Elevation of VEGFR-3, the primary mediator of lymphangiogenesis (i.e., new lymphatic vessel formation), is frequently associated with inflammation related to chronic disease and cancer. In the latter case, VEGFR-3 dependent lymphangiogenesis induced by inflamed tumors increases the incidence of distant metastasis, leading to decreased patient survival. However, the molecular mechanisms underlying inflammation-induced VEGFR-3 elevation and lymphangiogenesis are currently unknown. Two potential candidate genes that may regulate expression of VEGFR-3 are Prox1, the primary mediator of embryonic lymphangiogenesis, and NF-κB, the key intracellular regulator of inflammation-induced transcription. We hypothesized that the key inflammatory mediator, NF-κB, regulates transcription of key mediators of lymphangiogenesis, VEGFR-3 and Prox1. We further hypothesized that inflammation-induced elevation of VEGFR-3 and Prox1 are essential steps required for robust lymphangiogenesis in response to inflammation. The three primary goals of this study were to (1) delineate the time-course of events leading to inflammation-induced lymphangiogenesis in vivo; (2) clone and characterize the VEGFR-3 promoter and identify factors regulating VEGFR-3 expression in vitro; and (3) characterize the lymphatic phenotype of NF-κB p50 knockout mice. To begin testing these hypotheses, we used a mouse model of peritonitis to characterize induction of lymphangiogenesis and expression kinetics of NF-κB, Prox1 and VEGFR-3. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-κB followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Characterization of the VEGFR-3 promoter by luciferase-reporter and ChIP assays showed direct activation by Prox1, NF-κB p50 and p65 transcription factors. This also revealed that Prox1 and NF-κB p50 bind in close proximity and synergistically activate the VEGFR-3 promoter. Characterization of p50 knockout mice revealed significantly decreased lymphatic vessel density in several organs that corresponded to reduced VEGFR-3 and Prox1 expression. Activation of NF-κB by inflammatory stimuli also elevated expression of NF-κB, Prox1 and VEGFR-3 in cultured lymphatic endothelial cells, which enhanced proliferation and migration in response to the VEGFR-3-specific ligand, VEGF-C152S. Collectively, our findings suggest that induction of the NF-κB pathway by inflammatory stimuli activates Prox1, and both NF-κB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of pre-existing lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

Filarial infections (lymphatic filariasis and onchocerciasis) are amongst the major neglected tropical diseases, and together account for more than 120 million infections in tropical and subtropical regions. The gold-standard technique for the diagnosis of filariases relies on the detection of microfilariae (mf) either in blood smears (lymphatic filariasis) or in skin biopsies (onchocerciasis). The secretion of extracellular RNAs (exRNAs) by parasitic nematodes has opened new avenues for the development of novel biomarkers for helminthiases, including filariasis. However, rather little is known about the origin and regulation of these RNAs inside the nematodes. One outstanding question is whether the secretion of small RNAs is distinct across the developmental stages of parasitic nematodes. Similarly, it is not clear whether the secretion of miRNAs is affected by treatment with anthelminthic chemotherapy or their potential as biomarkers for infection. Litomosoides sigmodontis is a murine filarial nematode closely related to filarial nematodes of medical and veterinary importance, including Onchocerca spp. and Brugia spp. L. sigmodontis has been extensively used to decipher multiple aspects of filarial biology, including parasite development, vaccine, and host-pathogen interactions. Therefore, we decided to use this model to address fundamental questions regarding the secretion of small RNAs and their biomarker potential. Our in vitro studies demonstrate that some extracellular miRNAs are enriched in a sexand stage-specific manner in the Excretion/Secretion (ES) products from early larval and adult stages from the rodent filarial nematode Litomosoides sigmodontis. Moreover, our data demonstrates that the gravid adult female worms secrete a plethora of miRNAs enriched in the secretome of this developmental stage when compared to adult males or mf. Further characterization studies show that the miRNAs are likely to be secreted in association with extracellular vesicles (EVs), as previously reported for other parasitic nematodes, including the human pathogen Brugia malayi. Interestingly, Ivermectin, which is typically used to treat filarial infections, does not have consistent effects on the secretion of miRNAs by gravid adult female worms in vitro, requiring further in vivo experiments to determine the effect of IVM on detection of extracellular parasite-derived miRNAs. In vivo experiments, using murine models of infection with L. sigmodontis (gerbils and BALB/c mice), as well as human samples from patients infected with Onchocerca volvulus and cattle infected with Onchocerca ochengi, demonstrated the presence of filarial-derived miRNAs, including female-specific miRNA markers, in biofluids from infected hosts. Further statistical analysis showed that two parasite-derived miRNAs, miR-71 and miR-100d, can significantly discriminate infected animals from naïve controls with high sensitivity/specificity (> 80%/100%). The results presented in this PhD thesis provide an initial framework to understand the secretion of small RNAs throughout nematode development, the potential interactions between anthelminthic chemotherapy and small RNA trafficking and secretion, as well as the use of parasite-derived miRNAs for the development of a new generation of biomarkers for filarial infections.

Breast cancer is the second leading cause of cancer-related death in women in the United States. Complications that lead to mortality of cancer patients are associated with tumor metastasis. Specifically, lymphatic metastasis in breast cancer patients strongly correlates with poor patient survival and this process is facilitated by the formation of new tumor lymphatic vessels termed lymphangiogenesis. Previously, our lab reported that lymphangiogenesis was promoted by a distinct subset of bone marrow (BM)-derived myeloid cells that co-express lymphatic-specific markers designated as myeloid-derived endothelial cell progenitors (M-LECPs). Furthermore, our lab has generated M-LECP in vitro from a mouse macrophage cell line (RAW264.7) by LPS stimulation. Taken together, these data suggest that chronically inflamed sites drive M-LECP differentiation and that these cells can contribute to the formation of new lymphatic vessels and promote lymph node metastasis. Evidence supporting this hypothesis was indicated by high levels of circulating M-LECP in peripheral blood of breast cancer patients but undetectable levels in healthy donors, cancer-free donors. Additionally, the generation of M-LECP was prompted through TLR4-signaling pathway, and de novo expression of VEGFR-3 and VEGF-C. This co-expression produces an autocrine loop essential for pro-lymphatic reprogramming in both primary human monocytes and the immature monocytic cell line, THP-1. Taken together, these data indicate the major regulatory role of TLR4 in inflammation-driven lymphangiogenesis involves the recruitment and differentiation of M-LECP, a process that may promote lymphatic metastasis.

Submitted by CHESTON CÉSAR HONORATO PEREIRA null (cheston@bol.com.br) on 2016-01-19T22:39:51Z No. of bitstreams: 1 Tese_Cheston_Cesar_Honorato_Pereira.pdf: 2607975 bytes, checksum: 4fe741cf6ad1a893c8fbc6017c62699a (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-01-20T16:40:14Z (GMT) No. of bitstreams: 1 pereira_cch_dr_jabo.pdf: 2607975 bytes, checksum: 4fe741cf6ad1a893c8fbc6017c62699a (MD5) / Made available in DSpace on 2016-01-20T16:40:14Z (GMT). No. of bitstreams: 1 pereira_cch_dr_jabo.pdf: 2607975 bytes, checksum: 4fe741cf6ad1a893c8fbc6017c62699a (MD5) Previous issue date: 2015-12-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Estudou-se a anatomia do timo em 30 filhotes de avestruzes, 13 machos e 17 fêmeas com idade variando entre 10 a 20 dias, mediante fixação de cada ave em solução aquosa de formol a 10%. As cadeias de lobos tímicos localizaram-se no terço distal da região ventral do pescoço, dispondo-se de forma linear e paralela, desde a décima primeira até a décima quinta vértebra cervical, relacionando-se com o nervo vago e veia jugular. O número de lobos variou de 1 a 3 e 1 ou 2, para os antímero direito e esquerdo respectivamente. A média das dimensões dos lobos foram 2,09 x 0,71 x 0,41 cm para o lobo cranial, 0,69 x 0,38 x 0,27 cm para o lobo médio e 0,55 x 0,33 x 0,29 cm para o lobo caudal do antímero direito. No antímero esquerdo foram registrados de 2,14 x 0,71 x 0,37 cm para o lobo cranial e 0,60 x 0,31 x 0,22 cm para o lobo caudal. Os lobos tímicos direitos e esquerdos mostraram-se nutridos, em todos os casos, por vasos oriundos, direta e exclusivamente, das artérias comuns dos nervos vagos, esofágicas ascendentes e cervicais cutâneas ascendentes direitas e esquerdas. Os lobos tímicos do antímero direito receberam, independentemente de sua origem, de um a cinco ramos e do antímero esquerdo, houve uma variação de um a quatro ramos. A morfologia e vascularização arterial do timo apresentaram comportamentos bem definidos, possuindo características comuns que identificam a espécie que foi objeto de nosso estudo. / It was studied the morphology and the biometry of the thymus in 30 young ostriches, 13 males and 17 females aged from 10 to 20 days, by setting each bird in aqueous solution of 10% formaldehyde. Chains of thymic lobes were located in the third distal part of the ventral neck, offering up linear and parallel, from the eleventh to the fifteenth cervical vertebra, relating to the vagus nerve and jugular vein. The number of lobes varied from 1 to 3 and 1 or 2, to right and left antimeres respectively. The average dimensions of the lobes were 2.09 x 0.71 x 0.41 cm to the cranial lobe, 0.69 x 0.38 x 0.27 cm to the middle lobe and the 55 x 0.33 x 0.29 cm to the caudal lobe of the right side. On left antimere were found the average dimensions of the lobes of 2.14 x 0.71 x 0.37 cm for cranial lobes and 0.60 x 0.31 x 0.22 cm to the caudal lobes. Thymic right and left lobes shown to be nourished , in all cases , for vessels coming directly and exclusively , common arteries of the vagus nerves , skin and cervical esophageal ascending upward right and left . Thymic lobes of the right side received , regardless of its source , from one to five branches and left antimere , there was a variation of one to four branches. The morphology and thymus biometrics showed well-defined behaviors, possessing common characteristic who characterized the specie that was the subject of our study.

Made available in DSpace on 2014-06-11T19:24:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-25Bitstream added on 2014-06-13T20:12:58Z : No. of bitstreams: 1 silva_rh_me_botfm.pdf: 1035047 bytes, checksum: 7d6063580b3eebf28e90334d7650ea1c (MD5) / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / A presença de edema prejudica o fluxo sanguíneo, retardando o processo de cicatrização das feridas, visto interferir na oxigenação e nutrição dos tecidos em formação. Neste sentido, a oxigenação e a perfusão tissular são condições essenciais para ocorrer à cicatrização. Quando s perfusão é inadequada todo processo de cicatrização é prejudicado, visto que a deficiência de oxigênio impede a síntese do colágeno, diminui a proliferação e migração celular e reduz a resistência dos tecidos à infecção. Deste modo a drenagem linfática manual pode ser um recurso terapêutico de grande importância na contribuição do reparo tecidual. A drenagem linfática tem como objetivo drenar os líquidos excedentes que banham as células, diminuindo o edema e mantendo o equilíbrio hídrico dos espaços intersticiais, aumento da oxigenação, desintoxicação, aumento da ação antiinflamatória, fortalecimento do sistema imunológico e evacuação dos dejetos provenientes do metabolismo celular. O efeito da drenagem linfática manual é direcionar e aumentar o fluxo linfático promovendo, assim, uma remoção mais rápida de excesso de líquido intersticial, contribuindo dessa forma para a hemostase e regeneração tecidual. Sendo assim, a fisioterapia pode desempenhar um papel indispensável na melhoria da qualidade de vida desses pacientes. Avaliar os efeitos da drenagem linfática manual em pacientes portadores de feridas venosas crônicas em tratamento com bioativos. Fez parte deste estudo um quantitativo de 22 pacientes portadores de feridas crônicas de etiologia venosa. Redução da ferida em 73% dos pacientes do GII e em 34% do GII. Melhora do edema em 87% dos pacientes do GI e 66% do GII. Ganho da amplitude articular do tornozelo em 67% dos pacientes pertencentes ao GI. Melhora da pele perilesional em 67% dos pacientes do GI e em 33% do GII... / The presence of edema affect blood flow, slowing the healing process of wounds, as interfering with oxygenation and nutrition of tissues in training. In this sense, oxygenation and tissue perfusion are essential for healing to occur. When s perfusion is inadequate throughout the healing process is hampered, since the oxygen deficiency prevents the synthesis of collagen, reduces cell proliferation and migration and reduces the resistance of tissues to infection. Thus the manual lymphatic drainage can be a therapeutic resource of great importance in the contribution of tissue repair. Lymphatic drainage is intended to drain the excess fluid that bathes the cells, reducing edema and maintaining the water balance of the interstitial spaces, increase oxygenation, detoxification, increased anti-inflammatory action, strengthening the immune system and disposal of waste from cell metabolism . The effect of manual lymphatic drainage is to direct and increase lymphatic flow, thus facilitating a faster removal of excess interstitial fluid, thereby contributing to hemostasis and tissue regeneration. Thus, physical therapy can play a vital role in improving the quality of life of these patients. To evaluate the effects of manual lymphatic drainage in patients with chronic venous wounds treated with bioactive. It was part of a quantitative study of 22 patients with chronic wounds of venous etiology. Reduction of the wound in 73% of patients in GII and 34% of the GII. Improvement of edema in 87% of GI patients and 66% of GII. Gain range of motion of the ankle in 67% of patients in the IM. Improvement of perilesional skin in 67% of GI patients and in 33% of the GII ... (Complete abstract click electronic access below)

Orientador: Maria Salete Costa Gurgel / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T11:22:09Z (GMT). No. of bitstreams: 1 Rezende_LauraFerreirade_D.pdf: 9192920 bytes, checksum: b761ed1e8c55e161d7c896509a841411 (MD5) Previous issue date: 2008 / Resumo: O objetivo deste estudo foi descrever o padrão linfocintilográfico e avaliar as compensações linfáticas do membro superior no pós-operatório de câncer de mama com dissecção axilar. Sujeitos e métodos: Foram inicialmente analisadas 37 pacientes que realizaram a linfocintilografia pré-operatória, sendo que 32 delas realizaram também o estudo no membro contralateral. Através de critérios para homogeinezação da amostra, 23 pacientes tiveram seus estudos pré-operatórios comparados com a linfocintilografia realizada com 60 dias de pós-operatório. O protocolo de exame consistiu na realização de imagens estáticas de cada membro superior em semiflexão e do tórax, após 10 minutos, 1 e 2 horas da injeção subcutânea de 1 mCi (37 MBq) de dextran-99mTc no dorso da mão. Foram feitas análises comparativas da captação hepática do radiofármaco, do grau de captação dos linfonodos axilares (a ¿ acentuada, b ¿ moderada, c ¿ discreta e d - ausente) e da velocidade de aparecimento destes (I ¿ aos 10 minutos, II ¿ à 1 hora, III ¿ às 2 horas e IV ¿ não visíveis) antes e após a cirurgia. Resultados: Das pacientes analisadas no pré-operatório, apenas quatro apresentaram o padrão considerado normal (Ia) no estudo do membro ipsilateral e seis no contralateral, enquanto três apresentaram total comprometimento (IVd) no membro ipsilateral e duas no contralateral. Entre o grupo com 23 pacientes, duas foram classificadas como Ia no pós-operatório, que foi considerada a categoria ideal e três pacientes apresentaram classificação IVd. Comparando os padrões de drenagem no pré e pós-operatório foram encontradas pacientes com piora, pacientes que não apresentaram modificação e pacientes com melhora. Todas as pacientes apresentaram manutenção ou aumento da captação hepática após o tratamento cirúrgico. Conclusão: Este estudo encontrou alterações relevantes nas linfocintilografias pré e pós-operatórias, demonstrando a existência de diferenças funcionais do sistema linfático do membro superior. Alterações no padrão de drenagem linfática já podem ser percebidas com 60 dias de pós-operatório, assim como o indício de presença de anastomose linfo-venosa / Abstract: The aim of this study was to describe lymphoscintigraphic pattern and evaluate upper limb lymphatic compensation following breast cancer surgey with axillary dissection. Subjects and methods: From September 2006 to June 2007, 37 patients who had performed preoperative lymphoscintigraphy were initially analyzed in the Division of Gynecologic Oncology and Breast Pathology of the Women¿s Integral Healthcare Center at the Universidade Estadual de Campinas. Thirtytwo of these patients also performed contralateral limb study. Using criteria for sample homogenization, preoperative studies of 23 patients were compared to lymphoscintigraphy performed within 60 days of surgical treatment. The examination protocol consisted in performing static imaging of each upper limb in semi-flexion and thoracic imaging, after 10 minutes, and 1 and 2 hours after subcutaneous injection of 1 mCi (37 MBq) of Tc-99m-dextran into the dorsum of the hand. Comparative analyses of hepatic radiopharmaceutical uptake, velocity of axillary lymph node visualization (I ¿ visible at 10 minutes, II ¿ visible at 1 hour, III ¿ visible at 2 hours and IV ¿ not visible) and degree of nodal uptake (a ¿ marked, b ¿ moderate, c ¿ mild and d - absent) before and after surgery were made. Results: Of the patients analyzed preoperatively, only four (11%) had an optimal pattern (Ia) in the ipsilateral limb study and six (19%) had an optimal pattern in the contralateral limb study, while total lymphatic compromise (IVd) was observed in three (8%) patients in the ipsilateral limb and two (6%) patients in the contralateral limb. Among the group with 23 patients, two (9%) were classified postoperatively as Ia, which was considered the optimal category and three patients (13%) were classified as IVd. Comparing preoperative and postoperative drainage, 11 (48%) patients worsened, 5 (22%) patients showed no modification and 7 (30%) patients improved. All maintained or increased hepatic uptake after sugical treatment. Conclusion: This study encountered relevant changes in preoperative and postoperative lymphoscintigraphy evaluations, demonstrating the existence of functional differences in the upper limb lymphatic system. Alterations in lymphatic drainage patterns can already be perceived within 60 days of surgery, as well as signs of lymphovenous shunts / Doutorado / Ciencias Biomedicas / Doutor em Tocoginecologia

Orientador: Edgard Graner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-21T21:26:22Z (GMT). No. of bitstreams: 1 Bastos_DeboraCampanella_D.pdf: 2999887 bytes, checksum: 8cc36631bdfe28b13c872fff12fdf00e (MD5) Previous issue date: 2012 / Resumo: A enzima ácido graxo sintase (FASN) é responsável pela síntese endógena de ácidos graxos e tem alta expressão em diversas neoplasias malignas. Cerulenina e orlistat são inibidores farmacológicos da atividade de FASN e têm sido descritos como potenciais agentes anti-tumorais. O sistema linfático constitui a via primária de metástases de diversas neoplasias malignas, dentre elas o melanoma. Em estudos prévios, realizados no Laboratório de Patologia Bucal da FOP-UNICAMP, a inibição da FASN com orlistat foi capaz de reduzir significantemente as metástases linfonodais em modelo murino de melanoma e no modelo ortotópico de carcinoma espinocelular oral. Além disso, o meio condicionado por células de melanoma humano tratadas com inibidores da FASN inibiu a formação de capilares in vitro em matrigel, efeito este mediado pela isoforma anti-angiogênica VEGF-A165b. A correlação entre a FASN e a linfangiogênese ainda não está descrita na literatura, neste trabalho investigamos os efeitos da inibição farmacológica da FASN sobre os fatores de crescimento endotelial vascular (VEGF-C e -D) em células de melanoma murino (B16-F10) e humano (SK-Mel-25) através de RT-PCR quantitativo e ELISA e verificamos os efeitos do bloqueio de FASN sobre a linfangiogênese por meio de culturas tridimensionais de células endoteliais linfáticas humanas (HDLEC) e culturas primárias de endotélio linfático de camundongo. Avaliamos também, o efeito da exposição de células SK-Mel-25 os inibidores da FASN sobre a proliferação de xviii células HDLEC. O tratamento de células B16-F10 e SK-Mel-25 com cerulenina ou orlistat modulou de forma diferencial a expressão de VEGF-C e -D, inibindo o primeiro ao mesmo tempo em que estimulou o último. Além disto, estas mesmas drogas reduziram em células endoteliais linfáticas a viabilidade, a proliferação, migração e formação de capilares em ensaio ex vivo, além de provocar apoptose. O tratamento com inibidores farmacológicos de FASN em células SK-Mel-25 também induziu um fenótipo anti-linfangiogênico, observado em experimentos com meio condicionado. Em conjunto, nossos resultados sugerem que a inibição farmacológica da FASN inibe a linfangiogênese por atuar ao mesmo tempo nas células dos vasos linfáticos e de melanoma. Esses resultados explicam, pelo menos em parte, a redução do número de metástases linfonodais em modelos animais tratados com orlistat e reforçam a teoria de que o bloqueio da FASN é um alvo terapêutico em potencial para os melanomas / Abstract: Fatty acid synthase (FASN) is responsible for de novo synthesis of long-chain fatty. FASN has emerged as a potential therapeutic target for human cancers since it's over expression is associated with depth of invasion and poor prognosis. Cerulenin and orlistat are pharmacological inhibitors of FASN and have been described as potential anti-tumor agents. Lymphatic vessels are the primary route of metastasis in several malignancies including melanomas. In previous studies performed in our laboratory, we demonstrated that FASN activity is essential for melanoma and oral squamous cell carcinoma progression, as its pharmacological inhibition with orlistat reduces lymphatic metastasis in both experimental intraperitoneal and subcutaneous melanomas and orthotopic tongue carcinomas. We also reported that cell culture medium previously conditioned by human melanoma cells in the presence of orlistat induces an anti-angiogenic phenotype mediated by VEGFA165b. Therefore, in order to understand a possible correlation between FASN and lymphangiogenesis, here we investigate the effects FASN inhibitors on the secretion of vascular endothelial growth factors (VEGF) -C and -D by murine (B16-F10) and human (SK-Mel-25) melanoma cells with the aid of qRT-PCR and ELISA assays. We also analyzed the effect of FASN blockage on tridimensional cultures of human lymphatic endothelial cells (HDLEC) and primary cultures of murine lymphatic endothelium. In addition, we also evaluated an indirect effect of the treatment with cerulenin and orlistat in SK-Mel-25 cells on HDLEC xx proliferation. The incubation of B16-F10 or SK-Mel-25 cells with cerulenin or orlistat modulated VEGF-C and -D expression by significantly inhibiting the former and increasing the latter. In addition, these drugs reduced the viability, proliferation, and migration as well as promoted apoptosis in human lymphatic endothelial cells (HDLEC). These compounds also inhibited lymphatic capillary formation in a murine ex vivo assay. Finally, conditioned media from orlistat-treated human melanoma cells resulted in an anti-lymphangiogenic phenotype. These data suggest that FASN inhibitiors reduce lymphangiogenesis by acting simultaneously in the lymphatic endothelium and melanoma cells. Taken together, the studies here presented explain, at least in part, the anti-metastatic effect of orlistat observed in experimental tumors and further suggest that FASN is a potential therapeutic target for melanomas / Doutorado / Patologia / Doutora em Estomatopatologia

Brugia malayi is one of three species of nematode known to cause lymphatic filariasis (LF) in humans. LF infects over 120 million people, causing debilitating disease. Various global programs have been launched in the past 20 years to eliminate LF. These programs have greatly scaled up the resources and efforts allocated to halting the transmission and reducing disease burden. Only a few drugs are used to treat LF, and resistance is thus a devastating possibility. Research aimed at identifying new drug targets could therefore prove essential in elimination of LF. Genetic manipulation of B. malayi has been limited to transient transfections. A transfection system allowing for stable integration of transgenic sequences into the nuclear genome of this parasite would enable more robust studies that could lead to identification of novel drug targets and vaccine candidates. The piggyBac (pB) transposon system has been successfully applied to develop a stable transfection system in a variety of species. This system involves two plasmids, a helper and a donor. The donor plasmid contains the target DNA and a selectable marker flanked by specific inverted terminal repeat (ITR) regions. The helper plasmid expresses the pB transposase that will catalyze the precise integration of any DNA report tools necessary to adapt the pB system in B. malayi. Three versions of the donor plasmid were constructed, each containing a Gaussia Luciferase (GLuc) selectable marker but differing only by the fluorescent protein expressed. The construct containing a YFP gene was used to transfect embryos via biolistics to test whether YFP and GLuc are expressed.

No description available.

Cardiovascular disease continues to be the leading cause of morbidity and mortality worldwide with an estimated 17 million annual deaths. A majority of cases are attributed to disease affecting the vascular system including arterial, venous and lymphatic vessels. Despite progress in understanding the molecular bases of vascular development and disease, the role of chromatin modifying enzymes in vascular processes remains ill defined. Here we show that the histone-modifying enzyme Hdac3 is a critical regulator of lymphatic vascular development. Endothelial specific loss of Hdac3 in mice affects the development of lymphovenous and lymphatic valves resulting in aberrant blood lymph separation, lymphedema and complete lethality. We demonstrate that Hdac3 functions in a flow responsive manner to regulate the expression of Gata2, a transcription factor essential for lymphatic valve development. In response to flow, transcription factors Tal1, Ets1/2 and Gata2 recruit Hdac3 to an evolutionarily conserved intragenic enhancer of Gata2 gene. In turn, Hdac3 recruits p300, a histone acetyl transferase, to render activation of the Gata2 enhancer, and thus promotes Gata2 transcription. Together, our findings demonstrate the molecular basis by which cell extrinsic and intrinsic cues cooperate to regulate lymphatic development.