields of Makeup and Computer Vision. / Inicialmente, o reconhecimento de características faciais era apenas utilizado de forma intuitiva, ou seja, um indivíduo reconhecia outro por meio de certas características relevantes para uma própria identificação. Com o passar do tempo, e com o avanço tecnológico, outros métodos foram criados para este propósito. Porém, a adição de características artificiais pode influenciar negativamente o processo de reconhecimento facial. Por este motivo, a área de processamento e análise de imagens com aplicação de cosméticos foi escolhida, com o propósito de se explorar com mais detalhes tanto os efeitos no reconhecimento quanto também o processo de detecção de maquiagem na face. Para esta finalidade, o modelo de cor CMYK foi escolhido, devido ao seu desempenho satisfatório na detecção de pele. O objetivo deste trabalho é colocar ênfase na viabilidade da aplicação do modelo de cor CMYK em procedimentos de Visão Computacional e Análise de Imagem, em comparação a outro modelo amplamente utilizado, que é o HSV. Para o processo de classificação de maquiagem foi escolhida uma variante das Rede Neurais Artificiais, conhecida como Rede Neural Convolucional, que se baseia no córtex visual dos gatos. Primeiramente foi comprovada a influência negativa da maquiagem no reconhecimento facial, por meio do descritor LBP. Na sequência, seis redes neurais foram treinadas para detecção de maquiagem, sendo alcançada uma acurácia de 97 pontos percentuais na região dos olhos, 95 pontos percentuais na face inteira e 80 pontos percentuais nos lábios, no modelo de cor CMYK, e 91 pontos percentuais na região dos olhos, 92 pontos percentuais na face inteira e 73 pontos percentuais nos lábios, no modelo de cor HSV. Com isto, comprova-se que o CMYK é um espaço de cor que merece atenção nas áreas de Detecção de Maquiagem e Visão Computacional.