Indiana University-Purdue University Indianapolis (IUPUI) / The pancreas is a vital organ that secretes key metabolic hormones and digestive enzymes. In pancreatic ductal adenocarcinoma (PDAC), one of the leading causes of cancer-related death in the world, limited advances in diagnosis or therapies have been made over decades. Key features of PDAC progression include an elevated matrix sti ness and an increased deposition of extracellular matrices (ECM), such as hyaluronic acid (HA). Understanding how cells interact with components in the tumor microenvironment (TME) as PDAC progresses can assist in developing diagnostic tools and therapeutic treatment options. In recent years, hydrogels have proven to be an excellent platform for studying cell-cell and cell-matrix interactions. Utilizing chemically modi ed and naturally derived materials, hydrogel networks can be formed to encompass not only the components, but also the physicochemical properties of the dynamic TME. In this work, a dynamic hydrogel system that integrates multiple click chemistries was developed for tuning matrix physicochemical properties in a manner similar to the temporally increased matrix sti ness and depositions of HA. Subsequently, these dynamic hydrogels were used to investigate how matrix sti ening and increased HA presentation might a ect survival of PDAC cells and their response to chemotherapeutics.

Unaccustomed, strenuous exercise can cause skeletal muscle damage that subsequently induces an acute inflammatory response in the tissue which is marked by an infiltration of leukocytes into the damaged muscle. To try and suppress the initial pro-inflammatory response in skeletal muscle of horses performing a single exercise stress test, a commercial sodium hyaluronate (HA) treatment was administered and tested for anti-inflammatory properties. Unfit, adult Thoroughbreds were intravenously injected three times with HA or received no injection at all (CON) over a 3-week period before performing a single submaximal exercise test. Gluteal muscle biopsies were collected before and 1 h after the completion of exercise for RNA-Seq and staining. The results indicated that HA treatment in horses down regulated genes associated with lymphocyte activation and cytokine production (Il17RA, OSCAR, LYL1, TLR1, TLR2, TLR8, TLR10) but did not irreversibly down regulate these genes with the addition of exercise. Exercise as a stressor did cause an acute inflammatory response in muscle which was seen through global expression of macrophage and neutrophil surface markers (NCF2, ELANE, CD168I). These results determine that HA treatment does act as an anti-inflammatory in equine skeletal muscle but does not possess prolonged effects with the initiation of inflammation. / Master of Science / Horses subjected to an unaccustomed increase in exercise intensity can experience damage and subsequent acute inflammation within the skeletal muscle tissue that may hinder the performance of the horse by causing muscle swelling and soreness. Hyaluronic acid treatment may suppress this exercise-induced inflammatory response by acting as an anti-inflammatory in the muscle. Adult Thoroughbred horses were injected intravenously with a commercial sodium hyaluronate treatment in the weeks prior to performing an exercise stress test. Muscle biopsy samples were obtained before and after the exercise stress test was performed. The results indicate that horses receiving the hyaluronic acid treatment had decreased expression of inflammatory genes within skeletal muscle, but no genes remained suppressed after the induction of inflammation through exercise. These results demonstrate that hyaluronic acid treatment does act as an anti-inflammatory in skeletal muscle tissue but does not have long-term suppressive effects when inflammation does occur.

Glioblastoma Multiforme (GBM) is the most common and malignant form of adult brain cancer, with 95% of patients succumbing to the disease within 5 years of diagnosis. An important contributing factor to this poor prognosis is upregulation of the transmembrane protein system xc- (SXC) found on GBM cells. Approximately 50% of GBM patients have tumors with upregulated levels of SXC, and these patients experience faster disease progression than patients with tumors expressing moderate levels of SXC. SXC is a sodium-independent antiporter and is comprised of a light chain catalytic subunit (xCT) bound to a heavy chain regulatory subunit (4f2hc/CD98) via a disulfide bond. The xCT subunit is responsible for the equimolar exchange of extracellular cystine for intracellular glutamate. Clinical studies have shown areas immediately surrounding the tumor, known as the peritumoral region, reach glutamate concentrations over 100 times that of the normal brain, creating an excitotoxic environment in which neurons cannot survive. In addition to neuronal excitotoxicity, excess glutamate release has also been shown to promote GBM cell invasion, as well as contributing to the clinical presentation of seizures in patients. Moreover, cystine is a component of the antioxidant glutathione, which confers protection to the cells from alkylating therapeutics such as temozolomide (TMZ). In an effort to identify novel targets that regulate SXC function, I investigated the relationship between SXC and two signaling molecules known to promote GBM progression: CD44 and the epidermal growth factor receptor (EGFR). I experimentally manipulated the CD44-hyaluronic acid (HA) interaction and EGFR to determine if these two signaling molecules were involved in regulating SXC expression and function in two patient-derived GBM cell lines. Experimental data led me to conclude that the tumorigenic potential conferred to GBM cells by CD44 is not related to an interaction with SXC. However, I found that knocking down EGFR led to a significant reduction in SXC expression. These findings are important to the field, as combinatorial therapies become more actively pursued in clinical trials. Inhibition of EGFR may provide quality of life benefits to patients who suffer from tumor-associated epilepsy through downregulating xCT-mediated glutamate release. / Doctor of Philosophy / Glioblastoma multiforme (GBM) is an advanced and aggressive form of brain cancer. Incidence of this disease in the United States of America is approximately 3.19 per 100,000 individuals, which translates to more than 13,000 expected annual diagnoses. These tumors arise from genetic mutations that instruct cells to replicate and migrate abnormally. Despite an aggressive medical armamentarium that includes maximal surgical resection, chemotherapy, and radiation, GBM patients have an expected survival period of 12-15 months after diagnosis. Previous studies have shown that approximately 50% of GBM patients have unusually high expression levels of the System xc- (SXC) protein. SXC is a protein transporter located at the membrane of GBM cells, and facilitates the exchange of the excitatory neurotransmitter glutamate for the amino acid dimer cystine. SXC exports glutamate out of the tumor cell, where it can then bind to glutamate receptors on surrounding neurons. In the brain, the concentration of extracellular glutamate must be tightly regulated to prevent hyperexcitability of neurons, which may lead to cell death and the induction of seizures. In patients whose tumors highly express SXC, studies have shown that glutamate levels can rise to concentrations over 100 times greater than the levels seen in normal brain tissue. Additionally, glutamate has been shown to stimulate GBM cells to migrate within the brain and establish secondary tumor sites. The medical and scientific community is justifiably interested in discovering novel methods for regulating or inhibiting SXC-mediated glutamate release. While SXC inhibitors have been identified, clinical studies have determined they are not appropriate for the clinical treatment of GBM. Thus the focus of this project was to identify novel molecular regulators of SXC. To that end, I explored two signaling molecules that are known to promote GBM pathogenesis: CD44 and the epidermal growth factor receptor (EGFR). I found no evidence to support a role for CD44 in regulating SXC in GBM. However, I was able to determine, through genetic and pharmacologic manipulation of patient-derived GBM cells, that EGFR regulates SXC expression and function. The results of these experiments confirmed EGFR as a key signaling protein involved in orchestrating SXC-mediated glutamate release, and may inform future clinical studies investigating combinatorial therapies for GBM patients.

Physiological functions of a biomacromolecule seem to be closely related to its molecular conformations. The knowledge of any conformational changes due to changes in its environment may lead to a proper understanding of its functions. Hyaluronic acid, a biomacromolecule with unusually high molecular weight and some important biological functions is the subject of the present work. A temperature-dependent transition in hyaluronate solution of 120 mg/ml concentration was observed at physiological temperature. It is shown that this temperature-dependent behavior can be related to the orientational polarizability term in the Debye theory of polar molecules in liquids.

The fields of tissue engineering, regenerative medicine, and stem cell engineering are rapidly growing. However, these fields must overcome several obstacles before they can make a significant impact on treating cellular disorders. Two major hurdles that must be addressed are: determining how to control the pluripotency of stem cells and developing systems for high-throughput culture of stem cells. The prospect of using a cell source capable of differentiating into cells of any tissue in the body (embryonic stem cells) has received enormous interest in recent years. The pluripotent attribute of embryonic stem cells seems ideal but developing methods to drive embryonic stem cells to specific lineages, including the hematopoietic lineage, is a complex process dependent on multiple intrinsic and extrinsic factors including chemical, cellular, and environmental signaling. With regards to environmental signaling, the use of three-dimensional culture systems such as scaffolds and hydrogels, have been utilized in an attempt to drive lineage-specific differentiation in a synthetic, biomimetic microenvironment. To determine specific environmental factors responsible for hematopoietic differentiation a systematic biological and engineering process must be implemented. A biodegradable hydrogel composed of the hyaluronic acid, a polysaccharide abundant in the bone marrow microenvironment, and the synthetic polymer, poly(ethylene glycol) was formulated to culture mouse embryonic stem cells (mESCs). Photoencapsulation of mESCs did not significantly decrease cellular viability or proliferation. The FACS data was inconclusive however, from gene expression studies, it was determined that the hydrogel culture system promoted differentiation of mESCs as evidenced by a down-regulation of the gene encoding for stem cell maintenance transcription factor, Oct-3/4. Furthermore, embryoid bodies, necessary for in vitro differentiation were observed in the hydrogel systems. Although an increase in the gene encoding for the cell surface marker, c-kit was up-regulated, the surface marker, sca-1 was not up-regulated. Up-regulation of both c-kit and sca-1 is necessary for the development of hematopoietic progenitor cells. Results indicate that the differentiation of mESCs into the hematopoietic lineage was unsuccessful but differentiation in these hydrogel systems did occur. Future cell marker and gene expression studies are necessary to determine which cell lineage the encapsulated mESCs are differentiating into before the effects of incorporating other environmental, cellular, and chemical factors can be investigated. / text

Hyaluronic acid (HA) is one of the major components of the extracellular matrix; and may exhibit different biological functions, dependent on polymer molecular weight (MW). The signalling events performed by HA are mediated through interactions with its main cell receptors: CD44 and RHAMM. However, the direct effect between the HA MW and the expression of CD44 and RHAMM remains unclear. This study aimed to investigate whether different HA polymer MW alters the proliferation of tumour-derived cell lines, and whether different HA-sized has an effect on the regulation of the expression of CD44 and RHAMM. In order to determine size-specific responses of tumour cells of defined fragment MW, this investigation was undertaken using HA-tethered culture surfaces. Four surfaces were constructed, coated with polymers of different MWs. HA (4, 234, 2590 kDa) and an oligomer mixture were tethered onto an aminosilane (AHAPTMS)-treated glass surfaces using a carbodiimide reaction. Surfaces were analysed using a toolbox of in situ characterisation techniques, including wettability measurements, QCM, AFM and confocal microscopy. Using the constructed surfaces was demonstrated that HA-polymer MW modulates cell proliferation of human bladder (RT112 and T24) and prostate (PC3 and PNT1A) cell lines, with low HA MW (HA4) increasing proliferation, whereas a decrease is seen in the presence of medium (HA234) and high MW fragments (HA2590). The proliferation stimulus performed by HA was found to be phenotype dependent, with HA4 surfaces stimulating an increased proliferation in those less invasive cell lines (T24 and PNT1A), while HA234 and HA2590 inducing a sharper decrease in the most malignant tumour cell lines (RT112 and PC3). It was also demonstrated that the regulation of CD44 and RHAMM transcripts expression appears to be phenotype dependent but not HA-MW dependent. HA down-regulates CD44 and RHAMM in the most malignant cell lines; with up-regulation of the expression of the cell receptors in the less invasive cell lines. In addition, the presence of exogenous HA was shown to be involved in the regulation of the expression of CD44 variants expression. The results obtained for the CD44 and RHAMM protein expression were also found to be correlated with the obtained transcripts expression. However, the significance of these findings in tumourigenesis remains unclear. Findings from this investigation may help in the design and development of biocompatible implants with controlled surface properties to be used in cancer therapeutics; with medium and large HA polysaccharides being potential biopolymer candidates, useful for the development of novel therapies for highly invasive cancer. In addition, implications from this work can serve as a base for future research, and can lead to ideas for drugs and methods to be used in cancer therapeutic approaches.

L’administration intraveineuse d’ADN thérapeutique rencontre de nombreux obstacles liés à sa dégradabilité, ainsi qu’à sa difficulté à pénétrer les cellules en raison de sa taille importante et de son hydrophilie. Des lipoplexes conjugués à de l’acide hyaluronique (HA) de haut poids moléculaire ont été développés afin de délivrer de l’ADN plasmidique à l’intérieur de cellules cancéreuses exprimant le récepteur membranaire CD44, récepteur clé du développement tumoral. L’emploi d’HA conjugué au phospholipide DOPE (HA-DOPE) et d’un plasmide modèle GFP a permis d’obtenir des lipoplexes d’environ 250 nm, chargés négativement, protégeant efficacement l’ADN contre les nucléases et activant peu la fraction C3 du système du complément. Dans un modèle cellulaire exprimant CD44, la transfection optimale a été obtenue par l’utilisation de lipides avec 10% d’HA-DOPE complexés à de l’ADN selon un rapport 2:1. Ces lipoplexes sont internalisés par la voie des cavéoles et de façon dépendante du récepteur CD44. Cette formulation a été appliquée à la vectorisation d’un gène thérapeutique, codant pour le récepteur des estrogènes β (ERβ), qui est un potentiel suppresseur de tumeur. Sur un modèle in vivo de xénogreffes de cellules humaines de cancer du sein estrogéno-dépendant et exprimant CD44, la diminution du volume tumoral, ainsi que de l’indice de prolifération Ki67 ont permis de montrer l’effet anticancéreux par voie intraveineuse des lipoplexes conjugués à l’HA. / Intravenous administration of therapeutic DNA faces many obstacles related to its degradability and its difficulty to penetrate into the cells due to its large size and its hydrophilicity. Lipoplexes conjugated with high molecular weight hyaluronic acid (HA) have been designed in order to deliver plasmid DNA inside cancer cells expressing the membrane receptor CD44, a key receptor in the development of tumors. The use of HA conjugated to the phospholipid DOPE (HA-DOPE) and of the GFP model plasmid lead to obtain lipoplexes around 250 nm, negatively charged, which efficiently protect the DNA against nucleases and slightly stimulate the C3 fraction of the complement system. In a cellular model expressing CD44, the optimal transfection was obtained by using lipids containing 10% of HA-DOPE complexed to DNA at a 2:1 ratio. Internalization of these lipoplexes is mediated by the caveolae pathway and involves the CD44 receptor. This formulation was applied to the delivery of a therapeutic gene encoding the estrogen receptor β (ERβ), which is a potential tumor suppressor. On an in vivo xenograft model of estrogen-dependent breast cancer cells expressing CD44, decrease of the tumor volume, as well as decrease of the Ki67 proliferation index, have shown the anticancer activity of the lipoplexes conjugated to HA following intravenous administration.

A ausência ou perda da papila interdental cria deficiência estética, problemas fonéticos, impactação alimentar e gera muita expectativa ao paciente. Até o momento, o tratamento da ausência ou perda da papila interdental tem sido mal sucedido e não há estudos que indiquem que a regeneração da papila é um resultado previsível. O objetivo deste estudo foi avaliar a efetividade da injeção de gel de ácido hialurônico de origem não animal na redução ou eliminação da deficiência de papila entre dentes naturais comparativamente ao tratamento por meio de enxerto de tecido conjuntivo subepitelial. Foram avaliados neste estudo 20 sítios de 6 pacientes de ambos os sexos, com idade variável de 29 a 62 anos, apresentando deficiência de papila entre dentes naturais, na região anterior superior, em pelo menos dois dentes. Os 20 sítios tratados foram aleatoriamente divididos em dois grupos, de acordo com o tratamento para correção da deficiência de papila por meio de enxerto de tecido conjuntivo subepitelial (grupo controle) ou por meio de injeção de gel de ácido hialurônico (grupo teste). Um examinador único, calibrado, avaliou a distância da ponta da papila ao ponto de contato com auxílio de sonda periodontal milimetrada antes e aos 1, 3 e 6 meses após o tratamento. Além disso, foram investigados, nos sítios tratados, as medidas de profundidade de sondagem, nível de inserção, índice de sangramento do sulco, índice de placa, distância do ponto de contato à crista óssea alveolar, distância da ponta da papila à crista óssea alveolar e largura da papila. Os resultados demonstraram que aos 6 meses de pósoperatório o percentual de mudança na altura da papila foi maior no grupo teste (14,94% ± 21,35%) do que no grupo controle (-1,39% ± 31,46%), entretanto sem diferenças significantes entre os grupos (p> 0.05). Não houve variação estatisticamente significante na largura da papila antes e aos 4 meses após o tratamento nos grupos teste (p= 0.09) e controle (p= 0.16), assim como não houve variação significativa na distância entre a ponta da papila e a crista óssea alveolar. Houve melhora significativa do Índice de Estética Rosa (IER) observado aos 6 meses de acompanhamento em comparação com a condição inicial no grupo teste (p= 0.0078; Wilcoxon), enquanto que não houve mudança significativa no IER observado no grupo controle aos 6 meses de acompanhamento (p= 0.35). Os resultados obtidos permitiram concluir que o tratamento da deficiência de papila por meio de injeção de gel de ácido hialurônico promove melhora da deficiência de papila, similar aos resultados obtidos com o tratamento por meio de enxerto de tecido conjuntivo subepitelial, porém com melhora estética significativa relacionada especialmente às características de cor e textura do tecido relativamente aos tecidos moles adjacentes. / The absence or loss of interdental papilla creates an esthetic deficiency, phonetic problems and food impaction and generates a lot of expectation for the patient. Until now, the treatment for absence or loss of interdental papilla is unsuccessful e and there are no researches that show that the papilla regeneration is a predictable outcome. The aim of this study was to evaluate the effectivity of a non-animal originated hyaluronic acid injection in the reduction or elimination of papilla deficiency between natural teeth in comparison to a sub epithelial connective tissue graft treatment. The analysis was made on 20 sites in 6 patients, both genders, 29 - 62 years, showing deficiency in the papilla between natural teeth in the upper anterior region in at least two teeth. The 20 sites treated were randomly divided into two groups, according to the treatment by subepithelial connective tissue graft (control group) or by hyaluronic acid injection (test group). A single calibrated examiner evaluated the distance between the tip of the papilla to the contact point using a graduated periodontal probe before the treatment and 1, 3 and 6 months after it. Besides, it were investigated probing pocket depth, clinical attachment level, gingival bleeding index, plaque index, distance from papilla to alveolar crest, distance from contact point to alveolar crest and width of the papilla. The results showed that 6 months after the procedure, the percentage of change in the papilla level was higher in the test group (14,94% ± 21,35%) than in the control group (-1,39% ± 31,46%), though not statistically significant (p>0.05). There was no significant difference variation in the width of the papilla before and 4 months after the treatment in test group (p=0.09) and control group (p=0.16), and there was no significant difference variation in the distance between the tip of the papilla and the alveolar bone crest. There was significant improvement of the Pink Esthetic Score (PES) after 6 months in comparison to the initial condition in test group (p=0.0078; Wilcoxon), while there was no significant difference in the PES in control group 6 months after treatment (p=0.35). The results allow to conclude that the treatment for of the papilla deficiency using hyaluronic acid injection promotes improvement, similar to the results of the sub epithelial connective tissue graft treatment, but with significant esthetic improvement related specially to the color and texture characteristics of the adjacent soft tissues.

Introdução: a cistite intersticial é doença crônica do trato urinário inferior cujos sintomas são: aumento da freqüência urinária, nictúria, dor pélvica ou perineal que piora com a repleção vesical e melhora com a micção. A etiopatogenia não é totalmente conhecida, mas há indícios de que os glicosaminoglicanos e proteoglicanos que revestem o urotélio vesical possam participar da sua gênese. A perda destes componentes protetores facilitaria o contato de íons e solutos presentes na urina com as porções mais profundas do urotélio desencadeando e perpetuando um processo inflamatório local. Para tentar entender seu metabolismo, investigamos o comportamento dos glicosaminoglicanos na urina e no tecido (biópsia do urotélio vesical) de pacientes portadoras de cistite intersticial e de incontinência urinária de esforço genuína. Casuística e métodos: o perfil e expressão gênica de glicosaminoglicanos no tecido, e o perfil dos glicosaminoglicanos da urina de 11 pacientes com cistite intersticial foram comparados aos de 11 pacientes com incontinência urinária de esforço. A análise estatística foi feita através de teste T e Anova, considerando significativos valores p<0,05. Resultados: verificamos que pacientes com cistite intersticial excretam menor concentração de glicosaminoglicanos na urina do que as portadoras de incontinência urinária de esforço (0,45 ± 0,11 x 0,62 ± 0,13 g/mg creatinina, p<0,05), porém sem redução do conteúdo de glicosaminoglicanos no urotélio. Na imunofluorescência o urotélio de pacientes com cistite intersticial mostrou maior marcação de TGF-beta, decorim (um proteoglicano de condroitim/dermatam sulfato), fibronectina e de ácido hialurônico. Foi identificada menor expressão gênica (PCR em tempo real) das sintases e uma hialuronidase do ácido hialurônico no urotélio das cistites intersticiais. Conclusão: a combinação desses resultados sugere que os glicosaminoglicanos podem estar relacionados ao processo contínuo de inflamação e remodelamento do urotélio disfuncional presente na cistite intersticial. O estudo da expressão gênica pode representar uma altenativa para o entendimento da doença. / Introduction: interstitial cystitis is a chronic disease of the lower urinary tract whose symptoms are: increased urinary frequency, nocturia, perineal or pelvic pain that worses with bladder filling and improves with urination. The pathogenesis is not fully known, but there is evidence that proteoglycans and glycosaminoglycans lining the bladder urothelium can participate in its genesis. The loss of these protective compounds facilitate the contact of ions and solutes in the urine with deeper portions of bladder wall triggering and perpetuating a local inflammatory process. We investigated GAG behavior in urine and tissue (biopsy of bladder urothelium) of patients with IC/PBS and genuine stress urinary incontinence (SUI) in an attempt to better understand its metabolism. Patients and Methods: gene expression and glycosaminoglycans profile in tissue, and glycosaminoglycans profile in urine of 11 patients with interstitial cystitis were compared to 11 patients with pure urinary stress incontinence. Statistical analysis were performed using t Student test and Anova, considering significant when p<0,05. Results: patients with interstitial cystitis excreted lower concentration of glycosaminoglycans in urine when compared to those with pure urinary stress incontinence (respectively 0.45 + 0.11 x 0.62 + 0.13 mg/mg creatinine, p< 0.05). However, there was no reduction of the content of glycosaminoglycans in the urothelium of both patients. The immunofluorescence study showed that patients with interstitial cystitis had a stronger staining of TGF-beta, decorin (a proteoglycan of chondroitin/dermatan sulfate), fibronectin and hyaluronic acid. We were able to indentify by real-time PCR lower gene expression of hyaluronic acid synthases and hyaluronidase in the urothelium of patients with interstitial cystitis. Conclusion: the results suggest that glycosaminoglycans may be related to the ongoing process of inflammation and remodeling of the dysfunctional urothelium that is present in the interstitial cystitis. The study of the gene expression may represent an alternative to understand the disease

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisors: Patricia A. Martin-DeLeon and Deni S. Galileo, Dept. of Biological Sciences. Includes bibliographical references.