Background and Aims: During positive energy balance, excess lipid storage in subcutaneous adipose tissue (SAT) is associated with increased lipolysis. Elevated circulating fatty acid (FA) concentrations from both SAT lipolysis and dietary fat intake may result in visceral adipose tissue (VAT) accumulation, impairment of glucose metabolism, altogether increasing obesity-associated metabolic risks. We aimed to test the hypothesis that FA composition of red blood cell total phospholipids (RBC-TPL) and SAT is associated with body fat centralisation (VAT/SAT ratio) and insulin sensitivity (SI) in black South African women with obesity. Methods: Participants’ (n = 41) body fat composition and distribution, SI, and RBC-TPL, abdominal and gluteal SAT (gSAT) FA composition (gas-liquid chromatography) were measured. Results: RBC-TPL contained higher proportions of saturated fatty acids (SFAs) than SAT (p < 0.001), which were associated with lower SI (p < 0.05). Mono-unsaturated fatty acids (MUFAs) and stearoyl-CoA desaturase-1 (SCD1)-16 were lower, while poly-unsaturated fatty acids (PUFAs), and delta-5 and delta-6 desaturase indices were higher in RBC-TPL than SAT (p < 0.001). Interestingly, FA profiles differed between SAT depots with higher SFAs and lower MUFAs, SCD1-16 and SCD1-18 indices in abdominal compared to gluteal SAT (p < 0.01). In both SAT depots, higher SFAs and lower PUFAs (n-3 and n-6) correlated with lower VAT/SAT ratio; and lower PUFAs (n-3 and n-6) and higher total MUFA correlated with higher SI. Conclusion: Our findings confirm the relationships between the FA composition of RBC-TPL and SAT and metabolic risk in black women with obesity, which are dependent on both the FA class, and the tissue type/blood compartment in which they are distributed.

<p>Biotherapeutics, such as monoclonal antibodies (mAbs), represent a primary mechanism for treatment of human disease, and there has been a steady increase in Food and Drug Administration approvals since the first one in 1982. Subcutaneous (SC) injection of protein-based therapeutics is a convenient and clinically established drug delivery method that increases the convenience and reduces cost compared to other delivery methods. However, progress is needed to optimize bioavailability via this route. This dissertation describes the methods for evaluation of mass transport of high molecular weight proteins, such as mAbs, following SC injection using <em>in vitro</em> and <em>ex vivo</em> modeling developed to describe the factors relevant for optimal distribution prior to uptake into systemic circulation. The first chapter describes a novel collagen and hyaluronic acid (HA) based hydrogel for investigation of macromolecule transport based on the physiochemical properties of the diffusing molecule and the tissue matrix. This initial study demonstrated that, in collagen alone, collagen combined with HA, and HA alone, the molecules demonstrated different transport paradigms dependent primarily on molecule size, matrix viscosity, and electrostatic charge, respectively. This showed that the local tissue heterogeneity and therapeutic properties could be determining factors for molecule transport and bioavailability. The second, third, and fourth chapters describe two novel platforms for the investigation of injection plume formation in SC tissue utilizing three-dimensional X-ray tomography. Injection plume analysis has been studied comprehensively in the context of insulin transport using co-injection of radiopaque dyes to track the protein distribution. However high molecular weight therapeutics have vastly different physiochemical properties than insulin and are injected under different rates, concentrations, volumes, and viscosities due to dosing considerations. To address the gap mAb distribution, we first developed a novel protein conjugated to an x-ray contrast agent to directly track injection plume formation and investigated the effects of injection rate and tissue location through injections into ex vivo porcine tissue, described in chapters three and four. Ex vivo tissue analysis showed that the rate did not influence the distribution, however, plume volume was lower in porcine belly compared to neck tissue. Whereas porcine tissue is an excellent model to represent the structural features of human injection, the large heterogeneity between animal subjects and collected samples is a disadvantage. Therefore, the fourth chapter describes the fabrication of a gelatin hydrogel-based injection platform representing the dermal and subcutaneous tissue layers for controlled injection plume analysis. In summary, all three models represent useful platforms for the assessment of macromolecular mass transport, pharmaceutical autoinjector performance, as well as the potential impact of tissue properties and intersubject heterogeneity on plume formation. Overall, the findings in these studies might better inform drug designers and clinicians on how to most optimally engineer an injection to deliver the most efficient patient outcomes through better dosing and increased cost savings. </p>

<p>The overall time and cost for a drug to go from the drug discovery to the consumer market is  significant,  showing  a  need  for  improved  drug  testing  and  discovery  methods.    Work  on nonlinear  optical  methods  for  both  small  active  pharmaceutical  ingredient  drug  formulation analysis and large biological therapeutic stability testing has been shown to improve testing times for formulation, stability and dissolution testing.  Herein, we review the existing and conventional approaches to address stability testing that the pharmaceutical industry uses, and how leveraging nonlinear optical (NLO) methods can improve the current challenges.  The specificity, sensitivity and low limit of detection of second harmonic generation is discussed in application to crystal formation in small-molecule active pharmaceutical ingredients.  The nonlinear optical methods second harmonic generation and two-photon excited ultraviolet fluorescence are directly compared to  ‘gold  standard’  powder  X-ray  diffraction,  which  is  commonly  used  for  measuring  crystal formation and growth of active pharmaceutical ingredients in amorphous solid dispersions.  In addition, the existing FRAP method (with multiple limitations) is improved upon with the ability to  perform  recovered  diffusion  coefficient  data  analysis  in  the  spatial  Fourier  domain.    The collective results discussed in this thesis are just a small subset of the total breadth of investigations marrying the new challenges in the pharmaceutical industry with the new NLO tools tailored to meet them</p>

<p dir="ltr">Subcutaneous injection for self-administration of biotherapeutics, such as monoclonal antibodies (mAbs), is becoming increasingly prominent within the pharmaceutical sector due to its benefits in patient compliance and cost-effectiveness. The success of this drug delivery process depends on the coupled mechanical and transport phenomena within the subcutaneous tissue, both during and after the injection. Yet, the details of these processes are not well-elucidated, sparking a surge in computational efforts to fill this knowledge gap. Remarkably, there are very few computational studies on subcutaneous injection into three-dimensional porous media that account for large tissue deformations, drug transport and absorption, the use medical devices, and human factors. Here, we develop a high-fidelity computational framework to study large-volume subcutaneous injection of mAbs. Our investigation begins with a linear poroelastic model without drug transport, which we employ to study the effect of tissue deformation on injection dynamics. We progressively enhance this model, advancing to a nonlinear porohyperelastic framework that include drug transport and absorption. To capture the anisotropy of subcutaneous tissue, we employ a fibril-reinforced porohyperelastic model. Furthermore, we integrate the multi-layered structure of skin tissue by creating data-driven geometrical models of the tissue layers derived from histological data. Our analysis explores the impact of different handheld autoinjectors on the injection dynamics for various patient-applied forces. We investigate the effect of different pre-injection techniques, such as the pinch and stretch methods, on the drug transport and absorption. Additionally, we evaluate the impact of several physiological variables, including flow rate, injection depth, and body mass index. Our simulations yield crucial insights essential for comprehending and improving subcutaneous drug administration of mAbs. Additionally, they offer a deeper understanding of the human aspect of the injection procedure, thereby paving the way for advancements in the development of patient-centered injection devices and techniques.</p>

Las técnicas ablativas basadas en energía por radiofrecuencia (RF) se emplean con el fin de lograr un calentamiento seguro y localizado en el tejido biológico. En los últimos años ha habido un rápido crecimiento en el número de nuevos procedimientos médicos que hacen uso de dichas técnicas, lo cual ha ido acompañado de la aparición de nuevos diseños de electrodos y protocolos de aplicación de energía. Sin embargo, existen todavía muchas incógnitas sobre el verdadero comportamiento electro-térmico de los aplicadores de energía, así como de la interacción energía-tejido en aplicaciones concretas. El principal propósito de esta Tesis Doctoral es adquirir un mejor conocimiento de los fenómenos eléctricos y térmicos involucrados en los procesos de calentamiento de tejidos biológicos mediante corrientes de RF. Esto permitirá, por un lado, mejorar la eficacia y seguridad de las técnicas actualmente empleadas en la clínica en campos tan diferentes como la cirugía cardiaca, oncológica o dermatológica; y por otro, sugerir mejoras tecnológicas para el diseño de nuevos aplicadores. La Tesis Doctoral combina dos metodologías ampliamente utilizadas en el campo de la Ingeniería Biomédica, como son el modelado computacional (matemático) y la experimentación (ex vivo e in vivo). En cuanto al área cardiaca, la investigación se ha centrado, por una parte, en mejorar la ablación intraoperatoria de la fibrilación auricular por aproximación epicárdica, es decir, susceptible de ser realizada de forma mínimamente invasiva. Para ello, se ha estudiado mediante modelos matemáticos un sistema de medida de la impedancia epicárdica como método de valoración de la cantidad de grasa previo a la ablación. Por otra parte, se ha estudiado cómo mejorar la ablación de la pared ventricular por aproximación endocárdica-endocárdica (septo interventricular) y endocárdica-epicárdica (pared libre del ventrículo). Con este objetivo, se han comparado mediante modelado por computador la eficacia de los modos de ablación bipolar y unipolar en términos de la transmuralidad de la lesión en la pared ventricular. En lo que respecta al área de cirugía oncológica, la investigación se ha centrado en la resección hepática asistida por RF. Las técnicas de calentamiento por RF deberían ser capaces de minimizar el sangrado intraoperatorio y sellar vasos y ductos mediante la creación de una necrosis coagulativa por calentamiento. Si este calentamiento se produce en las cercanías de grandes vasos, existe un problema potencial de daño a la pared de dicho vaso. En este sentido, se ha evaluado con modelos matemáticos y experimentación in vivo si el efecto del flujo de sangre dentro de un gran vaso es capaz de proteger térmicamente su pared cuando se realiza una resección asistida por RF en sus cercanías. Además, se ha realizado un estudio computacional y experimental ex vivo e in vivo del comportamiento electro-térmico de aplicadores de RF bipolares internamente refrigerados, puesto que representan una opción más segura frente a los monopolares en la medida en que las corrientes de RF fluyen casi exclusivamente por el tejido biológico situado entre ambos electrodos. Respecto al área dermatológica, la investigación se ha centrado en mejorar el tratamiento de enfermedades o desórdenes del tejido subcutáneo (tales como lipomatosis, lipedema, enfermedad de Madelung y celulitis) mediante el estudio teórico de la dosimetría correcta en cada caso. Para ello, se han evaluado los efectos eléctricos, térmicos y termo-elásticos de dos estructuras diferentes de tejido subcutáneo durante el calentamiento por RF, y se ha cuantificado el daño térmico producido en ambas estructuras tras dicho calentamiento / González Suárez, A. (2014). Computational Models and Experimentation for Radiofrequency-based Ablative Techniques [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/36502

<p dir="ltr">Biopharmaceuticals, such as insulin, monoclonal antibodies, growth hormones, and vaccines, have emerged as a major class of therapeutic molecules. Subcutaneous administration of biotherapeutics is a convenient drug delivery method that is less invasive, requires shorter clinic times, improves patient compliance, and reduces cost to the healthcare system compared to intravenous administration. The mass transport of a therapeutic injected into the subcutaneous tissue is dictated by physiochemical properties of the molecule such as size and electrostatic charge. The bioavailability and efficacy of the therapeutic formulation depend on efficient transport of the molecule from the injection site to lymphatic or blood vessels. The injected biotherapeutic needs to traverse complex structures of the subcutis and the extracellular matrix (ECM) before it arrives at the uptake site. In vitro transport screening platforms provide insights into the effects of tissue and therapeutic properties on macromolecular transport through biological barriers.</p><p dir="ltr">In this work, we develop an in vitro Transwell macromolecular recovery platform, an economical and high-throughput method that can be used to systematically evaluate effects of ECM components on mass transport properties of macromolecules. In Chapters 2-3, we engineer subcutaneous tissue models based on collagen type I ((Col I), the most abundant fibrillar protein in the subcutaneous ECM) and hyaluronic acid ((HA), an anionic and highly viscous polysaccharide). In Chapter 2, we optimize protocols to reproducibly fabricate Col I and combined Col I and HA (ColHA) hydrogels. In Chapter 3, we establish a workflow to characterize collagen material from different sources (animal sources, different vendors, and between batches of identical material) since inherent variabilities can occur.</p><p dir="ltr">Next, we develop and optimize a high throughput Transwell platform, and we screen the transport of macromolecules, which are representative of current therapeutics used in subcutaneous injections. We demonstrate that macromolecular transport within Col type I (Col I), blended collagen I and II (Col I/II), blended Col I and III (Col I/III), and combined Col I and HA hydrogels (ColHA) hydrogels is inversely related to the hydrodynamic radius of the diffusing macromolecules. Blending col I/II and I/III gels results in altered fibril morphologies (smaller fibrils), which decrease mass recovery rates. Increasing HA concentration within the Col I hydrogels decreases macromolecular recovery. This decrease is mainly a consequence of increased viscosity within the matrix. Recovery rates of large molecules such as immunoglobulin G (IgG), a molecule similar in size to therapeutic antibodies, were highly sensitive to HA concentration in col hydrogels. Smaller molecules, such as myoglobin and lysozyme, that are similar in size to insulin experience electrostatic effects as HA concentration increases within col gels. Recovery of macromolecules in an HA solution was a function of both electrostatic and steric interactions. The results from these studies were highly reproducible and highlighted the robustness of the optimized assay.</p><p dir="ltr">Our results thus demonstrate that the Transwell platform can be utilized for systematic evaluation of therapeutic transport as a function of molecular characteristics. The results presented can inform desirable physiochemical properties for efficient biotherapeutic transport within the subcutaneous tissue. </p><p dir="ltr">In the last main portion of the thesis, we work with elastin, another biologically derived material. In this portion, we developed an optimized method for expression and purification of elastin-like polypeptide proteins. We then present a method to chemically alter the material to introduce underwater adhesive properties to the material.</p>

Hipodermóclise (HDC) é uma importante técnica alternativa para a administração de medicamentos e fluidos pela via subcutânea. É usada com frequência para o controle dos sintomas em pacientes em cuidados paliativos com dificuldade de acesso venoso e que são incapazes de tolerar medicação oral. No entanto, raros estudos abordaram o uso da HDC de uma forma global, para reposição hidroeletrolítica e terapia medicamentosa, tanto na forma contínua quanto intermitente, observando detalhes e complicações do seu uso. Os objetivos deste estudo incluíram caracterizar o uso da HDC para administração de medicamentos, soluções e eletrólitos e avaliar as possíveis complicações locais, identificando também outros fatores que influenciam sua ocorrência. Estudo observacional prospectivo com coleta de dados em prontuário e acompanhamento diário de pacientes internados com câncer avançado, da equipe de Cuidados Paliativos do Instituto do Câncer do Estado de São Paulo (ICESP) em uso de HDC, verificando local de punção, medicamentos administrados e possíveis complicações, acompanhando os detalhes de seu uso. A análise estatística não-paramétrica e método de regressão logística foram realizados. Foram acompanhados 99 pacientes com 243 punções, das quais 166 (68,3%) em coxa e 46 (18,9%) em abdome. Os medicamentos mais utilizados foram morfina em 122 (50,2%) punções, seguido de dipirona em 118 (48,6%) e dexametasona em 86 (35,4%). A solução mais prescrita foi a glicofisiológica em 38 (15,6%) punções, pelo seu aporte calórico. 13,6% das punções (33 de 243) tiveram complicações, sendo apenas seis casos maiores (edema). Complicações ocorreram mais frequentemente até o segundo dia da punção e foram associadas com o número (p=0,007) e o volume (p=0,042) de medicamentos administrados e também com a solução glicofisiológica (p=0,003) e os eletrólitos cloreto de potássio (p=0,037) e cloreto de sódio (p=0,013). Este estudo permitiu o conhecimento de fatores associados a complicações e propõe algumas recomendações, como: individualização da terapia, especialmente relacionada com o volume de escolha, número de medicamentos administrados e evitar a adição de eletrólitos na solução glicofisiológica / Hypodermoclysis (HDC) is an important alternative technique for the administration of drugs and fluids into the subcutaneous tissue. It is frequently used for symptom control in palliative care patients, with difficult venous access and unable to tolerate oral medications. However, few studies address the use of HDC as a whole to fluid replacement and drug therapy, both continuous and intermittent mode, observing details and complications of its use. The objectives of this study included characterizing the use of HDC to administer drugs, solutions and electrolytes and to evaluate possible local complications also identifying other factors influencing their occurrence. Prospective observational study with data collection in medical records and daily monitoring of advanced cancer inpatients of the palliative care team of São Paulo State Cancer Institute (ICESP) in use of HDC, checking infusion site, administered drugs and possible complications, following the details of its use. Non-parametrical statistical analysis and logistic regression were performed. Were followed 99 patients with 243 infusion sites, which 166 (68.3%) in the thigh and 46 (18.9%) in the abdomen. The most commonly used drugs were morphine in 122 (50.2%) infusion sites, followed by dipyrone in 118 (48.6%) and dexamethasone in 86 (35.4%). The most prescribed solution was dextrose 5%/0,9% saline in 38 (15.6%) infusion sites because of its caloric intake. 13.6% of punctures (33 of 243) had complications, only six larger cases (edema). Complications occurred mainly up to the second day of the infusion sites and were associated with the number (p=0,007) and volume (p=0,042) of drugs used as also with 5% dextrose/0.9% saline solution (p=0,003) and NaCl (p=0,037) and KCl (p=0,013) electrolytes. This study has allowed the knowledge of factors associated with complications and proposes some recommendations as: individualization of therapy especially related to the volume of choice, number of drugs administered, and avoid adding electrolytes to the 5% dextrose/0.9% saline solution

Diabetes mellitus tipo 2 (DM2) está associado a um estado de inflamação crônica e ativação do estresse do retículo endoplasmático (ERE). Nesse contexto, são necessários estudos para encontrar alternativas que melhorem o quadro inflamatório, como os ácidos graxos poli-insaturados ômega 3 (n-3 PUFA), um conhecido agente anti-inflamatório. Esse estudo teve por objetivo avaliar o efeito da suplementação do ácido alfa-linolênico (ALA, um n-3 PUFA) no estresse do retículo endoplasmático e no estado inflamatório no tecido adiposo subcutâneo abdominal (TASC) em pacientes com DM2. Foi conduzido um estudo duplo-cego, prospectivo, placebo-controlado. Vinte pacientes com DM2 foram randomizados para suplementação com 3g/dia de ALA ou placebo durante 60 dias. O tecido adiposo foi coletado através de punção aspirativa por agulha fina do abdome antes e após a suplementação e os genes e proteínas foram avaliados através de PCR em tempo real e western blot. Foi encontrada, após suplementação, uma redução da expressão gênica do XBP1 (20%), sXBP1 (70%) e aumento da expressão gênica do GRP78 (150%), confirmado na expressão proteica. Além disso, foi encontrado aumento da expressão gênica da adiponectina (90%) e redução da expressão gênica do IL-6 (80%) e IRS-1 (60%), sem correlação com a expressão proteica, no tempo pós-suplementação com ALA. Portanto, foi demonstrado que o ALA pode modular o ERE através da via da IRE1/XBP, levando ao aumento das chaperonas (BIP/GRP78), além de um efeito adicional na expressão gênica da adiponectina, IL-6 e IRS-1, o que pode demonstrar um potencial terapêutico do ALA em pacientes com DM2. / Type 2 diabetes mellitus (T2DM) is a state of chronic inflammation and activation of endoplasmic reticulum stress (ERS). In this context, studies are necessaries to find new possibilities to improve this inflammation such as the n-3 polyunsaturated fatty acid (n-3 PUFA) acting as an anti-inflammatory agent. In this study, we aimed to evaluate the effect of n-3 PUFA alpha-linolenic acid (ALA, a n-3 PUFA) supplementation in T2DM patients on the molecular expression of ERS genes in abdominal subcutaneous adipose tissue (SAT). We performed a placebo-controlled study, in a double-blind design with 20 T2DM patients, receiving, randomly, 3g/day of ALA or placebo for 60 days. The adipose tissue was collected by fine-needle aspiration in abdomen before and after the supplementation and the genes and proteins were evaluated by real-time PCR and western blot. It was seen, after the supplementation, a reduction in XBP1 (20%), sXBP1 (70%) and an increase in Grp78 (150%) gene expression, likewise same results in protein concentration. Furthermore, it was observed an increase in adiponectina (90%) gene expression and reduction in IL-6 (80%) and IRS-1 (60%) gene expression, with no correlation to protein expression after supplementation of ALA. Therefore, we have provided evidence that ALA may modulate ERS by the IRE1/XBP pathway leading to an increase in chaperones (BIP/GRP78), additionally its effect on adiponectina, IL-6 and IRS-1 gene expression can demonstrate a therapeutic potential in T2DM.

Desde o início de 1990, a enxertia de gordura na prega vocal é descrita como um método para reparar a insuficiência glótica. O objetivo deste estudo é avaliar os efeitos da aplicação tópica de mitomicina-C no processo cicatricial de enxertos autólogos de gorduras inseridos em pregas vocais de coelhos através da medida da deposição de colágeno. Vinte e oito coelhos foram submetidos a enxertia de gordura em ambas pregas vocais. As pregas vocais direitas recebeu previamente a aplicação tópica de mitomicina-C (0,4mg/ml) durante cinco minutos enquanto que as pregas vocais esquerdas formavam o grupo controle (sem mitomicina-C). Quatro grupos com 6 coelhos cada foram sacrificados com 7, 14, 30 e 90 dias após a cirurgia de enxertia. As pregas vocais foram removidas para estudo histológico com a intenção de quantificar a deposição de colágeno através da coloração por Picrossírius Red sob microscopia polarizada. A deposição de colágeno foi menor em todos os grupos de pregas vocais que receberam aplicação tópica de mitomicina-C quando comparada com as pregas vocais do grupo controle. No presente estudo, a aplicação tópica de mitomicina-C antes da enxertia de gordura reduziu significativamente a deposição de colágeno (p = 0,05). / Since the early 1990s, fat implantation in the vocal fold is described as a method of repairing glottal insufficiency. The aim of this study was to evaluate the effect of topical application of mitomycin in the healing process with collagen deposition measurement around of autologous fat implants inserted in rabbits vocal folds. Twenty-eight rabbits were submitted to a fat implant in the both vocal folds. The right vocal folds received previously topical application of mitomycin (0,4mg/ml) for five minutes and the left vocal folds were the control group (without mitomycin). Four groups of 6 rabbits each were sacrificed 7, 14, 30 and 90 days after the implantation. The samples of the vocal folds were collected for histological analysis with the purpose of quantifying the collagen deposition by Picrosirius Red stain under polarization microscopy. The collagen deposition was lower in all groups of vocal folds with topical application of mitomycin than in control groups. In the present study, the topical application of mitomycin before the fat grafts reduced significantly the collagen deposition (p = 0,05).

Micro (mi)RNAs have recently been found to play an important role in cancer biology. In order to further understand how miRNAs affect prostate tumour progression, we evaluated miRNA expression in two invasive prostate tumour lines, PC3 and DU145. We then focused our evaluation on a novel miRNA, miR-105, whose levels were significantly decreased in both tumour cell lines as compared to normal prostate epithelial cells. As miR-105 levels were reduced in prostate tumour cell lines, we restored its expression following transfection of cells with mimic constructs to over-express miR-105 in both cell lines, in order to determine its effect on various tumourigenic properties. Over-expression caused decreased tumour cell proliferation, anchorage-independent growth and invasion in vitro and inhibited tumour growth in vivo. We further identified CDK6 as a putative target of miR-105, which likely contributed to its inhibition of tumour cell growth. Our results suggest that miR-105 inhibits tumour cell proliferation and may be an interesting target to regulate tumour growth or potentially used as a biomarker to differentiate between less and more aggressive tumours in patients.