Cell fate specification during Caenorhabditis elegans male tail development

The cells of the specialized mating structures of the nematode Caenorhabditis elegans adult male tail develop from sex-specific divisions of postembryonic blast cells. One male-specific blast cell, B, is the precursor to all the cells of the copulatory spicules. Both cell interactions and autonomous fate specification mechanisms are utilized in the B lineage to specify fate.

During development the anterior daughter of B, B.a, generates four distinct pairs of cells. Cell ablation experiments indicate that the cells of each pair respond to positional cues provided by other male-specific blast cells. F and U promote anterior fates, Y.p promotes some posterior fates, and the B.a progeny promote posterior fates. The cells within each pair may also interact.

The lin-3/let-23 signalling pathway, identified for its function in C. elegans hermaphrodite vulval induction, mediates the signal from F and U. Reduction-of-function mutations in lin-3 (EGF-like signal), let-23 (receptor), sem-5 (adaptor), let-60 (ras), or lin-45 (raf) disrupt the fates of the anterior cells, and mimic F and U ablation. In addition, ectopically expressed lin-3 disrupts the fates of the posterior cells, and can promote anterior fates even in the absence of F and U.

A genetic screen of over 9000 mutagenized gametes recovered 22 mutations in 20 loci that disrupt fate specification in male tail lineages. Seven of these mutations may represent new genes that play a role in male tail development.

The first division of the B cell is asymmetric. The gene vab-3 is required for specification of B.a fates, and it may represent a factor whose activity is localized to the B.a cell via the gene lin-17. lin-17 acts both at the first division of the B cell and at specific other cell divisions in the lineage.

Structural requirements for catalysis: studies on RTEM-1 β-lactamase

β-lactamases are a group of enzymes that confer resistance to penam and cephem antibiotics by hydrolysis of the β-lactam ring, thereby inactivating the antibiotic. Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Asp 132, a strictly conserved residue among the class A β-lactamases, appears to be involved in substrate binding, catalysis, or both. To study the contribution of residue 132 to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at position 132. Phenotypic screening of all mutants indicated that position 132 is very sensitive to amino acid changes, with only N132C, N132D, N132E, and N132Q showing any appreciable activity. Kinetic analysis of three of these mutants showed increases in K_M, along with substantial decreases in k_(cat). Efforts to trap a stable acyl-enzyme intermediate were unsuccessfuL These results indicate that residue 132 is involved in substrate binding, as well as catalysis, and supports the involvement of this residue in acylation as suggested by Strynadka et al.

Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Lys 73 and Glu 166, two strictly conserved residues among the class A β-lactamases, appear to be involved in substrate binding, catalysis, or both. To study the contribution of these residues to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at positions 73 and 166. Then all 400 possible combinations of mutants were created by combinatorial mutagenesis. The colonies harboring the mutants were screened for growth in the presence of ampicillin. The competent colonys' DNA were sequenced, and kinetic parameters investigated. It was found that lysine is essential at position 73, and that position 166 only tolerated fairly conservative changes (Aspartic acid, Histidine, and Tyrosine). These functional mutants exhibited decreased kcat's, but K_M was close to wild-type levels. The results of the combinatorial mutagenesis experiments indicate that Lysis absolutely required for activity at position 73; no mutation at residue 166 can compensate for loss of the long side chain amine. The active mutants found--K73K/E166D, K73KIE166H, and K73KIE166Y were studied by kinetic analysis. These results reaffirmed the function of residue 166 as important in catalysis, specifically deacylation.

The identity of the residue responsible for enhancing the active site serine (Ser 70) in RTEM-1 β-lactamase has been disputed for some time. Recently, analysis of a crystal structure of RTEM-1 β-lactamase with covalently bound intermediate was published, and it was suggested that Lys 73, a strictly conserved residue among the class A β-lactamases, was acting as a general base, activating Ser 70. For this to be possible, the pK_a of Lys 73 would have to be depressed significantly. In an attempt to assay the pK_a of Lys 73, the mutation K73C was made. This mutant protein can be reacted with 2-bromoethylamine, and activity is restored to near wild type levels. ^(15)N-2-bromoethylamine hydrobromide and ^(13)C-2-bromoethylamine hydrobromide were synthesized. Reacting these compounds with the K73C mutant gives stable isotopic enrichment at residue 73 in the form of aminoethylcysteine, a lysine homologue. The pK_a of an amine can be determined by NMR titration, following the change in chemical shift of either the ^(15)N-amine nuclei or adjacent Be nuclei as pH is changed. Unfortunately, low protein solubility, along with probable label scrambling in the Be experiment, did not permit direct observation of either the ^(15)N or ^(13)C signals. Indirect detection experiments were used to observe the protons bonded directly to the ^(13)C atoms. Two NMR signals were seen, and their chemical shift change with pH variation was noted. The peak which was determined to correspond to the aminoethylcysteine residue shifted from 3.2 ppm down to 2.8 ppm over a pH range of 6.6 to 12.5. The pK_a of the amine at position 73 was determined to be ~10. This indicates that residue 73 does not function as a general base in the acylation step of the reaction. However the experimental measurement takes place in the absence of substrate. Since the enzyme undergoes conformational changes upon substrate binding, the measured pK_a of the free enzyme may not correspond to the pK_a of the enzyme substrate complex.

Species Profiles: Life Histories and Environmental Requirements of Coastal Fishes and Invertebrates (Pacific Northwest): Amphipods

This report wi11 focus largely on the suborders Gammaridea, Caprellidea, and Hyperiidea because of their importance in coastal areas of the northeast Pacific Ocean. (PDF contains 27 pages)

Species Profiles: Life Histories and Environmental Requirements of Coastal Fishes and Invertebrates (Pacific Southwest): Amphipods

(PDF contains 24 pages)

Species Profiles: Life Histories and Environmental Requirements of Coastal Fishes and Invertebrates (Mid-Atlantic): Bay anchovy

The bay anchovy occurs along the Atlantic and Gulf of Mexico coasts, from Cape Cod, Massachusetts, to Yucatan, Mexico (Hildebrand 1963), except for the Florida Keys where it is apparently absent (Daly 1970). (PDF contains 22 pages)

Species Profiles: Life Histories and Environmental Requirements of Coastal Fishes and Invertebrates (Gulf of Mexico): Bay anchovy and Striped Anchovy

(PDF contains 24 pages)

Species Profiles: Life Histories and Environmental Requirements of Coastal Fishes and Invertebrates (Pacific Southwest): Northern anchovy

Three genetically distinct groups: British Columbia to northern California, Southern California to the northern Baja peninsula, and central and southern Baja California. (PDF contains 21 pages)

IBM PC Data Acquisition and Processing Software Evaluation

Commercially available software packages for IBM PC-compatibles are evaluated to use for data acquisition and processing work. Moss Landing Marine Laboratories (MLML) acquired computers since 1978 to use on shipboard data acquisition (Le. CTD, radiometric, etc.) and data processing. First Hewlett-Packard desktops were used then a transition to the DEC VAXstations, with software developed mostly by the author and others at MLML (Broenkow and Reaves, 1993; Feinholz and Broenkow, 1993; Broenkow et al, 1993). IBM PC were at first very slow and limited in available software, so they were not used in the early days. Improved technology such as higher speed microprocessors and a wide range of commercially available software made use of PC more reasonable today. MLML is making a transition towards using the PC for data acquisition and processing. Advantages are portability and available outside support.

Formal methods for control synthesis in partially observed environments : application to autonomous robotic manipulation

Modern robots are increasingly expected to function in uncertain and dynamically challenging environments, often in proximity with humans. In addition, wide scale adoption of robots requires on-the-fly adaptability of software for diverse application. These requirements strongly suggest the need to adopt formal representations of high level goals and safety specifications, especially as temporal logic formulas. This approach allows for the use of formal verification techniques for controller synthesis that can give guarantees for safety and performance. Robots operating in unstructured environments also face limited sensing capability. Correctly inferring a robot's progress toward high level goal can be challenging.

This thesis develops new algorithms for synthesizing discrete controllers in partially known environments under specifications represented as linear temporal logic (LTL) formulas. It is inspired by recent developments in finite abstraction techniques for hybrid systems and motion planning problems. The robot and its environment is assumed to have a finite abstraction as a Partially Observable Markov Decision Process (POMDP), which is a powerful model class capable of representing a wide variety of problems. However, synthesizing controllers that satisfy LTL goals over POMDPs is a challenging problem which has received only limited attention.

This thesis proposes tractable, approximate algorithms for the control synthesis problem using Finite State Controllers (FSCs). The use of FSCs to control finite POMDPs allows for the closed system to be analyzed as finite global Markov chain. The thesis explicitly shows how transient and steady state behavior of the global Markov chains can be related to two different criteria with respect to satisfaction of LTL formulas. First, the maximization of the probability of LTL satisfaction is related to an optimization problem over a parametrization of the FSC. Analytic computation of gradients are derived which allows the use of first order optimization techniques.

The second criterion encourages rapid and frequent visits to a restricted set of states over infinite executions. It is formulated as a constrained optimization problem with a discounted long term reward objective by the novel utilization of a fundamental equation for Markov chains - the Poisson equation. A new constrained policy iteration technique is proposed to solve the resulting dynamic program, which also provides a way to escape local maxima.

The algorithms proposed in the thesis are applied to the task planning and execution challenges faced during the DARPA Autonomous Robotic Manipulation - Software challenge.

Desarrollo de software para la realización de ensayos dinámicos de mecanismos de cinemática paralela

[ES]El presente Trabajo de Fin de Grado tiene como objetivo contribuir al desarrollo de un proyecto de investigación mediante la programación y control del movimiento de mecanismos de cinemática paralela para la realización de ensayos dinámicos. Dicho proyecto está enmarcado dentro de una línea de investigación del grupo de investigación CompMech de la UPV-­‐EHU que gira en torno al desarrollo y estudio de este tipo de mecanismos. Esto es; este trabajo, más allá de la utilidad que pudiera tener por sí mismo, está pensado para formar parte de un proyecto de mayor envergadura, para cuyo éxito será imprescindible la colaboración con otros investigadores y la integración de este trabajo con los realizados por ellos. Consiste en la creación de un software para el control y movimiento de mecanismos, generando vibraciones para la realización de ensayos dinámicos. Para ello, se programarán sobre la plataforma LabVIEW la interfaz de usuario y el motor de cálculo. Una vez se compruebe que el programa funciona correctamente, se integrará dentro de un programa principal, un control articular que será el encargado de comunicarse con la máquina. Posteriormente, se procederá a la realización de ensayos experimentales sobre los propios robots, en taller. Se tomarán medidas mediante acelerómetros y otros dispositivos, determinando las medidas más adecuadas para su correcta validación. Finalmente, se generalizará el trabajo realizado para posibilitar su empleo futuro en diferentes mecanismos