Platinum-based chemotherapeutics are used for the treatment of a wide range of cancers and a number of attempts have been made toward developing compounds with better cellular stability and similar or enhanced cytotoxicity as compared to their predecessors. The first part of the work reported here focuses on the cellular effects of the metabolically stable dinuclear platinum compound, BBR3610-DACH. Comet assay showed this compound to form interstrand crosslinks, a highly toxic DNA lesion in HCT116 cells, at equimolar concentrations to its parental compound, BBR3610. Cell cycle studies showed that BBR3610-DACH causes G1/S and G2/M cell cycle arrest with S phase depletion, which was p21 dependent and partially p53 dependent in contrast to BBR3610 which showed initial S phase accumulation followed by a classical G2/M arrest. BBR3610-DACH-induced G1/S and G2/M cell cycle arrest interestingly was found to be independent of the DNA damage response mediated via the activation of ATM and ATR kinases. Also, the cell cycle arrest culminated in apoptosis, although apparently through a non-canonical pathway. The second project explores the cellular effects of trans-4-NBD which is a fluorescent derivative of transplatin. Like cisplatin, trans-4-NBD induced interstrand crosslinks in HCT116 cells as detected by the comet assay. Treatment with trans-4-NBD showed a G2/M arrest in HCT116 cells and a transient S phase accumulation in A2780 cells, with a marked increase in p53 and p21 protein levels. A robust apoptotic response is also seen via caspase activation and PARP cleavage in both the cell lines. Finally, the focus is shifted toward the nucleolar targeting platinum complex, TriplatinNC. Confocal studies in TriplatinNC-treated HCT116 and A2780 cells showed disruption of rRNA transcription as an early event followed by a robust G1 cell cycle arrest. Apoptotic induction was observed with the onset of cellular morphological changes and apparent caspase activation which was independent of the p53 status of the cells. Overall, these studies explore novel platinum based compounds that show promising anti-cancer activities by affecting various facets of cellular signaling.

Hereditary equine regional dermal asthenia (HERDA) is a recessive connective tissue disorder of Quarter Horse lineages. This study correlates previously identified decreases in skin tensile strength in HERDA with abnormal dermal collagen cross linking patterns that are also identified in urine from HERDA horses. Dermal collagen from HERDA horses has significantly less pyridinoline and significantly more deoxypyridinoline than control or carriers. Concentrations of hydroxylysine, the rate limiting substrate for these crosslinks were significantly lower in HERDA versus control and carriers. These characteristics of HERDA skin parallel humans with a similar syndrome of skin fragility, Ehlers Danlos Syndrome TypeVIA. This is the first biochemical evidence explaining the clinical skin fragility that characterizes HERDA and suggests that altered collagen lysine metabolism may be physiologically relevant to the clinical manifestation of HERDA. Evaluations of mature scars indicate that lesion and nonlesioned skin should not be viewed as biologically equivalent in HERDA investigations.

No projeto desenvolvido, ligações cruzadas foram formadas no polietileno de alta densidade e alto peso molecular, grade HS5103, através dos processos reticulação por peróxidos e silanos, com o objetivo de se avaliar o efeito da introdução de ligações cruzadas nas propriedades térmicas e mecânicas deste PEAD. Misturas entre o HS5103 e os peróxidos orgânicos, 2,5-Dimetil-2,5-di(terc-butilperoxi)hexano (DHBP) e peróxido de dicumila (DCP), foram produzidas e analisadas para a avaliação do efeito dos tipos de peróxido na reticulação e propriedades do PEAD e para determinação da concentração e do tipo de peróxido a ser utilizado como agente iniciador de reticulação por silano. Ensaios de índice de fluidez (MFI), reometria capilar, extração de polímero por xileno (teor de gel), análise termogravimétrica (TGA), calorimetria diferencial de varredura (DSC) e tração foram realizados para caracterização das misturas com peróxidos. Os resultados indicaram aumento da viscosidade com o aumento da concentração de peróxido, sendo o DHBP o que apresentou maior índice de aumento; não houve mudanças relevantes nas propriedades mecânicas e, ocorreu aumento do grau de cristalinidade, sendo mais significativo nas amostras com DCP. Após avaliação dos resultados citados, para as amostras a serem reticuladas via silano, foi promovida a graftização de diferentes concentrações de viniltrimetóxisilano (VTMS) na presença de 0,01%p/p de DCP com a adição de 0,05%p/p de catalisador, posteriormente a reticulação foi promovida em água. As amostras produzidas foram caracterizadas por ensaios de teor de gel, análise dinâmico-mecânica (DMA), espectroscopia de absorção no infravermelho (FTIR), TGA, ensaios de desgaste por deslizamento e tração. Nas amostras com silano a formação de ligações cruzadas foi gradativa, apresentando de 8% de gel para amostra com 0,5%p/p de VTMS a 57 % para amostra com1,5% p/p de silano, maior concentração utilizada. A análise dinâmico-mecânica (DMA) realizada evidenciou que houve um aumento densidade de ligações cruzadas e do módulo de armazenamento após temperatura de fusão com o teor de silano, concordando com os resultados de teor de gel. As análises de FTIR mostraram que houve a graftização e a formação de ligações cruzadas no PEAD HS5103. Não se observou um aumento significativo para o limite de resistência para o PEAD modificado. E os testes de desgaste por deslizamento indicaram um aumento da resistência ao desgaste das amostras reagidas com VTMS. / In the project carried out, crosslinks were formed in high density and high molecular weight polyethylene, grade HS5103, through peroxide and silane reticulation processes, aiming to evaluate the effects of the introduction of crosslinks in thermal and mechanical properties of this PEAD. Mixtures between HS103 and organic peroxides, 2,5-Bis(t-butylperoxy)-2,5-dimethylhexane (DHBP) and dicumyl peroxide (DCP) have been produced and analyzed for the evaluation of the effect of types of peroxide in reticulation and PEAD properties and to determine both concentration and type of the peroxide to be used as silane reticulation initiation agent. Melt flow index (MFI), capillary rheometry, xylem polymer extraction (gel content), thermogravimetric analysis (TGA), differential canning calorimetry (DSC) and traction tests were carried out for peroxide mixtures characterization. The results indicated increase of viscosity with the increase of the concentration of peroxide, being DHBP the one with the highest increase level; there have been no relevant changes concerning mechanical properties and there has been an increase in the crystallinity levels, mostly in DCP samples. After the assessment of the former results, for the samples to be reticulated through silane, graphitization of different concentrations of Vinyltrimethoxysilane (VTMS) was carried out, together with 0.01%p/p of DCP, adding 0.05%p/p of a catalyst; later, reticulation was carried out in water. The samples produced were characterized by gel content tests, dynamic-mechanical analysis (DMA), Fourier transform infrared spectroscopy (FTIR), TGA, sliding and traction wear tests. In the silane samples, formation of crosslinks was gradual, showing 8% of gel to the sample with com 0.5%p/p of VTMS to 57 % to the sample with 1.5% p/p of silane, the highest concentration used. The dynamic-mechanical analysis (DMA) showed an increase of density in crosslinks and storing mode after fusion temperature with silane content, converging with gel content results. FTIR analysis showed there has been graphitization and crosslink formations in PEAD HS5103. A significant increase has not been observed concerning the limit of resistance for the modified PEAD. Besides, sliding wear tests indicated an increase of resistance to the wear of samples reacted to VTMS.

No projeto desenvolvido, ligações cruzadas foram formadas no polietileno de alta densidade e alto peso molecular, grade HS5103, através dos processos reticulação por peróxidos e silanos, com o objetivo de se avaliar o efeito da introdução de ligações cruzadas nas propriedades térmicas e mecânicas deste PEAD. Misturas entre o HS5103 e os peróxidos orgânicos, 2,5-Dimetil-2,5-di(terc-butilperoxi)hexano (DHBP) e peróxido de dicumila (DCP), foram produzidas e analisadas para a avaliação do efeito dos tipos de peróxido na reticulação e propriedades do PEAD e para determinação da concentração e do tipo de peróxido a ser utilizado como agente iniciador de reticulação por silano. Ensaios de índice de fluidez (MFI), reometria capilar, extração de polímero por xileno (teor de gel), análise termogravimétrica (TGA), calorimetria diferencial de varredura (DSC) e tração foram realizados para caracterização das misturas com peróxidos. Os resultados indicaram aumento da viscosidade com o aumento da concentração de peróxido, sendo o DHBP o que apresentou maior índice de aumento; não houve mudanças relevantes nas propriedades mecânicas e, ocorreu aumento do grau de cristalinidade, sendo mais significativo nas amostras com DCP. Após avaliação dos resultados citados, para as amostras a serem reticuladas via silano, foi promovida a graftização de diferentes concentrações de viniltrimetóxisilano (VTMS) na presença de 0,01%p/p de DCP com a adição de 0,05%p/p de catalisador, posteriormente a reticulação foi promovida em água. As amostras produzidas foram caracterizadas por ensaios de teor de gel, análise dinâmico-mecânica (DMA), espectroscopia de absorção no infravermelho (FTIR), TGA, ensaios de desgaste por deslizamento e tração. Nas amostras com silano a formação de ligações cruzadas foi gradativa, apresentando de 8% de gel para amostra com 0,5%p/p de VTMS a 57 % para amostra com1,5% p/p de silano, maior concentração utilizada. A análise dinâmico-mecânica (DMA) realizada evidenciou que houve um aumento densidade de ligações cruzadas e do módulo de armazenamento após temperatura de fusão com o teor de silano, concordando com os resultados de teor de gel. As análises de FTIR mostraram que houve a graftização e a formação de ligações cruzadas no PEAD HS5103. Não se observou um aumento significativo para o limite de resistência para o PEAD modificado. E os testes de desgaste por deslizamento indicaram um aumento da resistência ao desgaste das amostras reagidas com VTMS. / In the project carried out, crosslinks were formed in high density and high molecular weight polyethylene, grade HS5103, through peroxide and silane reticulation processes, aiming to evaluate the effects of the introduction of crosslinks in thermal and mechanical properties of this PEAD. Mixtures between HS103 and organic peroxides, 2,5-Bis(t-butylperoxy)-2,5-dimethylhexane (DHBP) and dicumyl peroxide (DCP) have been produced and analyzed for the evaluation of the effect of types of peroxide in reticulation and PEAD properties and to determine both concentration and type of the peroxide to be used as silane reticulation initiation agent. Melt flow index (MFI), capillary rheometry, xylem polymer extraction (gel content), thermogravimetric analysis (TGA), differential canning calorimetry (DSC) and traction tests were carried out for peroxide mixtures characterization. The results indicated increase of viscosity with the increase of the concentration of peroxide, being DHBP the one with the highest increase level; there have been no relevant changes concerning mechanical properties and there has been an increase in the crystallinity levels, mostly in DCP samples. After the assessment of the former results, for the samples to be reticulated through silane, graphitization of different concentrations of Vinyltrimethoxysilane (VTMS) was carried out, together with 0.01%p/p of DCP, adding 0.05%p/p of a catalyst; later, reticulation was carried out in water. The samples produced were characterized by gel content tests, dynamic-mechanical analysis (DMA), Fourier transform infrared spectroscopy (FTIR), TGA, sliding and traction wear tests. In the silane samples, formation of crosslinks was gradual, showing 8% of gel to the sample with com 0.5%p/p of VTMS to 57 % to the sample with 1.5% p/p of silane, the highest concentration used. The dynamic-mechanical analysis (DMA) showed an increase of density in crosslinks and storing mode after fusion temperature with silane content, converging with gel content results. FTIR analysis showed there has been graphitization and crosslink formations in PEAD HS5103. A significant increase has not been observed concerning the limit of resistance for the modified PEAD. Besides, sliding wear tests indicated an increase of resistance to the wear of samples reacted to VTMS.

Das Verständnis natürlicher biologischen Materialien für die Entwicklung neuer biomimetischer Materialien ist von großem Interesse in der Chemie und den Materialwissenschaften. In vielen komplexen biomolekularen Materialien ist die Etablierung der Struktur-Funktionsbeziehungen von Proteinbausteine notwendig, um die Eigenschaften der daraus aufgebauten weichen, biologischen Materialien zu verstehen, wie z. B. die extrazelluläre Matrix. Inspiriert durch bekannte Faltungsmotive von ECM-Proteinen, wurden vereinfachte Modellpeptide entwickelt, um deren Funktion zu untersuchen oder diese als biomimetische Bausteine für synthetische Biomaterialien zu verwenden. Ziel dieser Arbeit war die Synthese von hybriden Hydrogelen, die aus einem synthetischen Polymer und ECM-inspirierten Modellpeptiden zusammengesetzt sind. Insbesondere Kollagen-mimetische Peptide und Coiled-Coil-formende Peptide wurden benutzt, um das biokompatible und hydrophile Polymer Polyethyleneglykol zu vernetzen. Dabei wurde von der Fähigkeit dieser Peptide zur dynamischen Selbstassemblierung Gebrauch gemacht. Unter Verwendung von Kollagen-mimetischen Peptiden mit langsamer Dissoziationskinetik wurden Hydrogele synthetisiert, die weichen, glasartigen Materialien mit einem gestauchten exponentiellen Relaxationsverhalten entsprechen und auch einen Alterungsprozess zeigen. Darüber hinaus wurde gezeigt, dass Netzwerkkonnektivität ein bis dato selten gebrauchter Designparameter ist, um die rheologischen Eigenschaften von Hydrogelen nach Wunsch zu kontrollieren. Die Kombination molekular einstellbarer Vernetzer mit einem Fluoreszenz-Reportersystem, welches deren Zustand auslesen kann, kann detaillierte Einblicke in das Reaktionsvermögen solcher Netzwerke auf mechanische Stimuli ermöglichen. Das Verständnis molekularer Prozesse erlaubt langfristig die Synthese von ECM-inspirierten Biomaterialien, deren Eigenschaften nach Wunsch einstellbar sind und die selbst ihren mikroskopischen und mesoskopischen Zustand anzeigen. / Understanding natural biological materials for the development of novel biomimetic materials has drawn enormous attention from the areas of chemistry and material science. In many complex biomolecular materials, establishing molecular structure-function relationships of proteins forms the basis for understanding the emerging properties of various biological soft materials, such as the extracellular matrix (ECM). Inspired by common association motifs of ECM proteins, simplified model peptides have been developed for functional studies and as biomimetic building blocks for synthetic biomaterials. The aim of this thesis was to utilize ECM-inspired and molecularly controlled model peptides for the synthesis of peptide-polymer hybrid hydrogels. Specifically, collagen-mimetic peptides (CMPs) and coiled coil (CC)-forming peptides were utilized to crosslink the biocompatible and hydrophilic polymer poly(ethylene glycol) (PEG), making use of the ability of these peptides to dynamically self-assemble. Employing CMPs with slow dissociation kinetics, hydrogels have been synthesized that resemble soft glassy materials with compressed exponential relaxation and aging. Furthermore, network connectivity has been shown to be an underutilized design parameter for tuning the rheological properties of hydrogels. Combining molecularly controlled crosslinks with a fluorescence reporter system that allows to read out crosslink status will ultimately allow for more detailed insights into the response of such networks to mechanical perturbation and thus aid the synthesis of ECM-inspired biomaterials with tunable and self-reporting properties.