Two stage abe fermentation with in situ pervaporation and high cell density

Dissertation presented to Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa for obtaining the master degree in Membrane Engineering

Spontaneous regional heading of extensive skin lesions in diffuse cutaneous leishmaniasis (DCL)

The authors report a case of diffuse cutaneous leishmaniasis, with longstanding evolution and presenting with diffuse infiltrated lesions rich in amastigotes in the absence of mucosal involvement. In situ characterization with monoclonal antibodies revealed Leishmania amazonensis. Large regional lesions have presented spontaneous healing without specific therapy. Considering that DCL presents with a defect in the cellular immune response, thisfact demonstrate that this patient may develop a regional cellular immune response enough to destroy the parasites and to produce clearing of some lesions.

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology

Cell-to cell Communication in Pectobacterium wasabiae

Dissertation presented to obtain the PhD degree in Biology

Burkitt's lymphoma of the duodenum in a patient with AIDS

Non-Hodgkin's lymphoma of B-cell type is the second most common neoplasm after Kaposi's sarcoma, among patients with human immunodeficiency virus infection. Most non-Hodgkin's lymphoma cases that are associated with acquired immunodeficiency syndrome involve extranodal sites, especially the digestive tract and the central nervous system. We report a case of primary lymphoma of the duodenum in a patient with AIDS. Upper gastrointestinal endoscopy revealed pseudopolypoid masses found in the second portion of the duodenum. A complete diagnostic study including histological, immunohistochemical and virological analyses showed high-grade B-cell Burkitt's lymphoma. The Epstein-Barr virus genome was detected in biopsies by immunohistochemical and in situ hybridization.

Novel Cryopreservation Strategies for Cell‐Therapies and Pre‐Clinical Research

The expanding need for complex biologics for therapeutic applications, in‐vitro pharmacology and toxicology studies and fundamental research demands the production of banks of well‐characterized and safety‐tested stocks of a large number of cell/tissue samples. This implies the development of effective cryopreservation methodologies that can cope with process scalability and automation and must reflect the biological and physical properties of the cells as these can be significantly altered by the process.

Immunopathogenesis and neurological manifestations associated to HTLV-1 infection

The human T lymphotropic virus type-1 (HTLV-1) was the first human retrovirus identified. The virus is transmitted through sexual intercourse, blood transfusion, sharing of contaminated needles or syringes and from mother to child, mainly through breastfeeding. In addition to the well-known association between HTLV-1 and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), several diseases and neurologic manifestations have been associated with the virus. This review was conducted through a PubMed search of the terms HTLV-1, immune response and neurological diseases. Emphasis was given to the most recent data regarding pathogenesis and clinical manifestations of HTLV-1 infection. The aim of the review is to analyze the immune response and the variety of neurological manifestations associated to HTLV-1 infection. A total of 102 articles were reviewed. The literature shows that a large percentage of HTLV-1 infected individuals have others neurological symptoms than HAM/TSP. Increased understanding of these numerous others clinical manifestations associated to the virus than adult T cell leukemia/lymphoma (ATLL) and HAM/TSP has challenged the view that HTLV-1 is a low morbidity infection.

Burkitt-like lymphoma in an infant: a case report

Childhood non-Hodgkin's lymphomas, including Burkitt and Burkitt-like, are rarely diagnosed in infants. A case of B-cell lymphoma in a 13-month-old girl with extensive abdominal disease, ascites, pleural effusion, and tumor lysis syndrome is reported. Phenotypic analysis showed a germinal center B-cell phenotype, and a B-cell clonality was confirmed by polymerase chain reaction. There was no evidence of Epstein-Barr and HIV infection. The case herein reported emphasizes the need for considering the diagnosis of lymphoma even in very young children.

Algorithms and tools for in silico design of cell factories

PhD thesis in Bioengineering

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

Expression of histone methyltransferases as novel biomarkers for renal cell tumor diagnosis and prognostication

Renal cell tumors (RCTs) are the most lethal of the common urological cancers. The widespread use of imaging entailed an increased detection of small renal masses, emphasizing the need for accurate distinction between benign and malignant RCTs, which is critical for adequate therapeutic management. Histone methylation has been implicated in renal tumorigenesis, but its potential clinical value as RCT biomarker remains mostly unexplored. Hence, the main goal of this study was to identify differentially expressed histone methyltransferases (HMTs) and histone demethylases (HDMs) that might prove useful for RCT diagnosis and prognostication, emphasizing the discrimination between oncocytoma (a benign tumor) and renal cell carcinoma (RCC), especially the chromophobe subtype (chRCC). We found that the expression levels of three genes-SMYD2, SETD3, and NO66-was significantly altered in a set of RCTs, which was further validated in a large independent cohort. Higher expression levels were found in RCTs compared to normal renal tissues (RNTs) and in chRCCs comparatively to oncocytomas. SMYD2 and SETD3 mRNA levels correlated with protein expression assessed by immunohistochemistry. SMYD2 transcript levels discriminated RCTs from RNT, with 82.1% sensitivity and 100% specificity (AUC=0.959), and distinguished chRCCs from oncocytomas, with 71.0% sensitivity and 73.3% specificity (AUC: 0.784). Low expression levels of SMYD2, SETD3, and NO66 were significantly associated with shorter disease-specific and disease-free survival, especially in patients with non-organ confined tumors. We conclude that expression of selected HMTs and HDMs might constitute novel biomarkers to assist in RCT diagnosis and assessment of tumor aggressiveness.

Estudos citológicos em Hemíoteros da família Coreidae

A more or less detailed study of the spermatogenesis in six species of Hemiptera belonging to the Coreid Family is made in the present paper. The species studied and their respective chromosome numbers were: 1) Diactor bilineatus (Fabr.) : spermatogonia with 20 + X, primary spermatocytes with 10 + X, X dividing equationaliv in the first division and passing undivided to one pole in the second. 2) Lcptoglossus gonagra (Fabr.) : spermatogonia with 20 + X, primary spermatocytes with 10 + X, X dividing equationally in the first division and passing undivided to one pole in the second. 3) Phthia picta (Drury) : spermatogonia with 20 + X, primary spermatocytes with 10 + X, X dividing equationally in the first division and passing undivided to one pole in the second. 4) Anisocelis foliacea Fabr. : spermatogonia with 26 + X fthe highest mumber hitherto known in the Family), primary .spermatocytes with 13 + X, X dividing equationally in the first division an passing undivided to one pole in the second. 5) Pachylis pharaonis (Herbtst) : spermatogonia with 16 + X, primary spermatocytes with 8 + X. Behaviour of the heteroehromosome not referred. 6) Pachylis laticornis (Fabr.) : spermatogonia with 14 + X, primary spermatocytes with 7 + X, X passing undivided to one pole in the first division and therefore secondary spermatocytes with 7 + X and 7 chromosomes. General results and conclusions a) Pairing modus of the chromosomes (Telosynapsis or Farasynapsis ?) - In several species of the Coreld bugs the history of the chromosomes from the diffuse stage till diakinesis cannot be follewed in detail due specially to the fact that lhe bivalents, as soon as they begin to be individually distinct they appear as irregular and extremely lax chromatic areas, which through an obscure process give rise to the diakinesis and then to the metaphase chomosomes. Fortunately I was able to analyse the genesis of the cross-shaped chromosomes, becoming thus convinced that even in the less favorable cases like that of Phthia, in which the crosses develop from four small condensation areas of the diffuse chromosomes, nothing in the process permit to interpret the final results as being due to a previous telosynaptic pairing. In the case of long bivalents formed by two parallel strands intimately united at both endsegments and more or less widely open in the middle (Leptoglossus, Pachylis), I could see that the lateral arms of the crosses originate from condensation centers created by a torsion or bending in the unpaired parts of the chromosomes In the relatively short bivalents the lateral branches of the cross are formed in the middle but in the long ones, whose median opening is sometimes considerable, two asymetrical branches or even two independent crosses may develop in the same pair. These observations put away the idea of an end-to-end pairing of the chromosomes, since if it had occured the lateral arms of the crosses would always be symetrical and median and never more than two. The direct observation of a side- toside pairing of the chromosomal threads at synizesis, is in foil agreement with the complete lack of evidence in favour of telosynapsis. b) Anaphasic bridges and interzonal connections - The chromosomes as they separate from each other in anaphase they remain connected by means of two lateral strands corresponding to the unpaired segmenas observed in the bivalents at the stages preceding metaphase. In the early anaphase the chromosomes again reproduce the form they had in late diafcinesis. The connecting threads which may be thick and intensely coloured are generally curved and sometimes unequal in lenght, one being much longer than the other and forming a loop outwardly. This fact points to a continuous flow of chromosomal substance independently from both chromosomes of the pair rather than to a mechanical stretching of a sticky substance. At the end of anaphase almost all the material which formed the bridges is reduced to two small cones from whose vertices a very fine and pale fibril takes its origin. The interzonal fibres, therefore, may be considered as the remnant of the anaphasic bridges. Abnormal behaviour of the anaphase chromosomes showed to be useful in aiding the interpretation of normal aspects. It has been suggested by Schrader (1944) "that the interzonal is nothing more than a sticky coating of the chromosome which is stretched like mucilage between the daughter chromosomes as they move further and further apart". The paired chromosomes being enclosed in a commom sheath, as they separate they give origin to a tube which becomes more and more stretched. Later the walls of the tube collapse forming in this manner an interzonal element. My observations, however, do not confirm Schrader's tubular theory of interzonal connections. In the aspects seen at anaphase of the primary spermatocytes and described in this paper as chromosomal bridges nothing suggests a tubular structure. There is no doubt that the chromosomes are here connected by two independent strands in the first division of the spermatocytes and by a single one in the second. The manner in which the chromosomes separate supports the idea of transverse divion, leaving little place for another interpretation. c) Ptafanoeomc and chromatoid bodies - The colourabtlity of the plasmosome in Diactor and Anisocelis showed to be highly variable. In the latter species, one may find in the same cyst nuclei provided with two intensely coloured bodies, the larger of which being the plasmosome, sided by those in which only the heterochromosome took the colour. In the former one the plasmosome strongly coloured seen in the primary metaphase may easily be taken for a supernumerary chromosome. At anaphase this body stays motionless in the equator of the cell while the chromosomes are moving toward the poles. There, when intensely coloured ,it may be confused with the heterochromosome of the secondary spermatocytes, which frequently occupies identical position in the corresponding phase, thus causing missinterpretation. In its place the plasmosome may divide into two equal parts or pass undivided to one cell in whose cytoplasm it breaks down giving rise to a few corpuscles of unequal sizes. In Pachylis pharaonis, as soon as the nuclear membrane breate down, the plasmosome migrates to a place in the periphery of the cell (primary spermatocyte), forming there a large chromatoid body. This body is never found in the cytoplasm prior to the dissolution of the nuclear membrane. It is certain that chromatoid bodies of different origin do exist. Here, however, we are dealing, undoubtedly, with true plasmosomes. d) Movement of the heterochromosome - The heterochromosome in the metaphase of the secondary spermatocytes may occupy the most different places. At the time the autosomes prient themselves in the equatorial plane it may be found some distance apart in this plane or in any other plane and even in the subpolar and polar regions. It remains in its place during anaphase. Therefore, it may appear at the same level with the components of one of the anaphase plates (synchronism), between both plates (succession) or between one plate and tbe pole (precession), what depends upon the moment the cell was fixed. This does not mean that the heterochromosome sometimes moves as quickly as the autosomes, sometimes more rapidly and sometimes less. It implies, on the contrary, that, being anywhere in the cell, the heterochromosome m he attained and passed by the autosomes. In spite of being almost motionless the heterochromosome finishes by being enclosed in one of the resulting nuclei. Consequently, it does move rapidly toward the group formed by the autosomes a little before anaphase is ended. This may be understood assuming that the heterochromosome, which do not divide, having almost inactive kinetochore cannot orient itself, giving from wherever it stays, only a weak response to the polar influences. When in the equator it probably do not perform any movement in virtue of receiving equal solicitation from both poles. When in any other plane, despite the greater influence of the nearer pole, the influence of the opposite pole would permit only so a slow movement that the autosomes would soon reach it and then leave it behind. It is only when the cell begins to divide that the heterochromosome, passing to one of the daughter cells scapes the influence of the other and thence goes quickly to join the autosomes, being enclosed with them in the nucleus formed there. The exceptions observed by BORING (1907) together with ; the facts described here must represent the normal behavior of the heterocromosome of the Hemiptera, the greater frequency of succession being the consequence of the more frequent localization of the heterochromosome in the equatorial plane or in its near and of the anaphase rapidity. Due to its position in metaphase the heterochromosome in early anaphase may be found in precession. In late anaphase, oh the contrary ,it appears almost always in succession. This is attributed to the fact of the heterochromosome being ordinairily localized outside the spindle area it leaves the way free to the anaphasic plate moving toward the pole. Moreover, the heterochromosome being a round element approximately of the size of the autosomes, which are equally round or a little longer in the direction of the movement, it can be passed by the autosomes even when it stands in the area of the spindle, specially if it is not too far from the equatorial plane. e) The kinetochore - This question has been fully discussed in another paper (PIZA 1943a). The facts treated here point to the conclusion that the chromosomes of the Coreidae, like those of Tityus bahiensis, are provided with a kinetochore at each end, as was already admitted by the present writer with regard to the heterochromosome of Protenor. Indeed, taking ipr granted the facts presented in this paper, other cannot be the interpretation. However, the reasons by which the chromosomes of the species studied here do not orient themselves at metaphase of the first division in the same way as the heterochromosome of Protenor, that is, with the major axis parallelly to the equatorial plane, are claiming for explanation. But, admiting that the proximity of the kinetochores at the ends of chromosomes which do not separate until the second division making them respond to the poles as if they were a single kinetochore ,the explanation follows. (See PIZA 1943a). The median opening of the diplonemas when they are going to the diffuse stage as well as the reappearance of the bivalents always united at the end-segments and open in the middle is in full agreement with the existence of two terminal kinetochores. The same can be said with regard to the bivalents which join their extremities to form a ring.

Reticulo-endotheliomatose maligna

A spindle-cell sarcoma (fig. 5) apparently originating from the dura (fig. 4) was found at the autopsy of a male, mulato, 17 years of age. The bones of the skull (occipital and both parietals) were penetrated and destroyed (fig. 1 and 2). The nervous tissue was not penetrated, the only change in the brain being a depressed area where the tumor was included. Metastatic nodules were found in the liver (fig. 3),hepatic lymphnodes (fig. 14), spleen (fig. 12) and suprarenal bodies (fig. 15). The structure, however, in all those different locations was that of a typical endothelioma (figs. 8, 11 and 13). The cells are of large and moderate size, of polyhedral form, with vesicular nuclei, diminutive nucleoli and clear cytoplasm. (Figs. 6 and 8). They are arranged about a central lumen which represents a rudimentary vessel (figs. 9 and 13). Other areas are composed of cells without concentric arrangement (figs. 4 and 10). In small areas, the colums of liver cells are marginated in one side by typical sinusoids, while in the other side tumor cells arranged about a narrow lumen are seen suggesting a pathological (neoplastic) sinusoid (figs. 7 and 9). The case is considered as a multiple diffuse endothelioma. The origin of the tumor is referred to the reticulo-endothelial apparatus of the liver, the spleen, the suprarenal bodies and the lymph nodes, the structure being rather uniform in those organs. In the dura, the endothelioma reproduces the structure and presents the general character of a fibroblastic sarcoma; in some places, however, the structure of endothelioma could be found (fig.6). It corresponds to the reticulo-endotheliomatosis maligna according to Puhr's grouping of progressive changes in the reticulo-endothelial apparatus which is a follows: 1. HYPERPLASTIC - 1. Mnnocytic leukemia. 2. a) Aleukemic reticulosis (Goldschmid and Isaac). b) Idiopathic sarcoma of skin (Kaposi). c) Cutaneous sarcoid (Spiegler). 3. Secretory reticulosis. a) Gaucher's disease. b) Generalized xanthomatosis. c) Spleno-hepatomegaly with lipoidic cells (Pick). II. BLASTOMATOSUS OR NEOPLASTIC - 1. Benign - a) Circumscribed tumors. a) Epulis sarcomatosa; b) Benign giant-cells sarcoma of the bone - marrow of long bones. b) Generalized brown tumors of osteitis fibrosa. 2. Malignant - a) Circumscribed haemangio - endothelioma (reticulo- endothelioma (maligum). of {liver, spleen, bone-marrow. b) Generalized haemangio-endotheliomatosis (reticulo-endotheliomatosis maligna) (Grabowski).

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.