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Subcutaneous anticoagulant medications like Heparin and Low-Molecular Weight Heparin are injections that readily cause bruising, pain, induration, and hematoma formation at the injection site. It is known that these adverse reactions can be correlated to the technique used to administer these medications; however, there is no established technique that reduces bruising, pain, induration, and hematoma formation at the site. Currently, the only protocol for subcutaneous Heparin and Low-Molecular Weight Heparin is that it is to be administered subcutaneously in the abdomen and when using a prefilled syringe, the air bubble should not be removed. The purpose of this study was to identify current nursing practice for the administration of these medications and to compare the results to researched techniques that resulted in less adverse site reactions. A total of 33 participants were recruited. The survey targeted six researched techniques found, after a comprehensive literature review, to have reduced site adverse effects associated with subcutaneous Heparin and Low-Molecular Weight Heparin. After completing the survey, it was found that current practice does not reflect techniques researched to reduce bruising, pain, induration, and hematoma formation at the site. In fact, very few completed one of the six research techniques that were questioned, which included: a two minute application of a cold compress/pack before and/or after the injection, an injection duration lasting 30 seconds, slow removal of the needle over five seconds, application of pressure after the injection for a minimum of 30 seconds, use of a hot pack/compress after the injection, and the use of a3 mL syringe. It was also found that there were inconsistencies in techniques that have been previously established as current protocol for these medications.

Evaluation using mouse subcutaneous tumour models is a key process in cancer drug development. Tumour material is implanted subcutaneously and tumour growth measured using callipers. However this methodology can have poor reproducibility and accuracy due to observer variation. Furthermore the physical pressure of using callipers can distress the mouse and lead to tumour damage. Non-invasive digital tumour imaging would reduce handling stresses and allow volume determination without physical contact. This thesis focusses on capturing 2D digital images of subcutaneous tumours, then using image processing and machine learning methods to determine 3D volume. The biggest challenge faced was lack of differentiation between tumour and surrounding skin, rendering tumour boundary identification difficult. Whilst image processing methods such as colour segmentation and edge detection were unsuccessful, machine learning proved more successful. Three convolutional neural networks, VGG-Face, VGG-19 and VGG-16 models were evaluated, with VGG-Face producing the best results. Using the layer FC7 before RELU activation for extraction in the VGC-Face model, a tumour recognition rate of 98.86% was achieved. This was increased to 100% through a semi-automatic step with detection repeated on cropped versions of negatively classified images. Finally, volume was determined through extracting image features using the VGG-Face model and conducting partial least squares regression (error of 0.1). This work has successfully demonstrated that with computational methods the volume of subcutaneous tumours can be evaluated through non-invasive digital imaging without need to have contact with the tumour itself, thus offering refinement benefits to the mice as well as eliminating observer bias.

Over the last several years, injectable drugs have been a growing area for the treatment of various therapeutic conditions and they are projected to comprise an even larger proportion among the drugs that will be available in the years to come. The injectable drugs are administered by various routes such as intramuscular (IM), intravenous (IV), subcutaneous (SC) and others, however, the majority of these drugs are administered subcutaneously. Even though subcutaneous delivery has been utilized for a number of years, very little is known about the processes governing the absorption of macromolecules from the interstitial space; and the resulting impact of these processes on the bioavailability (BA) and pharmacokinetic (PK) profiles. Also, there is no established In vitro - In vivo correlation (IVIVC) for subcutaneously administered immediate release (IR) peptide based drugs in a biorelevant manner. The contribution of IVIVC in drug development of orally administered drugs is very well known. For oral drugs, the in vivo process of drug absorption is often rate limited by the rate at which drug dissolves in the gastrointestinal tract. This can be simulated by measuring the rate of dissolution in an in vitro apparatus, which can be correlated with the in vivo absorption rate to produce an IVIVC. This research program involved efforts to develop biorelevant IVIVC methods and model for subcutaneously administered peptide based drugs. The in vivo component of this Program involves the use of clinical data from a bioequivalence (BE) study of Iplex™ [(IGF-I (Insulin like growth factor-I)/IGFBP-3 (Insulin like growth factor binding protein-3)], administered subcutaneously, that was conducted at the Center for Drug Studies (CDS), VCU School of Pharmacy in the year 2005 (Barr et. al., 2005). The PK parameters for Increlex™ (IGF-I) are calculated from the clinical data obtained from another study (Rabkin et. al., 1996). Literature research and molecular modeling research formed the basis of our hypotheses that unbound and bound IGF-I are absorbed from the blood capillaries and lymphatic capillaries respectively and that simulation of these physiologic variables is possible with the use of the modified Hanson Microette® device. The Hanson Microette® device is a vertical diffusion cell system that has been modified to simulate the pores in the capillaries with the use of a synthetic membrane. The flow and composition of circulatory fluid was simulated by the use of modified Hanks balanced salts solution (HBSS). A validated RP-HPLC (reversed-phase high performance liquid chromatography) method has been used for the analysis of IGF-1/IGFBP-3 in the in vitro samples. The in vitro permeation/release results gave the in vitro component to conduct IVIVC analysis. The General Electric (GE) healthcare sourced polycarbonate nucleopore track etched membranes were the only set of membranes that resulted in significant permeation in the in vitro experiments. IVIVC results demonstrated high inter and intra-membrane variability for the membranes (available from today’s technology) that were used to simulate the in vivo membrane characteristics. Currently, there are no validated biorelevant IVIVC methods for SC formulations. The methods described here are the basis for future in vitro method development that will be of significant value for (a) predicting the in vivo performance of SC formulations based on the in vitro data, and (b) provide a reproducible in vitro method as the basis of developing an IVIVC for other subcutaneously administered drugs. This will provide an important tool for both development and regulation of this growing class of drugs.

Obesity results from an excess of adipose tissue and is a major risk factor for type 2 diabetes, cardiovascular disease, and cancer. Adipose tissue exists in two main forms: white adipose tissue (WAT), which stores energy as triglycerides, and brown adipose tissue (BAT), which dissipates stored energy as heat. White adipose tissue is composed of several subcutaneous and visceral depots, each possessing distinct molecular and functional characteristics. Brown-like adipocytes can emerge in WAT depots in response to cold or beta-adrenergic stimulation. These cells have been called “beige” or “brite” (brown-in-white) cells. The reduction of obesity in mice treated with beta-adrenergic agonists is correlated with the emergence of beige cells. Beige cell development occurs most readily in subcutaneous depots, and to the least extent in visceral depots. Understanding the molecular mechanisms underlying beige cell development in different WAT depots may be important in discovering new therapies against obesity and related diseases. Our lab has previously discovered that Tyrosine Kinase 2 (Tyk2), an important mediator of cytokine signaling, promotes the development of classical brown adipose tissue. Due to the lack of functional BAT, Tyk2-knockout (Tyk2-/-) mice become grossly obese with age and develop several symptoms of the metabolic syndrome. In the present study, we have found a potential role of Tyk2 in the development of beige cells. Here, we show that mRNA expression of BAT-selective genes (UCP1, Cidea, Cox8b, and Elovl3) is significantly reduced in subcutaneous WAT of Tyk2-knockout (Tyk2-/-) mice compared to wild-type mice. Surprisingly, BAT-selective genes are induced in Tyk2-/- subcutaneous WAT by acute starvation. These findings suggest that Tyk2 is required for the development of beige cells under ambient conditions, and that the need for Tyk2 in beige cell development is bypassed during nutritional stress, a stimulus of the sympathetic response.

Thesis is focused on the analysis of intersexual differences in adiposity markers within urban population of Czech children from 11,00 to 15,99 years. This age range encompasses puberty which is one of the critical periods for the possible development of overweight and obesity. The sample PRAHA 2013 consists of 356 girls and 353 boys which were surveyed for basic anthropometric indicators - body height, body weight, BMI, mid- upper arm circumference and the thickness of four skinfolds. Intersexual differences discovered in measured parameters show statistical significance (p<0,01 resp. p<0,001) and it corresponds with the characteristic changes of pubertal development in both of genders. Testing file PRAHA 2013 against reference standards and its comparison with the results of the last Czechoslovak spartakiade in 1985 points to positive secular trend of increasing body height. However increase in weight parametres is still continuing. Probands from the year 2013 reach higher body weight values, BMI and skinfold thickness in comparison with children of the same age in 1985, 1991 resp. 2001. Significant differences were found out in the most of age categories in both of genders. Keywords: adiposity tissue, subcutaneous fat, skinfolds, body fat distribution, sexual dimorphism, obesity, secular trend

Une nouvelle plateforme hydrogel uniquement formée par l’association de nanocapsules lipidiques (NCLs) a été développée en s’inspirant de précédents travaux utilisant une gemcitabine modifiée. Afin de limiter la toxicité de l’hydrogel, la gemcitabine a été remplacée par la cytidine, rendue amphiphile par une chaîne aliphatique (Cyt-C16). Placée à l’interface huile/eau des NCLs, la Cyt-C16 permet la formation d’un réseau tridimensionnel de NCLs à l’origine de la gélification. Un plan de mélange a permis d’optimiser les procédés de formulation de 4 tailles de NCLs modèles. Les propriétés viscoélastiques des hydrogels sont modulables. Plus les concentrations en NCLs et Cyt-C16 sont élevées, plus le gel est « rigide », indépendamment de la taille des NCLs qui doit être supérieure à 50 nm pour permettre la gélification. Les hydrogels sont injectables et permettent une libération prolongée de NCLs (de taille mono-disperse), sans toxicité supplémentaire in vitro, du fait de la présence de la Cyt-C16. De plus, uniquement solubilisée dans l’huile,la Cyt-C16 permet d’obtenir un organogel, dont les propriétés viscoélastiques sont renforcées en augmentant sa concentration. L’injection sous-cutanée (SC) in vivo des deux gels est bien tolérée et entraine une réaction inflammatoire locale comparable à celle provoquée par un excipient pharmaceutiquement acceptable. Ces deux formes pourront être utilisées pour libérer de façon prolongée différents actifs. Deux applications précliniques des hydrogels ont été explorées, l’une utilisant la voie SC pour cibler les ganglions lymphatiques, la seconde permettant un traitement local des suites opératoires d’une résection de glioblastome. / An innovative hydrogel platform obtained by the association of lipid nanocapsules (LNCs) was based on the previous work on modified gemcitabine. To limit the inherent toxicity of the hydrogel, gemcitabine was replaced by cytidine, then modified by an aliphatic chain (Cyt-C16). The hydrogel network was allowed by H-bond interactions between cytidine moieties exposed at the oil/water interfaces of LNCs. An experimental plan provided the formulation processes for 4 optimized sizes of model LNCs. The gelation was only possible for LNC sizes higher than 50 nm, and the hydrogel viscoelastic properties are versatile. The hydrogel is more “rigid” when LNC and Cyt-C16 concentrations increase, independently of the LNC size. The hydrogels are injectable and allow a sustained release of LNCs (withmonodisperse size), without additional in vitrocytotoxicity due to Cyt-C16. Moreover, when solubilized in oil, Cyt-C16 alone produced an organogel platform, whose viscoelastic properties are strengthened increasing its concentration. Both types of gels showed a good biocompatibility after an in vivo subcutaneous (SC) injection, with a local inflammatory response similar to that of induced by an approved excipient. These two forms could be used to sustain the release of various drugs, and two preclinical applications of hydrogels have been explored : one using the SC route to target lymph nodes, and the second for local treatment after glioblastoma resection.

A bactéria Neisseria meningitidis é um diplococo Gram-negativo. Dentre os sorogrupos de N. meningitidis, o sorogrupo B é um dos principais causadores da doença meningocócica invasiva (DMI), de ocorrência mundial. Recém-nascidos e crianças são especialmente susceptíveis a este tipo de infecção, uma vez que os seus sistemas imunitários ainda estão em desenvolvimento, assim mais estudos sobre a utilização da imunização materna para a proteção contra esta doença, são necessários. O objetivo desse estudo foi avaliar o potencial imunogênico dos antígenos de membrana externa de N. meningitidis sorogrupo B em camundongos Swiss quanto ao efeito da imunização passiva na prole, além de analisar o efeito de diferentes adjuvantes na produção de anticorpos e transferencia materno-fetal destes. O hidróxido de alumínio (HA) e fragmentos de bicamada de brometo de dioctadecildimetilamônio (DODAB-BF) foram utilizados como adjuvantes junto às vesículas de membrana externa (OMVs) para a imunização de camundongos adultos fêmeas, o que resultou em um aumento dos títulos de anticorpos do isótipo IgG quando comparado a imunização apenas com o antígeno, tanto pela via de imunização subcutânea com booster intramuscular (SC/IM), quanto pela via intranasal com booster subcutâneo (IN/SC). Esses anticorpos foram transferidos para a prole, que apresentou títulos de anticorpos significantes até o período no qual os camundongos apresentavam cinco semanas de vida. A imunização com OMVs + DODAB-BF foi capaz de gerar o reconhecimento específico de um maior número de antígenos que OMVs + HA, nos camundongos adultos fêmeas imunizados pela via SC/IM. A imunização com OMVs + DODAB-BF pela via SC/IM também resultou em anticorpos na prole que foram capazes de reconhecer antígenos proteicos da cepa homóloga e de cepas heterólogas, o que não ocorre quando essa imunização é realizada pela via IN/ SC. Apesar da presença de títulos de anticorpos na prole, estes irão apresentar funcionalidade diferente em comparação aos anticorpos da prole de mães imunizadas pela via SC/IM, sugerindo que a via SC/IM é melhor para a imunização materna com OMVs + DODAB-BF. / The Neisseria meningitidis bacterium is a Gram-negative diplococcus. Among the serogroups of N. meningitidis, serogroup B is one of the main causes of invasive meningococcal disease (IMD), worldwide occurrence. Newborns and children are particularly susceptible to this type of infection, since their immune systems are still developing, so further studies about the use of the maternal immunization to protect against this disease, are needed. The aim of this study was to evaluate the potential immunogenic antigens from outer membrane of N. meningitidis serogroup B in Swiss mice as to the effect of passive immunization in the offspring, in addition to analyze the effect of different adjuvants in the production of antibodies and maternal-fetal transfer of these. The aluminium hydroxide (HA) and bilayer fragments from dioctadecyldimethylammonium bromide (DODAB-BF) were used as adjuvants with the outer membrane vesicles (OMVs) for immunization of adult female mice, which resulted in an increase of the antibodies titers of IgG isotype when compared to immunization with only the antigen, either by subcutaneous route of immunization with intramuscular booster (SC/IM), as by the intranasal route with subcutaneous booster (IN/SC). These antibodies were transferred to the offspring, which presented significant antibodies titers to the period in which the mice had five weeks of live. Immunization with OMVs + DODAB-BF was able to generate the specific recognition of a greater number of antigens than OMVs + HA, in adult female mice immunized by SC/IM route. Immunization with OMVs + DODAB-BF SC/IM route also resulted in antibodies in offspring that were able to recognize protein antigens of the homologous strain and heterologous strains, which does not occur when this immunization is carried out by IN/SC route. Despite the presence of antibodies in the offspring, they will present different functionality compared to antibodies of the offspring of mothers immunized by SC/IM route, suggesting that the SC/IM route is better for maternal immunization with OMVs + DODAB-BF.

O objetivo deste estudo foi avaliar a resposta do tecido conjuntivo subcutâneo de camundongos isogênicos após o uso da Terapia Fotodinâmica antimicrobiana (aPDT), utilizando dois fotossensibilizadores, Derivado fenotiazínico (Helbo Blue) e Curcumina, em diferentes tempos de aplicação de lasers (30 segundos, 1 minuto ou 2 minutos). Foram utilizados 141 camundongos isogênicos da linhagem BALB/c cujo tecido conjuntivo subcutâneo foi exposto aos dois fotossensibilizadores, e em seguida irradiado com laser diodo no grupo do Derivado Fenotiazínico e ao LED no grupo da Curcumina. Para cada fotossensibilizador foram utilizados três tempos de irradiação: 30 segundos, 1 minuto e 2 minutos. Ao final de cada um dos períodos experimentais (7, 21 e 63 dias), uma porção do tecido conjuntivo subcutâneo da área do centro da área em que foi aplicada a aPDT foi removida e submetida ao processamento histotécnico de rotina. Foi realizada a descrição do processo inflamatório de forma qualitativa e semi-quantitativa (por meio de escores). Adicionalmente, foi realizada a marcação imunohistoquímica para neutrófilos e macrófagos. Os dados numéricos foram analisados por meio do programa estatístico Sigma Plot 12.0®, utilizando o teste não paramétrico de Kruskal-Wallis, seguido pelo Pós-teste de Dunn, quando houve diferença significativa entre os grupos. O nível de significância adotado foi de 5%. Foi possível observar que, com relação aos parâmetros fibrosamento, espessura e infiltrado inflamatório, no período inicial de 7 dias, as alterações teciduais foram pequena magnitude. No período de 21 dias, apenas o parâmetro infiltrado inflamatório apresentou pequenas variações entre os grupos. No período final de 63 dias, a compatibilidade tecidual foi observada para os dois fotossensibilizadores (Derivado Fenotiazínico e Curcumina) que não apresentaram diferenças significativas nos parâmetros avaliados, independentemente do tempo de aplicação do laser. / The aim of this study was to evaluate the response of subcutaneous connective tissue of isogenic mice after Antimicrobial Therapy (aPDT), using two photosensitizers, Phenothiazine Derivative (Helbo Blue) and Curcumin, at different laser application times (30 seconds, 1 minute or 2 minutes). One hundred and forty one (141) BALB/c isogenic mice were used, which had the subcutaneous connective tissue exposed to the two photosensitizers, followed by irradiation with laser diode to the Phenothiazine derivatives group, and LED to the Curcumin group. Three irradiation times were used to each photosensitizer: 30 seconds, 1 minute and 2 minutes. At the end of each experimental period (7, 21 and 63 days), a sample of the subcutaneous connective tissue, was collected and histotechnical processing was performed. Inflammatory process was described by qualitative and semi-quantitative analysis, using scores. Additionally, immunohistochemical technique was performed to identify neutrophils and macrophages. Data obtained was analyzed by the statistical program Sigma Plot 12.0®, using the non-parametric Kruskal-Wallis test, followed by the Dunn\'s post-test, when significant difference was found between groups. The significance level adopted was 5%. It was also possible to observe that, in relation with the parameters: fiber collagen formation, tissue thickness and inflammatory infiltrate, at the initial period of 7 days, the tissue alteration were of small significance (p<0.05). At 21-days period, only the inflammatory infiltrate parameter presented variation between groups (p<0.05). In the later time point of 63 days, it was observed tissue compatibility regarding the two photosensitizers (Phenothiazine Derivative and Curcumin) with no differences in the evaluated parameters or the laser application times.

Recentes estudos vêm demonstrando os efeitos benéficos da infiltração percutânea de dióxido de carbono em diferentes campos da Medicina, inclusive como alternativa para tratamento de depósitos localizados de gordura formados após lipoaspiração da parede abdominal. É possível que os efeitos dessa terapia encontrem-se relacionados à capacidade do método em aumentar a circulação sangüínea local, bem como aos efeitos lipolíticos do CO2 sobre adipócitos do tecido envolvido. Entretanto, até a presente data, poucos estudos avaliaram os efeitos histológicos e citométricos sobre adipócitos, presentes no tecido subcutâneo da parede abdominal. O objetivo do presente estudo foi avaliar os efeitos da infusão de gás carbônico sobre a população e morfologia de adipócitos da parede abdominal. Quinze voluntárias, com média de idade de 34 anos, foram submetidas a sessões de infusão de CO2 durante três semanas consecutivas (duas sessões por semana com intervalos de dois a três dias entre cada sessão). O volume de gás carbônico infundido por sessão, em pontos previamente demarcados, foi sempre calculado com base na superfície da área a ser tratada, com volume de gás de 250 mL/100 cm de superfície tratada. Utilizou-se As áreas foram demarcados respeitando-se o limite eqüidistante 2 cm entre elas. Em cada ponto de punção se injetou 10 mL, por sessão, mantendo-se fluxo de gás de 80 mL por minuto. Foram colhidos, por biópsia cirúrgica, fragmentos de tecido celular subcutâneo da região anterior da parede abdominal para estudo histológico antes e após tratamento. Os números e as alterações histomorfológicas nos adipócitos, tais como área, diâmetro médio, perímetro, comprimento, largura e número de adipócitos por campos de observação foram mensurados por processamento de imagem assistida por computador, utilizando-se o programa Image-Pró-plus. Os resultados encontrados antes e após a infusão de gás carbônico foram analisados por meio de teste t pareado, adotando-se nível de significância de 5% (p<0,05). Encontrou-se redução significativa nos valores das variáveis histomorfológicas mensuradas após o uso da hipercapnia tais como redução de sua área (p= 0,00001), diâmetro (p= 0,00001), perímetro (p= 0,00001), comprimento (p= 0,00001), largura (p= 0,00001), maior espaçamento entre as mesmas (p<0,0001) e menor número de adipócitos por campo (p= 0,00001). Conclui-se que existe efeito na morfologia e população dos adipócitos do tecido adiposo subcutâneo após utilização terapêutica de hipercapnia. / Recent researches have demonstrated the beneficial effects of percutaneous infiltration of carbon dioxide in different fields of medicine. Currently, this therapy proves to be an alternative for treatment of localized fat deposits formed after lipectomy of the abdominal wall. The effects appear to be related to the ability of therapy to increase local blood circulation and lipolitic effects of CO2 on tissue involved. However few studies have evaluated histological and cytometric effects on subcutaneous adipocytes tissue subjected to this new therapeutic modality. The purpose of this study was to evaluate the effects of infusion of carbon dioxide on the population and morphology of adipocytes in the abdominal wall. Fifteen volunteers were subjected to sessions of CO2 infusion for three consecutive weeks with two sessions per week at intervals of two to three days between each session. The volume of carbon dioxide infused per session, at points previously marked, was always based on the surface of the area to be treated with gas volume of 250 mL/100 cm2 of surface treated. Areas were demarcated with the limit up to 2 cm equidistant between them. At each point of puncture was injected 10mL of CO2, per session, keeping the gas flow of 80 mL/minute. Fragments of subcutaneous tissue of the anterior abdominal wall were collected by surgical biopsy for histological study before and after treatment. The numbers and histomorphological changes in adipocytes, such as area, diameter, perimeter, length, width and number of adipocytes per field of view were measured by image processing computer assisted using the program Image-Pro Plus. The results before and after the infusion of carbon dioxide were analyzed by paired t test. Significance level of 5% (p<0.05) was adopted. A significant difference was found in the values of variables measured after the use of hypercapnia as reduction of its area (p = 0,00001), diameter (p = 0.00001), perimeter (p = 0,00001), length (p = 0,00001), width (p = 0,00001), greater spacing between them and lower number of adipocytes per field (p = 0,00001). It is concluded that there is effect of adipocyte morphology and population of the subcutaneous adipose tissue after therapeutic use of hypercapnia