Academic literature on the topic 'HIV pathogenicity'

Divalent cations are essential for life and are fundamentally important coordinators of cellular metabolism, cell growth, host-pathogen interactions, and cell death. Specifically, for human immunodeficiency virus type-1 (HIV-1), divalent cations are required for interactions between viral and host factors that govern HIV-1 replication and pathogenicity. Homeostatic regulation of divalent cations’ levels and actions appear to change as HIV-1 infection progresses and as changes occur between HIV-1 and the host. In people living with HIV-1, dietary supplementation with divalent cations may increase HIV-1 replication, whereas cation chelation may suppress HIV-1 replication and decrease disease progression. Here, we review literature on the roles of zinc (Zn2+), iron (Fe2+), manganese (Mn2+), magnesium (Mg2+), selenium (Se2+), and copper (Cu2+) in HIV-1 replication and pathogenicity, as well as evidence that divalent cation levels and actions may be targeted therapeutically in people living with HIV-1.

To analyze the mechanism of Tat-mediated HIV pathogenicity, we produced a Drosophila melanogaster strain transgenic for HIV-tat gene and induced the expression of the protein during Drosophila development. By in vitro and in vivo experiments, we demonstrated that Tat specifically binds to tubulin via the MAP-binding domain of tubulin, and that this interaction delays the polymerization of tubulin and induces a premature stop to microtubule-dependent cytoplasmic streaming. The delay in the polymerization of microtubules, the tracks for the transport of the axes determinants, alters the positioning of the dorso-ventral axis as shown by the mislocalization of Gurken and Kinesin in oocyte of Drosophila after Tat induction. These results validate the use of Drosophila as a tool to study the molecular mechanism of viral gene products and suggest that Tat-tubulin interaction is responsible for neurodegenerative diseases associated with AIDS.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is frequently attenuated after long-term culture in vitro. The attenuation process probably involves mutations of functions required for replication and pathogenicity in vivo. Analysis of attenuated HIV-1 for replication and pathogenicity in vivo will help to define these functions. In this study, we examined the pathogenicity of an attenuated HIV-1 isolate in a laboratory worker accidentally exposed to a laboratory-adapted HIV-1 isolate. Using heterochimeric SCID-hu Thy/Liv mice as an in vivo model, we previously defined HIV-1 env determinants (HXB/LW) that reverted to replicate in vivo (L. Su, H. Kaneshima, M. L. Bonyhadi, R. Lee, J. Auten, A. Wolf, B. Du, L. Rabin, B. H. Hahn, E. Terwilliger, and J. M. McCune, Virology 227:46–52, 1997). Here we further demonstrate that HIV-1 replication in vivo can be separated from its pathogenic activity, in that the HXB/LW virus replicated to high levels in SCID-hu Thy/Liv mice, with no significant thymocyte depletion. Restoration of the nef gene in the recombinant HXB/LW genome restored its pathogenic activity, with no significant effect on HIV-1 replication in the thymus. Our results suggest that in vitro-attenuated HIV-1 lacks determinants for pathogenicity as well as for replication in vivo. Our data indicate that (i) the replication defect can be recovered in vivo by mutations in the envgene, without an associated pathogenic phenotype, and (ii)nef can function in the HXB/LW clone as a pathogenic factor that does not enhance HIV-1 replication in the thymus. Furthermore, the HXB/LW virus may be used to study mechanisms of HIV-1nef-mediated pathogenesis in vivo.

Most aspects of glycobiology play important roles in the ‘life’ of viruses, for example in the correct folding of their envelope glycoproteins as well as in immune representation and escape. The pathogenicity of three major human pathogens, HCV (hepatitis C virus), HBV (hepatitis B virus) and HIV, which collectively infect more than 560 million people worldwide, is dependent on their glycoproteins. As the sugars (or glycans) that the viruses rely on are supplied by the host cell, we can exploit our knowledge of glycobiology to target this apparent Achilles' heel of these viruses.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of cellular and viral proteins. The functions of many of these interactions in the pathogenesis of HIV-1 have been identified. Deletion of the vpr gene reduces the virulence of HIV-1 dramatically, indicating the importance of this protein for the virus. This review describes the current findings on several established functions of HIV-1 Vpr and some possible roles proposed for this protein. Because Vpr exploits cellular proteins and pathways to influence the biology of HIV-1, understanding the functions of Vpr usually involves the study of cellular pathways. Several functions of Vpr are attributed to the virion-incorporated protein, but some of them are attributed to the expression of Vpr in HIV-1-infected cells. The structure of Vpr may be key to understanding the variety of its interactions. Due to the critical role of Vpr in HIV-1 pathogenicity, study of the interactions between Vpr and cellular proteins may help us to understand the mechanism(s) of HIV-1 pathogenicity.

Introduction: Blastocystis spp. is a protist found in humans. Although usually the most frequent protozoa found in stool samples of both symptomatic and healthy subjects, its pathogenic or rather opportunistic role is yet to be clearly elucidated. To attempt to fill this gap, a cross-sectional study was conducted to compare the frequency of Blastocystis spp. in HIV positive (HIV+) versus HIV negative (HIV-) individuals in four health facilities of the Center Region of Cameroon. Methodology: Stool samples were collected from 283 HIV positive and 245 HIV negative subjects and analyzed using direct diagnostic tests. Results: A total of 46 (8.7%) individuals were found infected with Blastocystis spp., including 6.7% HIV positive and 11.0% HIV negative. This species was more frequent in urban and semi-urban areas than in rural areas, but evenly distributed among genders and age groups as well as among all sectors of activity. The prevalence of Blastocystis spp. (11.3%) was higher in HIV+ patients with a CD4 count ≥ 500 cells / mm3, but no significant difference was found among HIV clinical stages. Likewise prevalence, the mean number of cysts per gram of stool was similar between HIV positive and HIV negative individuals. People infected with Blastocystis spp. showed diverse clinical signs, but only flatulence was significantly more prevalent. The frequencies of these clinical signs were not related to HIV status. Conclusion: No clear relationship links the infection with Blastocystis spp. to HIV, although its presence was associated with digestive disorder, suggesting that this parasite might not be opportunist.

Depuis la découverte du virus de l'Immunodéficience humaine, un lentivirus, comme agent pathogène responsable de l'épidémie du SIDA en 1983, beaucoup de progrès sur le sujet ont été réalisés. Il existe deux types de virus différents pouvant infecter l'Homme, le HIV-1 et le HIV-2. Ces deux virus se regroupent en différents groupes et sous-types qui témoignent d'une grande diversité inter et intra individus (notions de quasi-espèces). La découverte de lentivirus infectant naturellement au moins quarante-cinq espèces de primates en Afrique sub-saharienne, a permis un enrichissement des connaissances sur les origines des épidémies lentivirales humaines. Aujourd'hui , il est clairement admis que l'origine des épidémies d'HIV-1 et HIV-2 sont le résultat de transmissions zoonotiques de virus de chimpanzés/gorilles et de mangabeys enfumées, respectivement. La mise en évidence de nombreux SIV circulant chez ces primates non-humains indique bien le risque potentiel de nouvelles zoonoses dans la population humaine exposée, cependant, il peut paraître surprenant que jusqu'à maintenant, deux lignées lentivirales seulement ont été capables de franchir cette barrière d'espèce. Pour pouvoir se répliquer dans les cellules d'un nouvel hôte, un lentivirus doit pouvoir contrecarrer les différents facteurs de restriction exprimés par les cellules cibles tout en exploitant au maximum la machinerie cellulaire. La famille de protéines TRIM5, APOBEC3 et les protéines Tetherin/Bst2 et SAMHD1 sont capables de bloquer une infection rétrovirale. Dans ce travail, le rôle des protéines TRIM5 a été étudié ainsi que celui d'autres protéines interagissant avec des capsides rétrovirales, dans un contexte de transmission inter-espèces de lentivirus de primates. L'étude de TRIM5α humain a montré que cette protéine n'était capable de bloquer aucune des infections par les lentivirus primates testés dans cette étude, ni par les autres SIV. Nous avons pu mettre en évidence que la dépendance de la liaison à la Cyclophiline A pour l'infection des différents SIV était variable en fonction de la capside testée. Ainsi, si cette interaction est largement répandue parmi les différentes lignées de SIV, elle n'est toutefois pas universelle. La sensibilité des SIV à la déplétion de nucléoporines qui sont connues pour affecter l'infection par HIV-1, était également variable pour différents SIV, et la même diversité a été observée concernant les déplétions de RanBP2 et Nup153. De plus, nous avons découvert une capside de SIV soumise à une forte restriction de son infection dans les cellules humaines, ce phénotype a été nommé Ref2.Il a été suggéré qu'il existait une possible corrélation entre des variations de la capside de HIV-2 et la progression vers le SIDA, nous avons donc élaboré une étude afin de déterminer si les protéines TRIM5 étaient impliquées dans ce phénotype. La conclusion est que TRIM5α humaine ne restreint fortement aucune des capsides de HIV-2 testées provenant d'une cohorte d'individus à “progression rapide“ ou “lente“ vers le SIDA. Cependant nous avons observé une capacité d'infection qui corrélait avec la pathogénicité. Il est intéressant de noter que toutes les capsides d'HIV-2 testées étaient dépendantes de la présence de Cyclophiline A pour leur infection. Toutes ces capsides étaient sensibles à la déplétion de RanBP2, et l'interaction est très probablement médiée par le motif C-ter de RanBP2 qui a une forte homologie avec la Cyclophiline A. En conclusion, il est très probable que des SIV infectant naturellement des singes puissent utiliser les mêmes protéines que HIV-1, pour un éventuel passage inter-espèces. TRIM5α ne semble pas être une barrière efficace aux différents SIV, et l'interaction avec la Cyclophiline A est probablement très conservée par les lentivirus primates
Ever since HIV has been discovered to be the pathogenic agent that causes AIDS in 1983, much progress has been made in the field. Two different viruses are now known to infect humans, HIV-1 and HIV-2. These two distinct viruses have many sub-types and clades representing a high diversity inter and intra-individuals (quasi-species). The finding of HIV simian counterparts, the Simian Immunodeficiency Viruses (SIVs), has broadened the knowledge of primate lentiviruses and to date forty-five species of non-human primates are known to be infected with SIVs in sub-saharan Africa. It is now clear that HIV-1 and HIV-2 epidemics are the result of zoonosis from chimpanzees/gorillas and sooty mangabeys, respectively. With such a big diversity of SIVs in the wild and a frequent contact of SIV infected monkey species with humans, it is interesting that so far, only two lineages breached the species barrier and infected human populations. To be able to correctly infect a cell, a lentivirus has to overcome the installed cellular barriers known as restriction factors while at the same time correctly exploiting the established host cellular machinery. Proteins such as TRIM5, APOBEC3, Tetherin/Bst2, SAMHD1 are able to restrict retroviral infections in certain conditions. In this thesis, it has been evaluated the role of TRIM5 proteins and other capsid interacting proteins with a scope to the eventuality of a cross-species transmission infection. The results showed that human TRIM5alpha does not restrict any of the primate lentiviruses tested, and so far, no primate lentivirus is known to be restricted by it. Cyclophilin A binding and dependence is variable depending on the SIV capsid; this interaction is widespread among the primate lentiviruses phylogenetic tree but not a universal phenotype. Different capsids from SIVs have been tested for the sensitivity to the depletion of nucleoporins that are known to be used by HIV-1 in its infection; it has been concluded that the same diversity applies to the interaction with RanBP2 and Nup153. Additionally, we identified a SIV capsid that is highly restricted in human cells; this phenotype was called Ref2. With the report of a possible correlation between HIV-2 capsid variations and different levels of progression to AIDS, we devised a study aiming to identify if TRIM5 proteins were involved in this phenotype. We concluded that human TRIM5alpha does not restrict any HIV-2 capsid obtained from a HIV-2 cohort, in which individuals were presenting different levels of progression to AIDS. However, we observed a different viral fitness that correlated with pathogenicity. Moreover, Cyclophilin A dependence seems ubiquitous among all of the tested HIV-2 capsids. All of these capsids are sensitive to RanBP2 depletion and the interaction is much likely mediated by RanBP2's C-terminal motif that shares a high homology with Cyclophilin A. Summing up, it is much likely that some SIVs that still circulate in the wild can hijack the same specific cellular co-factors as HIV-1 to produce a new epidemic in humans. TRIM5α does not seem to be a potent barrier to an eventual cross-species transmission from lower primates to humans, and Cyclophilin A interaction seems to play a major role to the infection of some SIVs

Estudou-se prospectivamente o comportamento das calcificações, da atrofia, das alterações da substância branca e alterações vasculares nas imagens de tomografia computadorizada de crânio de 162 crianças e adolescentes infectados pelo vírus da imunodeficiência humana (HIV) por transmissão vertical e que estavam ou estiveram em acompanhamento clínico no Ambulatório de Infectologia Pediátrica do Instituto da Criança do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, entre 1992 e 2002. Analisaram-se as possíveis correlações entre estas alterações e seu aspecto evolutivo. Para tal finalidade, foram avaliadas 606 tomografias computadorizadas de crânio (média de 3,74 exames por paciente), as quais constituíram o grupo de estudo. Após a caracterização quanto à presença ou não das alterações supracitadas, e suas possíveis inter-relações, realizou-se a análise estatística dos resultados obtidos através do teste exato de Fisher com nível de significância de 5%. Posteriormente, os mesmo aspectos foram avaliados em função do seu comportamento evolutivo em um subgrupo de 61 pacientes (média, 4,18 exames por paciente, totalizando 321 exames tomográficos). Estes pacientes tinham, pelo menos, quatro estudos tomográficos seriados (com intervalo mínimo de noventa dias entre os exames subseqüentes e pelo menos dois anos de intervalo total entre o primeiro e o último exame). As alterações tomográficas foram abordadas individual e qualitativamente segundo o critério de presença e intensidade. Inicialmente, o conjunto dos resultados foi tratado de forma individual (para cada paciente) e, depois, em relação à totalidade do grupo em questão. As calcificações foram encontradas em 46,30% dos pacientes; a atrofia, em 37,65%; as alterações da substância branca, em 25,93%; as anomalias vasculares, em 25,19%. Constatou-se uma correlação significativa entre as alterações de substância branca e a atrofia, bem como entre as calcificações e as alterações vasculares. A análise evolutiva destas características demonstrou haver um acréscimo significativo das alterações entre o momento inicial e o quarto momento no conjunto das alterações, sobretudo para as calcificações e para as alterações vasculares. Concluiu-se que as calcificações e a atrofia foram as alterações mais freqüentes nesta série de crianças e adolescentes com HIV adquirido por transmissão vertical. A atrofia e as alterações da substância branca apresentaram uma inter-relação importante na amostra descritiva, assim como as alterações vasculares e as calcificações mostraram uma associação evolutiva significativa em relação à sua progressão
We prospectively studied the behavior of calcifications, atrophy, white matter and vascular abnormalities on the images of computed tomography (CT) of 162 children and adolescents infected with the human immunodeficiency virus (HIV) acquired by vertical transmission, who are or were clinically followed in the Ambulatory of Pediatric Infectology of the Children Institute at the Clinics Hospital of University of São Paulo Medical School, from 1992 to 2002. We analyzed the possible correlation between these abnormalities, as well as, their evolutive aspects. For this purpose, we evaluated 606 CT scans (mean 3.74 exams per patient), which composed the group of study. After the characterization according to the presence or not of the anomalies mentioned above, and their possible inter-relations, we performed a statistical analysis of the obtained results with the Fisher test with a level of significance below 5%. Later, these aspects were evaluated regarding its evolutive behavior in a subgroup of 61 patients (mean, 4.18 exams per patient, summing 321 exams). These patients had, at least, four serial cranial CT (with minimum interval of ninety days between the subsequent exams and, at least, two years of total interval between the first and the fourth exam). The cranial CT abnormalities presented were assessed individually as absent or present. Initially, the set results were assessed individually (for each patient) and, later in relation to the totality of the group. Calcifications were found in 46.30% of all patients, atrophy in 37.65%, white matter abnormalities in 25.93% and vascular anomalies in 25.19%. We found a significant correlation between white matter abnormalities and atrophy, as well as, between calcifications and vascular anomalies. Evolutive analysis of these characteristics demonstrated a significant increase of the abnormalities between the first and the fourth moment, with emphasis to the calcifications and vascular anomalies. We concluded that, calcifications and atrophy were the most frequent abnormalities in this series of children and adolescents with HIV acquired by vertical transmission. Atrophy and white matter abnormalities presented a significant correlation in the descriptive sample, as well as, vascular anomalies and calcifications that also demonstrated a significant evolutive association regarding its progression

Further functional investigation revealed that knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells, concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium Conversely, overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta 1 (IFN-beta1). In this connection, IFN-beta1-mediated 2', 5'-oligoadenylate synthetase (OAS) and ribonuclease L (RNase L) signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover, GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p= 0.019) as compared with their counterpart pre-treatment liver biopsies.
Hepatitis B virus (HBV) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although considerable progress has been made over the past decade, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive.
In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via IFN-beta1-OAS-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.
In this study, we applied a two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach to globally analyze the host early response to HBV by using an inducible HBV-producing cell line HepAD38. Twenty-three proteins were identified as differentially expressed, with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, as well as in HBV-infected human liver biopsies by immunohistochemistry.
Ma, Yan.
Adviser: Ming-Liang He.
Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 111-129).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

by Shuk-kei Lau.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.
Includes bibliographical references (leaves 141-148).
Abstracts in English and Chinese.
Acknowledgement --- p.i
Table of Contents --- p.ii
Abstract --- p.vi
論文摘要 --- p.viii
Abbreviations --- p.ix
List of Figures --- p.x
List of Tables --- p.xii
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- General introduction --- p.1
Chapter 1.2 --- HBV and its role in hepatocarcinogenesis --- p.3
Chapter 1.2.1 --- Current situation of HBV infection and the HCC incidencein the world --- p.3
Chapter 1.2.2 --- Current situation of HBV infection and the HCC incidencein Hong Kong --- p.4
Chapter 1.2.3 --- Genetic organization of HBV --- p.4
Chapter 1.2.4 --- Principle of hepatocarcinogenesis induced by HBV --- p.5
Chapter 1.2.4.1 --- Role of chronic hepatitis in hepatocarcinogenesis --- p.5
Chapter 1.2.4.2 --- Role of HBV in hepatocarcinogenesis --- p.6
Chapter 1.2.5 --- Current screening tests for HCC --- p.7
Chapter 1.2.6 --- Current therapies for HCC --- p.9
Chapter 1.3 --- Aim of the present study --- p.13
Chapter 1.4 --- "Combining Expressed Sequence Tag (EST), Suppression Subtractive Hybridization and cDNA microarray for rapid differentially by expressed genes screening" --- p.14
Chapter 1.4.1 --- Expressed Sequence Tag (EST) --- p.14
Chapter 1.4.2 --- cDNA subtraction --- p.15
Chapter 1.4.3 --- cDNA microarray --- p.16
Chapter Chapter 2 --- Materials and Methods
Chapter 2.1 --- PCR-select cDNA subtraction --- p.17
Chapter 2.1.1 --- Amplification of subtracted cDNA clones by PCR --- p.17
Chapter 2.1.2 --- Cycle sequencing of subtracted cDNA clones --- p.18
Chapter 2.1.3 --- Sequence analysis using BLAST server and Stanford Online Universal Resource for Clones and ESTs (SOURCE) --- p.19
Chapter 2.2 --- cDNA microarray analysis --- p.20
Chapter 2.2.1 --- Array fabrication --- p.20
Chapter 2.2.1.1 --- Amplification of cDNA clones by PCR --- p.20
Chapter 2.2.1.2 --- Purification of PCR products --- p.21
Chapter 2.2.1.3 --- Cycle sequencing for clones checking --- p.22
Chapter 2.2.2 --- Microarray printing --- p.22
Chapter 2.2.2.1 --- Preparation of cDNA target --- p.22
Chapter 2.2.2.2 --- Arraying --- p.22
Chapter 2.2.3 --- Screening of differentially expressed genes in hepatocellular carcinoma and its surrounding normal counterpart by cDNA microarray --- p.23
Chapter 2.2.3.1 --- Extraction of RNA --- p.23
Chapter 2.2.3.2 --- RNA labeling --- p.24
Chapter 2.2.3.3 --- Microarray hybridization --- p.26
Chapter 2.2.3.4 --- Collection of data --- p.27
Chapter 2.2.3.5 --- Data normalization and analysis --- p.28
Chapter 2.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.30
Chapter 2.3.1 --- Tissue distribution of T2L522 gene --- p.30
Chapter 2.3.1.1 --- Northern hybridization --- p.30
Chapter 2.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.33
Chapter 2.3.2 --- Expression level of T2L522 in HCC and its surrounding normal counterpart --- p.33
Chapter 2.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.35
Chapter 2.3.3.1 --- "Cloning of T2L522 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.35
Chapter 2.3.3.2 --- Transformation of yeast competent cells --- p.39
Chapter 2.3.3.3 --- Mating of T2L522-BD with pretransformed human liver cDNA library --- p.40
Chapter 2.3.3.4 --- Colony lift p-galactosidase filter assay --- p.42
Chapter 2.3.4 --- Subcellular localization of T2L522 gene by tagging with green fluorescence protein (GFP) --- p.43
Chapter 2.3.4.1 --- "Cloning of T2L522 gene into the eukaryotic GFP expression vector, pEGFP-Cl" --- p.43
Chapter 2.3.4.2 --- Transfection of pEGFP-T2L522 into HepG2 cell --- p.43
Chapter Chapter 3 --- Results
Chapter 3.1 --- PCR-select cDNA subtraction --- p.45
Chapter 3.1.1 --- The sequencing results of subtracted-HCC cDNA clones --- p.45
Chapter 3.1.2 --- Categorization of ESTs sequenced from subtracted-HCC library --- p.45
Chapter 3.2 --- Microarray analysis --- p.49
Chapter 3.2.1 --- Array fabrication --- p.49
Chapter 3.2.1.1 --- Amplification of cDNA microarray targets --- p.49
Chapter 3.2.2 --- Microarray printing --- p.52
Chapter 3.2.3 --- Microarray analysis of differentially expressed genesin hepatocellular carcinoma and its surrounding normal counterpart --- p.55
Chapter 3.2.4 --- Data collection --- p.57
Chapter 3.2.5 --- Image processing: spots finding and quantitation --- p.61
Chapter 3.2.6 --- Data normalization and analysis --- p.61
Chapter 3.3 --- Molecular cloning and characterization of a novel cDNA clone differentially expressed in HCC --- p.73
Chapter 3.3.1 --- Tissue distribution of T2L522 --- p.77
Chapter 3.3.1.1 --- Northern hybridization --- p.77
Chapter 3.3.1.2 --- Reverse-transcriptase polymerase chain reaction (RT-PCR) --- p.79
Chapter 3.3.2 --- Expression level of T2L522 in hepatocellular carcinoma and its surrounding normal counterpart --- p.81
Chapter 3.3.3 --- Identification of interacting partner of T2L522 using yeast two-hybrid assay --- p.85
Chapter 3.3.4 --- Subcellular localization of GFP tagged T2L522 --- p.87
Chapter Chapter 4 --- Discussion
Chapter 4.1 --- EST analysis on subtracted-HCC cDNA library --- p.89
Chapter 4.2 --- cDNA microarray analysis --- p.92
Chapter 4.2.1 --- Generation of reliable data using cDNA microarray --- p.92
Chapter 4.2.1.1 --- Reproducibility of signal and normalized ratio --- p.92
Chapter 4.2.2 --- Comparison of data between multiple slides --- p.96
Chapter 4.2.2.1 --- Assession of data quality and statistical significance --- p.96
Chapter 4.2.2.2 --- Interpretation of gene expression data from single and multiple hybridizarion --- p.97
Chapter 4.3 --- Candidate genes differentially expressed in HCC and its surrounding normal counterpart --- p.99
Chapter 4.3.1 --- Protein up-regulated in HCC --- p.99
Chapter 4.3.1.1 --- Extracellular matrix protein --- p.99
Chapter 4.3.1.2 --- Protein involved in other metabolism --- p.100
Chapter 4.3.1.3 --- Protein involved in transcription and translation --- p.100
Chapter 4.3.2 --- Protein down-regulated in HCC --- p.101
Chapter 4.3.2.1 --- Membrane associated protein --- p.101
Chapter 4.3.2.2 --- Protein involved in other metabolism --- p.102
Chapter 4.3.2.2 --- Secretory protein --- p.104
Chapter 4.3.3 --- Novel protein differentially expressed in HCC --- p.107
Chapter 4.4 --- "TBC1 domain containing protein, T2L522" --- p.108
Chapter 4.4.1 --- Possible involvement of T2L522 gene in HCC --- p.109
Chapter 4.4.2 --- Tissue distribution and expression pattern of T2L522 --- p.110
Chapter 4.4.3 --- Potential interacting partner of T2L522 --- p.110
Chapter 4.4.4 --- Subcellular localization of T2L522 --- p.112
Chapter 4.5 --- Summary --- p.113
Appendix --- p.114
References --- p.141

Human diseases caused by single-stranded, positive-sense RNA viruses, are among the deadliest of the 21st Century. In particular, there are two notable standouts: human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Detection of these disease-causing viral transcripts, by next-generation RNA sequencing (RNA-Seq), represents the most immediate opportunity for advances in diagnostic, therapeutic, and preventive applicability in infectious diseases (e.g., AIDS and COVID-19). Moreover, RNA-Seq technologies add significant value to public health studies by first, providing real-time surveillance of known viral strains, and second, by the augmentation of epidemiological databases, construction of annotations and classifications of novel sequence variants. This chapter intends to recapitulate the current knowledge of HIV and SARS-CoV-2 transcriptome architecture, pathogenicity, and some features of the host immune response. Additionally, it provides an overview of recent advances in diagnostic sequencing methodologies and discusses the future challenges and prospects on the utilization of RNA-Seq technologies.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium accountable for causing life-threatening infections in humans. According to the World Health Organization, P. aeruginosa classified as a critical pathogen. Specifically, P. aeruginosa in its colonized or biofilm state presents a major infection threat to immunocompromised (HIV) patients, Cystic fibrosis, burns, wounds and surgery associated infection. It is also a common pathogen responsible for causing hospital acquired/nosocomial infection and Urinary tract infections. P. aeruginosa biofilm is made up of bacterial self-synthesized biomolecules includes extracellular DNA, polysaccharides, proteins, RNA, siderophores and metabolites such as pyocyanin. This chapter will elaborate the manifold functions of P. aeruginosa secreted biomolecules in establishing and stabilizing biofilms, triggering virulence and pathogenicity in host, and resisting antibiotics and antibacterial agents.