Organotypic Modeling Platform for Adverse Outcome Pathways of Male Reproductive and Developmental Processes

Thesis (Master's)--University of Washington, 2016-06

Haemodynamic assessment following inferior vena cava resection without replacement

Background: Treatment of bulky retroperitoneal malignancy may require en bloc resection of the infrarenal inferior vena cava. A number of reconstructive options are available to the surgeon but objective haemodynamic assessment of the peripheral venous system following resection without replacement is lacking. The aim of the present paper was thus to determine the symptomatic and haemodynamic effects of not reconstructing the resected infrarenal inferior vena cava. Methods: A retrospective descriptive study was carried out at Princess Alexandra Hospital in Queensland. Five patients underwent resection of the thrombosed infrarenal inferior vena cava as part of retroperitoneal lymph node dissection for testicular cancer (n = 3), radical nephrectomy for renal cell carcinoma (n = 1) and thrombosed inferior vena cava aneurysm (n = 1). Clinical effects were determined via the modified venous clinical severity score and venous disability score. Haemodynamic data were obtained postoperatively using venous duplex ultrasound and air plethysmography. Results: None of the present patients scored >2 (out of 30) on the modified venous clinical severity score or >1 (out of 3) on the venous disability score. Haemodynamic studies showed only minor abnormalities. Conclusions: Not reconstructing the resected thrombosed infrarenal inferior vena cava results in minor signs and symptoms of peripheral venous hypertension and only minor abnormalities on haemodynamic assessment.

Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus)

The cane toad (Bufo marinus) was used as a model to study male anuran reproductive endocrinology and to develop a protocol for non-invasive sperm recovery. Circulating testosterone concentrations in 6-hourly samples did not vary significantly (P < 0.05) over a 24 h period although there was a tendency (P = 0.06) for testosterone to be elevated at 19:00 h relative to other times of the day, which may be related to the nocturnal activity pattern of this species. Testosterone secretion after intraperitoneal (IP) injection of either a GnRH agonist (5 mu g IP) or hCG (1000 IU) was also examined. While the GnRH agonist did not produce a significant increase above basal plasma testosterone (0.29, 95% C.I. of 0.05-1.10 ng/ml), injection of hCG resulted in an increase (P < 0.01) of plasma testosterone with peak concentrations at approximately 120 min (4.17, 95% C.I. of 2.69-7.44 ng/ml) after injection. Non-invasive pharmaceutical sperm recovery was attempted following IP injection of graded doses of GnRH agonist, hCG or FSH. Urine was collected at 3, 6 and 12 h after treatment to assess sperm quality and quantity. The optimal protocol for sperm recovery in cane toads was injection of either 1000 or 2000 IU hCG; there was no significant difference in the quality of the spermic urine samples obtained using either dose of hCG or with respect to collection time. The findings indicated that hCG can be used to assess testicular steroidogenic status and also to induce sperm recovery in the cane toad. The hCG protocols developed in this study will have application in studies on the reproductive biology of rare and endangered male anurans. (c) 2005 Elsevier B.V. All rights reserved.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Dppa3 is a marker of pluripotency and has a human homologue that is expressed in germ cell tumours

We identified a transcript named 11M2 on the basis of its strong male-specific expression pattern in the developing mouse gonad. 11M2 was found to be expressed by gonad primordial germ cells (PGCs) of both sexes and down-regulated in female PGCs as they enter prophase I of the first meiotic division, similar to the expression of Oct4. Mouse EST analysis revealed expression only in early-stage embryos, embryonic stem cells and pre-meiotic germ cells. 11M2 corresponds to a recently reported gene variously known as PGC7, stella or Dppa3. We have identified the human orthologue of Dppa3 and find by human EST analysis that it is expressed in human testicular germ cell tumours but not in normal human somatic tissues. The expression patterns of mouse and human DPPA3, in undifferentiated embryonic cells, embryonic germ cells and adult germ cell tumours, together suggest a role for this gene in maintaining cell pluripotentiality.

The isolation and characterization of tescalcin, a novel gene differentially expressed in the mouse testis during the early stages of gonadal differentiation

Gonadal development is an ideal model to study organogenesis because a variety of developmental processes can be studied during the differentiation of the bipotential primordium into testis or ovary. To better understand this process, Representational Difference Analysis of cDNA was used to identify genes that are differentially expressed in mouse gonads at 13.5 days post-coitus. The analysis led to the identification of three testis specific genes and a sequence that was only expressed in the ovary. The male genes identified: renin, Col9a3, and a novel gene termed tescalcin had patterns of expression that suggested a role in testis determination. ^ Studies of the tescalcin gene revealed that it is organized into eight exons and seven introns. The gene was located at 64 cM in mouse chromosome 5, where it spans approximately 35 Kb. Three mRNA variants resulting from alternative splicing of intron 5 were identified in mouse tissues. Gel mobility shift assays demonstrated that Sp1 and Sp3 from Y-1, msc-1, and MIN-6 cells nuclear extracts bind the GC-boxes within the tescalcin proximal promoter. Bisulfite sequencing analysis of tescalcin CpG island revealed that it is differentially methylated in male and female mouse embryonic gonads, and that hypermethylation of this region represses expression of tescalcin in the β-TC3 cell line. ^ The major tescalcin mRNA encodes a protein with 214 amino acids that contains a consensus EF-hand Ca2+-binding domain and an N-myristoylation motif. The amino acid sequence of tescalcin is highly conserved among various species, and it showed the highest homology with calcineurin B homologous proteins 1 and 2, and calcineurin B. Western blot analysis using antibodies generated against the tescalcin protein confirmed its presence in specific mouse tissues and cell lines. Immunohistochemical analysis of mouse embryos confirmed the pattern of expression of tescalcin mRNA in fetal testis. Using pull-down assays, glyceraidehydes-3-phosphate dehydrogenase was identified as an interacting and potential functional partner of tescalcin. ^ The identification and characterization of tescalcin as a novel embryonic testicular marker will contribute to the elucidation of the genetic pathways involved in testis development and likely to the understanding of pathological conditions such as sex reversal and infertility. ^

The Fate of Spermatogonial Stem Cells in the Cryptorchid Testes of RXFP2 Deficient Mice

The environmental niche of the spermatogonial stem cell pool is critical to ensure the continued generation of the germ cell population. To study the consequences of an aberrant testicular environment in cryptorchidism we used a mouse model with a deletion of Rxfp2 gene resulting in a high intra-abdominal testicular position. Mutant males were infertile with the gross morphology of the cryptorchid testis progressively deteriorating with age. Few spermatogonia were identifiable in 12 month old cryptorchid testes. Gene expression analysis showed no difference between mutant and control testes at postnatal day 10. In three month old males a decrease in expression of spermatogonial stem cell (SSC) markers Id4, Nanos2, and Ret was shown. The direct counting of ID4+ cells supported a significant decrease of SSCs. In contrast, the expression of Plzf, a marker for undifferentiated and differentiating spermatogonia was not reduced, and the number of PLZF+ cells in the cryptorchid testis was higher in three month old testes, but equal to control in six month old mutants. The PLZF+ cells did not show a higher rate of apoptosis in cryptorchid testis. The expression of the Sertoli cell FGF2 gene required for SSC maintenance was significantly reduced in mutant testis. Based on these findings we propose that the deregulation of somatic and germ cell genes in the cryptorchid testis, directs the SSCs towards the differentiation pathway. This leads to a depletion of the SSC pool and an increase in the number of PLZF+ spermatogonial cells, which too, eventually decreases with the exhaustion of the stem cell pool. Such a dynamic suggests that an early correction of cryptorchidism is critical for the retention of the SSC pool.

Enigmas monetarios en el derecho local. Los mencales y maravedís en los fueros castellanos

La historia de la moneda en la Castilla medieval ha estado siempre mediatizada por la convivencia no siempre armónica entre dos sistemas monetarios muy diferentes. Uno basado en la plata, de origen europeo, otro centrado en el oro, de raíces árabes. La necesidad de conectar y de establecer unas equivalencias entre ellos se convirtió pronto en una necesidad, máxime cuando las monedas de oro incrementaron sus variantes. En esta compleja situación aparecen mencales y maravedís citados conjuntamente en muchos fueros: Zorita, Uclés, Cuenca,... sin que su naturaleza quede del todo clara. Este artículo compara estas referencias y analiza las equivalencias que los unen. Como colofón al trabajo podemos afirmar que los maravedís citados en cada texto corresponden a monedas áureas de distinto peso y valor mientras el mencal es identificado con un ponderal de oro que los relaciona.

La voluntad como deseo consciente: Kuno Fischer entre Spinoza y Nietzsche

Es muy defendible afirmar que el volumen dedicado a Spinoza de la obra Historia de la filosofía moderna de Kuno Fischer es la mediación más importante –si bien no la única– para comprender la relación Spinoza-Nietzsche. A partir de la lectura de esta obra en 1881, Nietzsche reconoce a Spinoza como precursor e identifica una tendencia en común entre ambos: «hacer del conocimiento el afecto más potente». Los principales objetivos de este trabajo son destacar la exposición que Fischer lleva a cabo de algunos de los contenidos de la filosofía spinociana para poder valorar mejor su influencia en Nietzsche y resaltar la importancia que tiene tulo 22 de la edición de 1865, titulado «La voluntad humana», donde se interpreta la voluntad como deseo consciente, lo que tendrá sus consecuencias en la recepción de Nietzsche de la filosofía spinociana, además de permitir una discusión respecto del rol no siempre atendido de la voluntad en Spinoza.

Franquismo e literatura galega: "Non Chores, Sabeliña", a primeira obra da literatura galega de Posguerra. Entre o populismo e o rexoubeirismo

En este estudio se desarrolla, en clave filológico-didáctica, un análisis concienzudo sobre la gestación y la recepción de Non chores, Sabeliña, la primera obra escrita y publicada en gallego tras la Guerra Civil española. Nunca antes se había estudiado esta pieza, por lo que constituye una aproximación muy novedosa tanto el el Área de Filologías Gallega y Portuguesa como en el de Didáctica de la Lengua y la Literatura.

Desarrollo de un dispositivo de liberación para la industria aeroespacial basado en una aleación con memoria de forma de NiTi.

Los dispositivos liberadores son utilizados en los satélites para desplegar los paneles solares, antenas y otros apéndices en las etapas iniciales de su puesta en órbita. En la actualidad, muchos de estos dispositivos tienen actuadores activados con cargas pirotécnicas. Esto trae algunas desventajas: niveles de shock elevados sobre los componentes cercanos, no son reutilizables y no son seguros de manipular. Por ello, es que desde hace unos años se están realizando importantes esfuerzos para desarrollar dispositivos no-pirotécnicos. En este trabajo se realiza el desarrollo de un dispositivo liberador que utiliza como actuador un cilindro tubular de una Aleación de Memoria de Forma (SMA, del inglés Shape Memory Alloys) de NiTi. Las SMAs tienen la posibilidad de deformarse por debajo de determinada temperatura y luego al calentarse, recuperar su forma original. Al restringir mecánicamente la trayectoria de recuperación, la aleación genera un esfuerzo mecánico. En el presente trabajo se caracteriza esta aleación, recurriendo a diversos tratamientos térmicos para obtener las propiedades deseadas. La liberación del conjunto se produce cuando el elemento de unión entre las piezas, el cual consiste en un bulón con una entalla circunferencial, se fractura debido al estiramiento de un actuador de NiTi previamente comprimido. Dada la importancia del bulón, se estudiaron diversos materiales para el mismo. Además se analizó el efecto de la geometría de la entalla y la profundidad de la misma. Finalmente, se realizaron pruebas de integración entre el actuador de SMA, el bulón y otros elementos auxiliares. Se pudo probar el funcionamiento del conjunto con éxito.

Desarrollo de un dispositivo de liberación para la industria aeroespacial basado en una aleación con memoria de forma de NiTi.

Los dispositivos liberadores son utilizados en los satélites para desplegar los paneles solares, antenas y otros apéndices en las etapas iniciales de su puesta en órbita. En la actualidad, muchos de estos dispositivos tienen actuadores activados con cargas pirotécnicas. Esto trae algunas desventajas: niveles de shock elevados sobre los componentes cercanos, no son reutilizables y no son seguros de manipular. Por ello, es que desde hace unos años se están realizando importantes esfuerzos para desarrollar dispositivos no-pirotécnicos. En este trabajo se realiza el desarrollo de un dispositivo liberador que utiliza como actuador un cilindro tubular de una Aleación de Memoria de Forma (SMA, del inglés Shape Memory Alloys) de NiTi. Las SMAs tienen la posibilidad de deformarse por debajo de determinada temperatura y luego al calentarse, recuperar su forma original. Al restringir mecánicamente la trayectoria de recuperación, la aleación genera un esfuerzo mecánico. En el presente trabajo se caracteriza esta aleación, recurriendo a diversos tratamientos térmicos para obtener las propiedades deseadas. La liberación del conjunto se produce cuando el elemento de unión entre las piezas, el cual consiste en un bulón con una entalla circunferencial, se fractura debido al estiramiento de un actuador de NiTi previamente comprimido. Dada la importancia del bulón, se estudiaron diversos materiales para el mismo. Además se analizó el efecto de la geometría de la entalla y la profundidad de la misma. Finalmente, se realizaron pruebas de integración entre el actuador de SMA, el bulón y otros elementos auxiliares. Se pudo probar el funcionamiento del conjunto con éxito.

Síndrome de Goldenhar: a propósito de un caso

El síndrome de Goldenhar forma parte del espectro de anomalías del primer y segundo arco branquial. Constituye una entidad congénita poco frecuente, caracterizada por la asociación de anomalías oculares, auriculares, mandibulares y vertebrales, y cuya etiología permanece aún desconocida. A continuación describimos el caso de un recién nacido que presenta hallazgos clínicos compatibles, que incluyen la asociación de lipodermoide conjuntival unilateral, apéndices preauriculares, hipoplasia hemifacial e hipoplasia de pabellones auriculares. Así mismo, se hace una breve revisión basada en el conjunto de los casos que hasta el presente han aparecido en la literatura científica.