Oncolytic viruses promise to significantly improve current cancer treatments through their tumour-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapies is the intravenous delivery of the virus, as it can be cleared by neutralizing antibodies (nAbs) from the bloodstream before it reaches the tumour cells. In our group, we have succeeded in developing aptamers to vesicular stomatitis virus (VSV), as well as to rabbit anti-VSV polyclonal neutralizing antibodies (nAbs). We tested these aptamers’ biological activity with a cell-based plaque forming assay and found that the aptamers prevented in vitro neutralization of VSV by nAbs and increased the virus infection rate of transformed cells up to 77%. In line with this approach, we enhanced the delivery of oncolytic viruses by selecting aptamers to the CT26 colon carcinoma cell line. The binding of aptamer pools has been tested on flow cytometry and the best pools were subjected to high throughput sequencing. Selected aptamers were linked to anti-VSV aptamers and applied for target delivery of the virus to cancer cells. Development of this aptamer-based technology aims to improve viral anti-cancer therapies, with a potential to be applied as treatment for patients affected with cancer. Finally, in collaboration with a group from Erlangen University, we performed an aptamer selection using capillary electrophoresis and cell-SELEX. The target, the extracellular domain of human CD83, is a maturation marker for dendritic cells and is involved in the regulation of the immune system. Selected aptamer sequences bound selectively to mature dendritic cells, in comparison to immature dendritic cells, and thus hold promise to be applied for further studies leading to a better understanding of CD83’s mechanism of action.

Orientador: Maria Elvira Pizzigatti Corrêa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-28T03:50:50Z (GMT). No. of bitstreams: 1 Vieira_RaizaMeira_M.pdf: 2654950 bytes, checksum: 493dac1ecf0ca2acb92f0ff8829107aa (MD5) Previous issue date: 2015 / Resumo: Complicações orais estão presentes em cerca de 80% dos pacientes durante o Transplante de Células Tronco Hematopoéticas (TCTH), sendo a mucosite oral (MO) e as alterações salivares umas da que possuem maior impacto para a qualidade de vida do paciente. A utilização do perfil proteico salivar (PPS) na abordagem diagnóstica em diversas doenças tem sido frequente. A identificação de um PPS que auxilie o entendimento das manifestações agudas orais do TCTH poderia sobremaneira, influenciar nas decisões terapêuticas visando melhora no manejo do paciente. O objetivo deste trabalho foi identificar alterações do PPS do início do regime do condicionamento pré TCTH até a recuperação medula e correlacioná- las com dados clínicos orais. Para tanto, foi utilizado o banco de dados de proteomica salivar do grupo de Odontologia do Hemocentro, encontrada por Feio et AL (2013). Nesta avaliação foram incluídos 16 pacientes submetidos ao primeiro TCTH alogênico na Unidade de Transplante de Medula Óssea do Hospital de Clínicas da UNICAMP. As amostras de saliva total não estimulada (STNE) foram coletadas em dois momentos: previamente ao condicionamento (coleta A) e a segunda (coleta B), entre os dias D+8 e D+10 pós-TCTH. Dados sobre saúde oral, grau de MO foram coletados, além da avaliação de hiposalivação. O estudo do PPS foi realizado por espectometria de massas no LNBio. Os PPS das duas coletas, A e B foram tabelados no programa Excel® 2007 (Microsoft, WA, EUA), juntamente com os dados clínicos. O teste T pareado foi utilizado para a comparação entre os PPSs com os tempos das coletas. Os critérios clínicos de hiposalivação foram comparados com as divergências proteicas durante as duas coletas por ANOVA. A comparação entre os dados clínicos de cada coleta e seu respectivo PPS, foi feito pelo teste T pareado, sendo considerado significativo p<0,005. Sete proteínas apresentaram intensidades divergentes entre as coletas A e B: Prolactin-inducible protein, Alpha-Amylase 1, Cystatin-SN, Submaxillary gland androgen-regulated protein 3B, Sthaterin, cDNA FLJ60163, highly similar to Carbonic anhydrase 6 apresentaram-se em decréscimo na coleta B comparada a coleta A e a vitamin D-binding protein isoform 1 precursor se apresentou aumentada na coleta B quando comparada a coleta A. A mucosite oral foi correlacionada com as proteinas, Immunoglobulin J chain, Pulative uncharacterized protein DKFZp686N02209 que se demonstraram com intensidade diminuída naqueles pacientes que apresentaram graus III e IV em comparação àqueles com grau 0¿II de MO; a proteína Statherin esteve presente em maior intensidade naqueles pacientes que apresentaram MO com grau III e IV. Esses resultados podem estar relacionados principalmente à toxicidade do regime de condicionamento mieloablativo nas glândulas salivares e sugerem que as proteinas Statherin, Immunoglobulin J chain e Pulative uncharacterized protein DKFZp686N02209 podem ser candidatas a um painel de PPS para a MO no TCTH / Abstract: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for various hematological diseases. Oral Complications are present in about 80% of patients who undergo to HSCT. Oral mucositis (OM), oral infections and salivary changes, among others are the most common oral clinical founds in the acute phase of the HSCT. The use of salivary profile as a tool for diagnosis and management of treatment responses has been shown a promising future in different clinical settings. Thus the discovery of a salivary protein profile (SPP) of patients submitted to the allogeneic HSCT during the acute phase of the transplantation may be helpful on the management of the oral effects of the HSCT. The aim of this research was to identify the SPP changes during the acute phase of allogeneic HSCT and correlate them with the oral clinical manifestation of the acute phase of HSCT. This study enrrolled 16 patients with hematological diseases who, underwent to their first allogenic HSCT at the Bone Marrow Transplantation Unit ¿ UNICAMP. Unstimulated salivary samples were collected in two periods: first (collection A), it was performed prior the initiation of the conditioning regimen for the HSCT and collection B; performed between day D+8-10 days after HSCT. Oral health indices were obtained in both moments of the saliva collection. The severity of oral mucositis was collected on the collection B. The PPS analysis was performed by mass spectrometry on a previous study performed by Feio et al (2013). Student T test paired was used in order to between the 39 identified proteins equally between the collections A and B. Clinical correlations were also compared to the different PPS using one-way ANOVA analysis. For both comparisons, considered significant p < 0.05. Among the collections A and B, 7 proteins were presented with differing intensities: Prolactin-inducible protein, Alpha-Amylase 1, Cystatin SN, Submaxillary gland androgen-regulated protein 3B, Sthaterin, cDNA FLJ60163 Similar to highly Carbonic anhydrase 6 showed a decrease intensity in the collection B and the vitamin D-binding protein isoform 1 precursor quantity showed a increased in the collection B. Three proteins were shown altered intensity when correlated with the severity of OM: Immunoglobulin J chain, Pulative uncharacterized protein DKFZp686N02209v. These proteins showed a decreased of intensity in those patients grade III-IV OM when compared to patients with grade 0-II OM. The protein Statherin showed in increased intensity in patients with OM grade III-IV. These results indicate the influence of the toxicity of the conditioning regimens on the salivary protein profiles in the acute phase of the HSCT. The proteins: Immunoglobulin J chain, Pulative uncharacterized protein DKFZp686N02209v and Statherin might be a potential candidates for a panel of salivary biomarkers for OM / Mestrado / Estomatologia / Mestra em Estomatopatologia

Celiac disease is a common autoimmune disease triggered by gluten-containing foods (wheat, barley and rye) in genetically predisposed individuals. We present a patient with celiac disease complicated by severe aphthous stomatitis resulting in impairing swallowing, chewing and speaking. This led to weight loss, psychosocial problems as well as inability to perform her work. A variety of topical and systemic medications used resulted in either no improvement or only partial alleviation of the patient's symptoms. After informed consent, etanercept was initiated and resulted in complete remission of aphthous stomatitis, decrease in arthralgia and fatigue and considerable improvement in her quality of life. The use of newer biological agents for selected and severe manifestations of celiac disease may lead to improved morbidity in these patients, but more studies are needed to determine long-term efficacy as well as safety of these drugs in the mucosal and/or systemic complications of this disease.

Celiac disease is a common autoimmune disease triggered by gluten-containing foods (wheat, barley and rye) in genetically predisposed individuals. We present a patient with celiac disease complicated by severe aphthous stomatitis resulting in impairing swallowing, chewing and speaking. This led to weight loss, psychosocial problems as well as inability to perform her work. A variety of topical and systemic medications used resulted in either no improvement or only partial alleviation of the patient's symptoms. After informed consent, etanercept was initiated and resulted in complete remission of aphthous stomatitis, decrease in arthralgia and fatigue and considerable improvement in her quality of life. The use of newer biological agents for selected and severe manifestations of celiac disease may lead to improved morbidity in these patients, but more studies are needed to determine long-term efficacy as well as safety of these drugs in the mucosal and/or systemic complications of this disease.

Aus dem Patientengut einer Fachklinik für Klein- und Heimtiere wurden 11 nicht vorbehandelte Katzen zwischen einem und zwölf Jahren mit mittel- bis hochgradiger Gingivo-Stomatitis ausgewählt. Diese wurden für zwölf Wochen (84 Tage) stationär aufgenommen und nach einem standardisierten Therapiekonzept behandelt: Am ersten Tag erfolgte nach dentalem Röntgen eine umfassende Zahnsanierung. An den Tagen 0, 14, 28, 42 und 84 wurde Interferon (Virbagen Omega® des Herstellers Virbac S.A.®, Carros Cedex, Frankreich) unter Sedation lokal, d.h. submukosal mit 1 ME/kg KGW injiziert. An den Tagen 56, 58, 60 und 62 erfolgte die Interferongabe systemisch. Begleittherapien wurde nach Bedarf eingesetzt, jedoch ohne die Verwendung von Glukokortikoiden und Hormonpräparaten. Verfüttert wurde ausschließlich Futter des Herstellers Royal Canin®, Köln, in den ersten 14 Tagen das Feuchtfutter Royal Canin convalescence support®, ab Tag 15 Royal Canin intestinal® Feucht- und Trockenfutter. An allen Behandlungstagen wurden zur qualitativen Virusbestimmung Tupferproben der am stärksten entzündeten Bezirke entnommen, die Maulhöhle nach einem festen System abfotografiert und die Veränderungen in Formblättern (Stärke der Faucites, Gingivitis, Buccostomatitis, Größe der Fläche und Art der Veränderung) und Grafik-charts festgehalten. Am ersten und letzten Tag wurden außerdem Biopsien zur quantitativen Bestimmung der Viruslast entnommen. Die Entwicklungen in folgenden Bereichen wurden anhand fixer Kriterien 14-tägig festgehalten: Allgemeinzustand, Schmerzen bei der Maulöffnung, Halitosis/zäher Speichel, Größe der Mandibularlymphknoten, Appetit, Schmerzen bei Futter-aufnahme oder Gähnen, Hypersalivation, Aktivität, Putztrieb und Zugänglichkeit. Die klinischen Verbesserungen waren bei allen Tieren schon nach 14 Tagen augenfällig. Der Hauptvorstellungsgrund der Besitzer, Appetitlosigkeit und Schmerzen bei der Futteraufnahme waren einer fast ungestörten Futteraufnahme gewichen, diese konnte in den folgenden Wochen kaum noch optimiert werden. Die entzündlichen Ulzerationen und Proliferationen der Maulhöhle halbierten sich innerhalb der ersten 14 Tage, nach 84 Tagen war der Heilungsprozess bei acht der elf Katzen abgeschlossen. Die persistierenden Proliferationen der restlichen Katzen waren allerdings nicht entzündlich und beeinflussten die Futteraufnahme nicht. Allgemeinzustand, Aktivität, Putztrieb und Zugänglichkeit stiegen bei zehn von elf Katzen bis zum 42. Tag etwa linear auf artspezifisches Normalniveau an und blieben hier konstant. Hypersalivation und Schwellung der Mandibularlymphknoten legte sich, so vorhanden, bei allen Tieren bis auf zwei innerhalb von 28 Tagen, bei diesen beiden war bis zum 84. Tag nur eine geringgradige Verbesserung zu beobachten. Nach der systemischen Vier-Tages-Therapie wurde ein erneutes Aufflackern der Gingivo-Stomatitis etwa auf das Niveau des 56. Tages beobachtet, allerdings ohne Folgen für die Verhaltensparameter. Eine Reduktion der Viruslast konnte trotz der eindrucksvollen Verbesserungen im klinischen Bereich in keinem Fall festgestellt werden. Die FIV/FeLV-positiven Katzen sprachen langfristig wesentlich schlechter auf das Therapiekonzept an als die übrigen Probanden. Daher gilt es bei diesen Tieren vor Interferoneinsatz kritisch zwischen den nicht unerheblichen Kosten und der zweifelhaften Prognose abzuwägen. Generell kann das Therapiekonzept Zahnsanierung – Interferon – Begleittherapie nach erfolgtem FIV/FeLV-Test bei der felinen Gingivo-Stomatitis als klinisch erfolgreich betrachtet werden.

No description available.

Scope and Contents: Virus specific RNA components of the cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis virus were examined. Studies were carried out both in the presence and absence of defective particle interference. / Thesis / Master of Science (MSc)

No description available.

The vesicular stomatitis virus glycoprotein G is responsible for low pH mediated membrane fusion induced by the virus. Four linker insertion mutants (H2, H5, HIO, A4) of the G ectodomain were found to disrupt fusion and yet maintained all the requirements for proper folding and cell surface expression (Li et al., 1993). Site specific mutagenesis of residues 123 to 137, surrounding the H2 mutant, either blocked or shifted the pH optima and threshold of fusion to more acidic values with a concomitant reduction in cell-cell fusion efficiency (Zhang and Ghosh, 1994; Fredericksen and Whitt, 1995). The region is highly conserved among vesiculoviruses and was found to insert into lipid membranes by hydrophobic photolabelling (Durrer et al., 1995) suggesting a possible role for this domain as the fusion peptide. Site-directed mutagenesis of residues 190 to 210, surrounding the H5 insertion mutant, did not significantly affect fusion (Fredericksen and Whitt, 1995). Surrounding the H10 and A4 insertion mutants is a conserved region, residues 395 to 424, that does not interact with target membranes (Durrer et al., 1995). To determine the functional importance of this region, site-directed mutagenesis was employed. Substitution of conserved Gly 404, Gly 406, Asp 409, and Asp 411 with Ala, Ala, Asn, and Asn, respt:.ctively, both reduced fusion and caused a shift in the pH of fusion threshold to more acidic values (tested by Y. He as published in Shokralla et al., 1998). In this study, the Hl0/A4 region is further mutagenized and tested for fusion. Cell surface expression was examined by indirect immunofluorescence and lactoperoxidase catalyzed iodination. Rates of transport from the endoplasmic reticulum and oligomerization into trimers were tested by resistance to endoglycosidase H and sucrose density gradient centrifugation, respectively. Low-pH induced conformational changes were assayed by resistance to proteolytic digestion. Residues Gly 395, Gly 404, Gly 409 and Ala 418 were substituted with Glu, Lys, Asp, and Lys, respectively. All mutants, with the exception of A418K, were expressed at levels similar to or above wild-type. Mutants G404K and D409A completely abolished fusion. Mutant G395E reduced cell-cell fusion efficiency by 82% and shifted both the pH threshold and optimum of wild type fusion. Although all mutants were capable of trimer formation, alterations in the structure of mutants G404K, D409 A, and A418K were detected by slower transport rates. All Hl0/A4 mutants were more susceptible to trypsin than wild-tyr,e at the pH of6.5, and mutant G404K was completely susceptible at this pH Reductions in the extent of fusion, along with shifts in the pH optima and thresholds of fusion suggest that the Hl0/A4 region (residues 395 to 418) of vesicular stomatitis virus G protein is important for G mediated fusion. The region may influence low-pH induced conformational changes. Double mutants of the H2 and HI0/A4 regions were also tested for their effects on fusion. The extents of fusion mediated by double mutant G proteins were severely reduced with levels ranging from 28% wild-type fusion to complete fusion deficiency. Only mutant Gl31A G404A was capable of 83% wild-type fusion. Mutants Gl31A G395E, Gl31A G404A, Gl31A D4LIN, Dl37N G404A, and the fusion defective D137N D411N were expressed at levels above wild-type G protein at the cell surface. Mutants Fl25Y D411N and Pl26L D411N, although capable of very low levels of fusion were not detectable at the cell surface by immunoflorescence and were detected at low levels by lactoperoxidase catalyzed iodination of cell surface proteins. These two mutants, along with Gl31A G404A, also showed slower transport rates than wild-type G. All double mutants showed increased sensitivity to trypsin at the pH of 6.5 with mutant Fl25Y D411N showing complete susceptibility. They were also all capable of trimer formation by sucrose density gradient centrifugation. In comparing the fusion profiles of double mutants with those of their component single mutants, it was found that in most cases the pH threshold of fusion by double mutants was greater than the sum of the single mutants and that the pH optimum of fusion corresponded to that of the constituent H2 single mutant. Although, the regions are functionally independent, they may indirectly affect one another through alterations in protein structure. / Thesis / Master of Science (MS)

Relatively little is known about the vesicular stomatitis virus (VSV) glycoprotein G fusion mechanism. Vesicular stomatitis virus has a single type 1 integral membrane glycoprotein G embedded in the viral membrane. It is the only viral protein required for VSV induced low pH mediated fusion. Mutations in four regions (H2, A5, A4 and HI0) of the VSV G ectodomain have been shown to abolish the fusion activity of the viral glycoprotein (Li et al.,l993). One region H2 (a.a 117-139) has been suggested to be the fusion peptide (Zhang and Ghosh, 1994)(Fredericksen and Whitt, 1995). Amino acids 59-221 of the G protein, an area that encompasses the H2 region, has recently been shown to interact with liposomes through hydrophobic photolabeling experiments (Durrer et al., 1995), suggesting that the H2 region (fusion peptide)is able to interact with hydrophobic target bilayers at low pH. A soluble VSV G protein lacking the transmembrane anchor and cytoplasmic tail of VSV G is not fusogenic, suggesting that G must be anchored to the plasma membrane to promote syncytia (Florkiewicz and Rose, 1984). To better understand the steps involved in the fusion mechanism of VSV G it is important to identify domains within the protein that are involved in the fusion process. To determine the contributions of the transmembrane anchor and cytoplasmic tail to the VSV fusion mechanism chimeric G proteins were constructed. The transmembrane anchor alone or in conjunction with the cytoplasmic tail ofVSV G was replaced with equivalent domain from other viral proteins, HSV-1 glycoproteins gB and gD, adenovirus E3 11.6 K gene, that are not involved in low-pH fusion and the cellular protein CD4. All chimeras were expressed in COS-1 cells, glycosylated, oligomerized, transported to the cdl surface, showed a low-pH induced conformational change and were expressed on the cell surface at levels equivalent to wild-type G. The transmembrane hybrids show extensive syncytia formation at levels similar to wild-type G when induced at pH 5.6. The transmembrane-cytoplasmic tail hybrids showed reduced levels of syncytia as compared to wild-type Gat both pH 5.6 and 5.2. A glycosylphosphatidylinositollipid-anchored ectodomain of G (GGPI), which lacks both the transmembrane and cytoplasmic tail ofG, was expressed in COS-1 cells. The GGPI chimera was glycosylated, expressed on the cell surface,and oligomerized similar to wild-type G. However the chimera was fusion negative, could not promote lipid mixing and h~,d an altered tryptic digestion profile. A fusion negative chimera Gt12gBwas constructed by exchanging the TM of G with the equivalent domain from HSV-1 gB TM plus eight extra amino acids of the gB ectodomain. Deletion of the 11 extra gB amino acids (GgB3G) restored the fusogenic activity of this chimera. Another chimera G 10 DAF directly demonstrated that the fusion negative phenotype of GGPI, like chimera Gtii1Lll2gB, was a result of the 10 extra amino acids at the EC-TM interface. The ectodomain (EC)-transmembrane (TM) interface is highly conserved among 5 vesiculoviruses. Chimeras with a 9 amino acid insertion (GlODAF), deletion (G~9) or replacement (G~910DAF) were expressed in COS-1 cells. The expressed proteins were glycosylated, underwent a low-pH induced conformational change and were expressed on the cell surface at levels equivalent to wild type, but were fusion negative. Suggesting that both the sequence and spatial arrangement of amino acids at the EC-TM interface may affect VSV G fusion. Taken together the data suggests that the specific amino acid sequence of the transmembrane anchor of VSV G is not essential for fusion. Replacement of the TM of VSV G with equivalent domains from other viral and cellular proteins does not affect the fusion activity. The cytoplasmic tail of VSV G may form an entity alone or in conjunction with the transmembrane anchor that can regulate fusion. Another region in the ectodomain of VSV G renders the glycoprotein fusion sensitive in a cell-cell fusion assay and was characterized at the EC-TM interface. / Thesis / Master of Science (MS)