Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

Modifications to the Suzuki reaction and mechanistic insights on the NBS mediated cleavage of benzylidene acetals /

One of the most challenging tasks for a synthetic organic chemist today, is the development of chemo, regio, and stereoselective methodologies toward the total synthesis of macromolecules. r . The objective of my thesis was to develop methodologies towards this end. The first part of my project was to develop highly functionalized chirons from D-glucose, a cheap, chiral starting material, to be utilized in this capacity. The second part of the project dealt with modifying the carbon-carbon bond forming Suzuki reaction, which is utilized quite often as a means of combining molecular sub units in total synthesis applications. As previously stated the first area of the project was to develop high value chirons from D-glucose, but the mechanism of their formation was also investigated. The free radical initiated oxidative fragmentation of benzylidene acetals was investigated through the use of several test-case substrates in order to unravel the possible mechanistic pathways. This was performed by reacting the different acetals with N-bromosuccinimide and benzoyl peroxide in chlorobenzene at 70^C in all cases. Of the three mechanistic pathways discussed in the literature, it was determined, from the various reaction products obtained, that the fragmentation of the initial benzylic radical does not occur spontaneously but rather, oxidation proceeds to give the benzyl bromide, which then fragments via a polar pathway. It was also discovered that the regioselectivity of the fragmentation step could be altered through incorporation of an allylic system into the benzylidene acetal. This allows for the acquisition of a new set of densely functionalized. chiral, valuable synthetic intermediates in only a few steps and in high yields from a-Dglucose. The second part of the project was the utilization of the phosphonium salt room temperature ionic liquid tetradecyltrihexylphosphonium chloride (THPC) as an efficient reusable medium for the palladium catalyzed Suzuki cross-coupling reaction of aryl halides, including aryl chlorides, under mild conditions. The cross-coupling reactions were found to proceed in THPC containing small amounts of water and toluene using potassium phosphate and 1% Pd2(dba)3. Variously substituted iodobenzenes, including electron rich derivatives, reacted efficiently in THPC with a variety of arylboronic acids and afforded complete conversion within 1 hour at 50 ^C. The corresponding aryl bromides also reacted under these conditions with the addition of a catalytic amount of triphenylphosphine that allowed for complete conversion and high isolated yields. The reactions involving aryl chlorides were considerably slower, although the addition of triphenylphosphine and heating at 70 ^C allowed high conversion of electron deficient derivatives. Addition of water and hexane to the reaction products results in a triphasic system in which the top hexane phase contained the biaryl products, the palladium catalyst remained fully dissolved in the central THPC layer, while the inorganic salts were extracted into the lower aqueous phase. The catalyst was then recycled by removing the top and bottom layers and adding the reagents to the ionic liquid which was heated again at 50 ^C; resulting in complete turnover of iodobenzene. Repetition of this procedure gave the biphenyl product in 82-97% yield (repeated five times) for both the initial and recycled reaction sequences.

Intraspecific and interspecific social variation in the sweat bee Lasioglossum Malachurum and other members of the subgenus Evylaeus

Sweat bees exhibit a range of social behaviours, from solitary nesting, in which no workers are produced, to strong eusociality, in which workers exhibit a high degree of altruism, behaviour that is measured by the degree of personal reproductive sacrifice. Field studies were carried out for seven weeks during May-June 2000 in southern Greece in order to investigate intraspecific social variation, and test the hypothesis of a north-south cline of decreasing eusociality in the obligately eusocial sweat bee L. (E.) malachurum. A comparative study, using principal components analysis, was performed to determine if patterns of intraspecific social variation in L. malachurum reflect the patterns of social variation within the subgenus, Evylaeus, as a whole. The results of the field study reveal that, in Greece, two worker broods were produced followed by a third brood consisting of gynes, males and some workers, indicating that there was an overlap in worker and gyne production. There was strong caste distinction between queens and workers. Workers actively foraged and participated in nest construction as most workers (58%, n=303) had a high degree of mandibular wear. Workers did not participate in the oviposition of Brood 3 gynes since only 0.7% (n=278) of workers were mated. Furthermore, queen survival until the end of Brood 3 and a substantial size differential of 10.6% between queens and workers suggested that queen domination over worker behaviour during the early to mid-part of the colony cycle was plausible. Male production in Brood 3 by some workers was likely, since the timing of worker ovarian development corresponded with the timing of male production. These findings suggest that workers of the first two broods were primarily altruistic, but some (28%) Brood 1 (9%) and Brood 2 (19%) workers produced males, indicating that the degree of altruistic behaviour declined during the lifetime of the colony. In comparison with other L. malachurum populations in Europe, the Greek population of L. malachurum had a weaker social level as a result of the higher proportion of workers potentially involved in male production, thus 3 supporting the hypothesis of a southerly cline of decreasing eusociality. Furthermore, intraspecific variation in social level across Europe appears to be due to longer breeding seasons in more southerly locations that would promote the production of larger colonies and provide opportunities for workers to evade queen control. The comparative study using principal components analysis on 20 solitary (of the subgenera Evylaeus and Lasioglossum), eusocial and socially polymorphic Evylaeus species and populations reveals that six traits are closely associated with stronger eusociality in Evylaeus. These traits are: (1) a reduction in the proportion of males in the early brood(s); (2) a reduction in the proportion of females that mate; (3) an increase in the mean number of first brood workers; (4) a reduction in the proportion of females with developed ovaries; (5) an increase in size dimorphism between castes, and (6) nest guarding. These are traits that most significantly define principal component one and therefore distinguish social type as indicated by a clear separation of the eusocial and the solitary populations, with a socially polymorphic species falling in between. Furthermore, most of these traits are under foundress control and may suggest that the evolutionary loss or gain of eusociality is based on selection pressures on a founding female. Colony size and female ovarian development are common factors distinguishing social variation in L. malachurum and within the subgenus as a whole. The principal components analysis excluding the solitary species and the socially aberrant L. marginatum populations show the L. malachurum populations separated based on an increasing proportion of workers with developed ovaries as populations are found more south, lending further support to the hypothesis of a north-south cline of decreasing eusociality.

An Exploration Into Doctoral Students' Sense of Community and its Influence on the Formation of Informal Mentoring Relationships

This exploratory mixed method research project was designed to investigate an area of doctoral education that has received little attention in the past. This research focused specifically on the non-intellectual, hoped-for by-products of doctoral education; the dynamic processes of developing and maintaining both a sense of community and informal mentoring relationships. The design of the study captured the experiences of doctoral students and alumni at various time periods in the doctoral program. Participants represented a diverse group of students with differences in professional and academic backgrounds and life stages. A pilot study for this research suggested that the presence of a sense of community and informal mentoring may provide the necessary relationships to support this diversity. The primary question at the forefront of this study was: Do doctoral students feel connected to one another? Five subquestions were developed to address this research topic: Does a sense of community already exist and flourish in doctoral education? Are the programs and resources of the doctoral program organized to nurture the creation and maintenance of a sense of community? Is a sense of community a foundational element in the formation of naturally occurring relationships among doctoral students? What educational and socio-emotional benefits are associated with informal mentoring relationships during the doctoral experience? and Do doctoral students perceive a change in their development as stewards of their discipline over time? The principal methods used to investigate these research questions combined both quantitative and qualitative techniques in a concurrent time sequence. The quantitative portion of the study involved a questionnaire, while the qualitative portion involved two approaches; face-to-face interviews and an open-ended question at the end of the questionnaire. Findings from the study indicated that the presence of both sense of community and informal mentoring enhance the overall quality of doctoral education. Program elements that enhanced or hindered connection between students were identified. Both the dynamics and the emotional, social, and academic benefits of informal mentoring were elucidated. Over time participants perceived changes in their development of the qualities assqciated with stewardship. This study brought the "hoped-for by-products" associated with doctoral education from the background shadows to an illuminated position at the forefront of inquiry.

Représentation et recherche de motifs cycliques et structuraux d’ARN connus dans les structures secondaires

L'acide désoxyribonucléique (ADN) et l'acide ribonucléique (ARN) sont des polymères de nucléotides essentiels à la cellule. À l'inverse de l'ADN qui sert principalement à stocker l'information génétique, les ARN sont impliqués dans plusieurs processus métaboliques. Par exemple, ils transmettent l’information génétique codée dans l’ADN. Ils sont essentiels pour la maturation des autres ARN, la régulation de l’expression génétique, la prévention de la dégradation des chromosomes et le ciblage des protéines dans la cellule. La polyvalence fonctionnelle de l'ARN résulte de sa plus grande diversité structurale. Notre laboratoire a développé MC-Fold, un algorithme pour prédire la structure des ARN qu'on représente avec des graphes d'interactions inter-nucléotidiques. Les sommets de ces graphes représentent les nucléotides et les arêtes leurs interactions. Notre laboratoire a aussi observé qu'un petit ensemble de cycles d'interactions à lui seul définit la structure de n'importe quel motif d'ARN. La formation de ces cycles dépend de la séquence de nucléotides et MC-Fold détermine les cycles les plus probables étant donnée cette séquence. Mon projet de maîtrise a été, dans un premier temps, de définir une base de données des motifs structuraux et fonctionnels d'ARN, bdMotifs, en terme de ces cycles. Par la suite, j’ai implanté un algorithme, MC-Motifs, qui recherche ces motifs dans des graphes d'interactions et, entre autres, ceux générés par MC-Fold. Finalement, j’ai validé mon algorithme sur des ARN dont la structure est connue, tels que les ARN ribosomaux (ARNr) 5S, 16S et 23S, et l'ARN utilisé pour prédire la structure des riborégulateurs. Le mémoire est divisé en cinq chapitres. Le premier chapitre présente la structure chimique, les fonctions cellulaires de l'ARN et le repliement structural du polymère. Dans le deuxième chapitre, je décris la base de données bdMotifs. Dans le troisième chapitre, l’algorithme de recherche MC-Motifs est introduit. Le quatrième chapitre présente les résultats de la validation et des prédictions. Finalement, le dernier chapitre porte sur la discussion des résultats suivis d’une conclusion sur le travail.

Correlates of protective immunity against hepatitis C virus

Le virus de l’hépatite C (VHC) infecte ~185 millions d’individus dans le monde. Malgré le développement des nouvelles thérapies dirigées contre le VHC, on compte deux millions de nouvelles infections chaque année, en particulier dans les pays sous-développés et les populations marginalisées. Comme pour la plupart des virus à infection chronique, le développement d’un vaccin prophylactique efficace est limité par le manque de caractérisation des déterminants de la mémoire immunitaire protectrice lors des épisodes de réinfection naturelle chez les êtres humains. Le VHC représente un modèle unique au sein des virus à infection chronique pour étudier l’immunité protectrice. En effet ~30% des patients infectés par le VHC peuvent être guéris suite à un premier épisode d’infection spontanément. Dans cette thèse, nous avons étudié l’immunité protectrice contre le VHC dans une cohorte d’utilisateurs de drogues par injection qui sont à risque d’être infectés ou réinfectés. Notre hypothèse est que la majorité des patients qui ont résolu une première infection par le VHC sont protégés contre le développement d’une infection chronique s’ils sont réexposés. Cette immunité protectrice est associée à la présence des cellules T CD4 et CD8 polyfonctionnelles qui possèdent des fréquences, magnitudes et avidités élevées. La capacité protectrice des cellules T mémoire contre les séquences variables du VHC est dépendante de la diversité et flexibilité du répertoire de leurs récepteurs de cellules T (TCR), qui reconnaissent les séquences variables des épitopes ciblés. Notre premier objectif était de définir et détailler les déterminants de l’immunité protectrice conférée par les cellules T spécifiques du VHC. Nos résultats ont montré que la protection pendant l’épisode de réinfection était associée à une augmentation de la magnitude et du spectre des réponses spécifiques par les cellules T CD4 et CD8 polyfonctionnelles, ainsi que par l’apparition d’une population de cellules T tétramère+ CD8+ effectrices qui expriment faiblement le marqueur CD127 (CD127lo) lors du pic de la réponse. Chez les patients qui ont développé une infection chronique pendant l’épisode de réinfection, nous avons observé une expansion très limitée des cellules T CD4 et CD8. Le séquençage des épitopes ciblés par les cellules T CD8 chez ces patients qui sont non-protégés a montré que les séquences de ces épitopes sont différentes des séquences de référence qui étaient utilisées pour tous les essais immunologiques de cette étude. Le deuxième objectif était d’analyser la dynamique du répertoire des TCRs des cellules T CD8 spécifiques chez les patients protégés versus les patients non-protégés. Nos résultats ont montré que le répertoire des cellules T CD8 spécifiques est plus focalisé que chez les patients protégés. En plus, nous avons observé que les clonotypes qui forment le répertoire chez les patients protégés sont distincts de ceux chez les patients non-protégés. Ces clonotypes chez les patients protégés ont montré de plus grandes avidité et polyfonctionnalité que leurs homologues chez les patients non-protégés. En conclusion, nos résultats suggèrent que la protection contre le développement d’une infection chronique pendant l’épisode de réinfection par le VHC est associée à une augmentation de la magnitude, du spectre et de la fonctionnalité des réponses des cellules T spécifiques, ainsi qu’à un répertoire des TCRs plus focalisé composé des clonotypes distincts qui possèdent de plus grandes avidité et polyfonctionnalité que chez les patients non-protégés. L’homologie des séquences des souches virales entre les différents épisodes de l’infection est un déterminant majeur de l’établissement d’une protection efficace. Ces résultats ont donc des implications très importantes pour le développement d’un vaccin prophylactique contre le VHC et d’autres virus à infection chronique.

In vitro and in vivo effects of deoxynivalenol (DON) mycotoxin on porcine reproductive and respiratory syndrome virus (PRRSV) in piglets

Les récoltes de céréales sont souvent contaminées par des moisissures qui se développent pendant la récolte et l’entreposage et produisent des métabolites secondaires appelés mycotoxines. Le porc est reconnu pour être sensible au déoxynivalénol (DON). L’infection virale la plus importante chez le porc est causée par le virus du syndrome reproducteur et respiratoire porcin (VSRRP). Celui-ci provoque un syndrome grippal et des troubles de reproduction. L’objectif du présent projet était de déterminer l'effet in vitro de DON sur la réplication du VSRRP dans de lignées cellulaires permissives, MARC-145 et PAM, et déterminer in vivo l'impact de DON dans des aliments naturellement contaminés sur l’infection au VSRRP chez le porcelet. Tout d’abord, les cellules ont été incubées avec des doses croissantes de DON et ont été infectées avec du VSRRP pour évaluer la viabilité et la mortalité cellulaire, la réplication virale et l’expression de cytokines. Les résultats ont montré que les concentrations de DON de 560ng/ml et plus affectaient significativement la survie des cellules MARC-145 et PAM infectées par le VSRRP. En revanche, il y avait une augmentation significative de la viabilité et une réduction de la mortalité cellulaire à des concentrations de DON de 140 à 280 ng/ml pour les cellules PAM et de 70 à 280 ng/ml pour les cellules MARC-145 avec une réduction de l'effet cytopathique provoqué parle VSRRP. Au niveau in vivo, 30 porcelets divisés en 3 groupes de 10 porcelets et nourris pendant 2 semaines avec 3 différentes diètes naturellement ont été contaminées avec DON (0; 2,5 et 3,5 mg/kg). Les porcelets ont été subdivisés en 6 groupes, 3 groupes de 6 porcelets et ont été exposés au DON pendant 2 semaines et infectés par voie intratrachéale et intramusculaire avec le virus. Les 3 autres groupes de 4 porcelets servaient de contrôle non infectés. Les signes cliniques ont été enregistrés pendant 21 jours. La virémie a été évaluée par PCR. À la fin de l’expérimentation, les porcelets ont été euthanasiés et les lésions pulmonaires ont été évaluées. Les résultats ont montré que l’ingestion de DON à 3,5 mg/kg a augmenté l’effet du VSRRP sur la sévérité des signes cliniques, les lésions pulmonaires et la mortalité. L’ingestion de DON à 2,5 mg/kg a entrainé une augmentation de la virémie au jour 3 après l’infection mais sans impact sur les signes cliniques et les lésions pulmonaires. Mot clés: DON, VSRRP, MARC-145, PAM, effet cytopathique, cytokines, PCR

Application of Fermentation Techniques in the Utilization of Prawn Shell Waste

The present study aimed at the utlisation of microbial organisms for the production of good quality chitin and chitosan. The three strains used for the study were Lactobacillus plantarum, Lactobacililus brevis and Bacillus subtilis. These strains were selected on the basis of their acid producing ability to reduce the pH of the fermenting substrates to prevent spoilage and thus caused demineralisation of the shell. Besides, the proteolytic enzymes in these strains acted on proteinaceous covering of shrimp and thus caused deprotenisation of shrimp shell waste. Thus the two processes involved in chitin production can be affected to certain extent using bacterial fermentation of shrimp shell.Optimization parameters like fermentation period, quantity of inoculum, type of sugar, concentration of sugar etc. for fermentation with three different strains were studied. For these, parameters like pH, Total titrable acidity (TTA), changes in sugar concentration, changes in microbial count, sensory changes etc. were studied.Fermentation study with Lactobacillus plantarum was continued with 20% w/v jaggery broth for 15 days. The inoculum prepared yislded a cell concentration of approximately 108 CFU/ml. In the present study, lactic acid and dilute hydrochloric acid were used for initial pH adjustment because; without adjusting the initial pH, it took more than 5 hours for the lactic acid bacteria to convert glucose to lactic acid and during this delay spoilage occurred due to putrefying enzymes active at neutral or higher pH. During the fermentation study, pH first decreased in correspondence with increase in TTA values. This showed a clear indication of acid production by the strain. This trend continued till their proteolytic activity showed an increasing trend. When the available sugar source started depleting, proteolytic activity also decreased and pH increased. This was clearly reflected in the sensory evaluation results. Lactic acid treated samples showed greater extent of demineralization and deprotenisation at the end of fermentation study than hydrochloric acid treated samples. It can be due to the effect of strong hydrochloric acid on the initial microbial count, which directly affects the fermentation process. At the end of fermentation, about 76.5% of ash was removed in lactic acid treated samples and 71.8% in hydrochloric acid treated samples; 72.8% of proteins in lactic acid treated samples and 70.6% in hydrochloric acid treated samples.The residual protein and ash in the fermented residue were reduced to permissible limit by treatment with 0.8N HCI and 1M NaOH. Characteristics of chitin like chitin content, ash content, protein content, % of N- acetylation etc. were studied. Quality characteristics like viscosity, degree of deacetylation and molecular weight of chitosan prepared were also compared. The chitosan samples prepared from lactic acid treated showed high viscosity than HCI treated samples. But degree of deacetylation is more in HCI treated samples than lactic acid treated ones. Characteristics of protein liquor obtained like its biogenic composition, amino acid composition, total volatile base nitrogen, alpha amino nitrogen etc. also were studied to find out its suitability as animal feed supplement.Optimization of fermentation parameters for Lactobacillus brevis fermentation study was also conducted and parameters were standardized. Then detailed fermentation study was done in 20%wlv jaggery broth for 17 days. Also the effect of two different acid treatments (mild HCI and lactic acid) used for initial pH adjustment on chitin production were also studied. In this study also trend of changes in pH. changes in sugar concentration ,microbial count changes were similar to Lactobacillus plantarum studies. At the end of fermentation, residual protein in the samples were only 32.48% in HCI treated samples and 31.85% in lactic acid treated samples. The residual ash content was about 33.68% in HCI treated ones and 32.52% in lactic acid treated ones. The fermented residue was converted to chitin with good characteristics by treatment with 1.2MNaOH and 1NHCI.Characteristics of chitin samples prepared were studied and extent of Nacetylation was about 84% in HCI treated chitin and 85%in lactic acid treated ones assessed from FTIR spectrum. Chitosan was prepared from these samples by usual chemical method and its extent of solubility, degree of deacetylation, viscosity and molecular weight etc were studied. The values of viscosity and molecular weight of the samples prepared were comparatively less than the chitosan prepared by Lactobacillus plantarum fermentation. Characteristics of protein liquor obtained were analyzed to determine its quality and is suitability as animal feed supplement.Another strain used for the study was Bacillus subtilis and fermentation was carried out in 20%w/v jaggery broth for 15 days. It was found that Bacillus subtilis was more efficient than other Lactobacillus species for deprotenisation and demineralization. This was mainly due to the difference in the proteolytic nature of the strains. About 84% of protein and 72% of ash were removed at the end of fermentation. Considering the statistical significance (Pof demineralization and deproteinisation, we have taken 0.8N HCI for the demineralization study and 0.6M NaOH for deprotenisation study. Properties of chitin and chitosan prepared were analyzed and studied.

A critical study of major construction projects in kerala vis-a-vis effectiveness in project implementation

Chapter 1 presents a brief note on the state at which the construction industry stands at present, bringing into focus the significance of the critical study. Relevance of the study, area of investigation and objectives of the study are outlined in this chapter. The 2nd chapter presents a review of the literature on the relevant areas. In the third chapter an analysis on time and cost overrun in construction highlighting the major factors responsible for it has been done. A couple of case studies to estimate loss to the nation on account of delay in construction have been presented in the chapter. The need for an appropriate estimate and a competent contractor has been emphasised for improving effectiveness in the project implementation. Certain useful equations and thoughts have been formulated on this area in this chapter that can be followed in State PWD and other Govt. organisations. Case studies on project implementation of major projects undertaken by Government sponsored/supported organizations in Kerala have been dealt with in Chapter 4. A detailed description of the project of Kerala Legislature Complex with a critical analysis has been given in this chapter. A detailed account of the investigations carried out on the construction of International Stadium, a sports project of Greater Cochin Development Authority is included here. The project details of Cochin International Airport at Nedumbassery, its promoters and contractors are also discussed in Chapter 4. Various aspects of implementation which led the above projects successful have been discussed in chapter 5. The data collected were analysed through discussion and perceptions to arrive at certain conclusions. The emergence of front-loaded contract and its impact on economics of the project execution are dealt with in this chapter. Analysis of delays in respect of the various project narrated in chapter 3 has been done here. The root causes of the project time and overrun and its remedial measures are also enlisted in this chapter. Study of cost and time overrun of any construction project IS a part of construction management. Under the present environment of heavy investment on construction activities in India, the consequences of mismanagement many a time lead to excessive expenditure which are not be avoidable. Cost consciousness, therefore has to be keener than ever before. Optimization in investment can be achieved by improved dynamism in construction management. The successful completion of coristruction projects within the specified programme, optimizing three major attributes of the process - quality, schedule and costs - has become the most valuable and challenging task for the engineer - managers to perform. So, the various aspects of construction management such as cost control, schedule control, quality assurance, management techniques etc. have also been discussed in this fifth chapter. Chapter 6 summarises the conclusions drawn from the above criticalr1 of rhajor construction projects in Kerala.